mTOR inhibitor

Research progress of mTOR inhibitors

Yifan Chen, Xiaoping Zhou*
School of Pharmaceutical Sciences, Jilin University, Changchun, 130021, China

Abstract

Mammalian target of rapamycin (mTOR) is a highly conserved Serine/Threonine (Ser/Thr) protein kinase, which belongs to phosphatidylinositol-3-kinase-related kinase (PIKK) protein family. mTOR exists as two types of protein complex: mTORC1 and mTORC2, which act as central controller regulating processes of cell metabolism, growth, proliferation, survival and autophagy. The mTOR inhibitors block mTOR signaling pathway, producing anti-inflammatory, anti-proliferative, autophagy and apoptosis induction effects, thus mTOR inhibitors are mainly used in cancer therapy. At present, mTOR inhibitors are divided into four categories: Antibiotic allosteric mTOR inhibitors (first generation), ATP-competitive mTOR in- hibitors (second generation), mTOR/PI3K dual inhibitors (second generation) and other new mTOR in- hibitors (third generation). In this article, these four categories of mTOR inhibitors and their structures, properties and some clinical researches will be introduced. Among them, we focus on the structure of mTOR inhibitors and try to analyze the structure-activity relationship. mTOR inhibitors are classified according to their chemical structure and their contents are introduced systematically. Moreover, some natural products that have direct or indirect mTOR inhibitory activities are introduced together. In this article, we analyzed the target, binding mode and structure-activity relationship of each generation of mTOR inhibitors and proposed two hypothetic scaffolds (the inverted-Y-shape scaffold and the C-shape scaffold) for the second generation of mTOR inhibitors. These findings may provide some help or reference for drug designing, drug modification or the future development of mTOR inhibitor.

1. Introduction

Mammalian target of rapamycin (mTOR) is a highly conserved eukaryotic Serine/Threonine (Ser/Thr) protein kinase, which be- longs to phosphatidylinositol 3-kinase-related kinase (PIKK) family. In 1991, mTOR was found by Heitman et al. [1] when they were studying at the mechanism and drug resistance formation of a macrolide antibiotic called Rapamycin. mTOR control protein biosynthesis by controlling transcription, translation and ribosomal biosynthesis, and then control cell growth and size [2], therefore mTOR was called central controller of cell growth [3]. On the other hand, protein synthesis is important to cell division, thus inhibiting mTOR can cause cell cycle arrest at G1 phase [1], which provide a significant direction to new drug development at anticancer, anti- restenosis and immunosuppression etc. Recently many de- rivatives of rapamycin and other types of mTOR inhibitors have been reported, here we review mTOR inhibitors that have been reported at present, as for a reference.

2. An overview of mTOR

In 1900s when different laboratories separated mTOR, due to its target to cytoplasmic receptor FKBP12 and Rapamycin, mTOR was once called FKBP12-Rapamycin associated protein (FRAP) [4], Rapamycin and FKBP12 target (RAFT) [5] and Rapamycin target (RAPT) [6]. Nowadays we use mTOR, which was named by Sabers et al. [7] to represent this protein kinase.

2.1. Structure of mTOR

Human mTOR is encoded by a highly conserved DNA sequence containing 166,963 base pairs, which is located in 1p36.22 (NCBI Gene ID: 2475). mTOR gene express mature mTOR after tran- scription, splicing, translation and modification. Mature mTOR is composed of 2549 amino acid residues, with a molecular weight of 289,000 and at least 95% of similarity with mice and rats in the level of amino acid sequence [8].
Structurally, in C-terminal, mTOR shows a high homology with the catalytic domain of phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol 4-kinase (PI4K), therefore mTOR belongs to PIKK protein family [8,9]. In N-terminal there is a domain consists of 20 tandem HEAT (Huntingtin, EF3, the A subunit of PP2A, TOR1) repeats, which is divided into 2 groups, each HEAT unit consists of 40 amino acid residues and forms 2 a-helixes (Fig. 1A). When fol- ded, HEAT domain forms hydrophobic surface which may plays a role in protein interaction and membrane anchoring [10].

Moreover, many eukaryotic regulatory cytoplasmic proteins involved in cytoplasmic transportation contain HEAT repeats [10], suggesting that HEAT repeats are probably related to cytoplasmic transportation. Downstream of HEAT repeats is FAT(FRAP-ATM- TRAPP) domain, there is also a similar FAT domain in C-terminal called FATC (FAT C-terminal) domain. These two domains get close, expose functional sequences and form a scaffold-like stabilized interaction in protein complex when folded [11] (Fig. 1B). FRB (FKBP12-Rapamycin Binding) domain, Ser/Thr kinase domain and negative regulatory domain are between FAT domain and FATC domain. Ser/Thr kinase domain is the activity center of mTOR, which can phosphorylate substrate proteins at Ser/Thr sites and accomplish signal transduction or functional regulation when activated. FRB domain is FKBP12-Rapamycin complex binding site of mTOR, which is close to the ATP binding site of kinase domain (Fig. 1C). Rapamycin and a cytoplasmic receptor FKBP12 compose to a complex, then binds to FRB domain, which allosterically inhibits mTOR activity [2,12]. Negative regulatory domain is the down- regulatory region of mTOR, also called autoinhibitory, is located downstream of kinase domain. Overall, the domain distribution of mTOR from N-terminal is two groups of HEAT repeats, FAT domain, FRB domain, Ser/Thr kinase domain, negative regulatory domain and FATC domain (Fig. 1D).

Human mTOR can assemble with different proteins and form two types of polyprotein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Early studies showed that only mTORC1 is sensitive to Rapamycin [13e15]. However, follow-up research reported that prolonged Rapamycin treatment can inhibit mTORC2 assembly and function in some cell lines [16]. mTORC1 consists of 5 subunits including mTOR, Raptor (Regulatory associated protein of mTOR), PRAS40 (Proline-rich AKT substrate 40ku), mLST8 (Mammalian LST8, also called G protein b-subunit- like protein, GbL) and Deptor (DEP domain-containing-mTOR- interacting protein) [17]. mTORC2 consists of 6 subunits including mTOR, Deptor, mLST8, Rictor (Rapamycin insensitive companion of mTOR), mSin1 (mitogen-activated protein kinase-associated pro- tein 1) and Protor (Protein observed with Rictor) [18,20] (Fig. 1F). Raptor and Rictor are subunits that distinct mTORC1 and mTORC2, which directly bind to mTOR and act as a scaffold and binding site of substrate and regulatory subunits [18,21e27]. In mTORC1, PRAS40 binds to Raptor as a negative regulatory subunit [26,28], mLST8 and Deptor directly bind to mTOR as positive and negative regulatory subunit respectively [18,29]. In mTORC2, mLST8 and Deptor behave like those in mTORC1, Protor responsible for assisting complex assembly [27,30] and mSin1 responsible for locating [31,32] bind to Rictor. Recently there are evidences suggesting that mTORC1 and mTORC2 exist as dimers [17e20] (Fig. 1G; 1H).

2.2. Signaling pathway and regulation of mTOR
2.2.1. mTORC1 signaling, regulation and effect
2.2.1.1. Upstream signaling and regulation of mTORC1. mTORC1 as a signal processor, receives upstream signals from nu- trients,
growth factors, energy and stress, process these signals and submit to downstream effectors.

Amino acids are essential materials for protein biosynthesis, DNA biosynthesis, gluconeogenesis and energy supply. In some cultured cells, amino acids are essential and irreplaceable for normal mTORC1 signaling transduction [33,34]. Existent researches have proven that amino acids regulate mTORC1 by a small GTPase called Rag family [25,35] (Fig. 2A). When amino acids are present, Rag turns to active conformation, which is easy to interact with Raptor and recruit mTORC1 to the surface of late endosome and lysosome where the Rag gathers [25,35,36]. Subcellular relocation and redistribution of mTORC1 increases possibility of mTORC1 interact with another small GTPase called Rheb, which is an essential activator of mTORC1 controlled by growth factor [25,37,38]. And therefore, amino acids are essential activator of mTORC1.

For multicellular organisms, cells with different functions have different growth needs, therefore it is particularly important to coordinate the distribution of nutrients in the body. Mammals control growth through controlling growth factors and their intracellular signaling transduction. The quantity of growth factor receptors (GFRs) determines the intensity of signal received and the effect produced in different types of cell. Taking insulin as example, when insulin or insulin-like growth factor (IGF) bind to its re- ceptors, it causes conformation changes of intracellular part, which activates PI3K signaling pathway and the phosphorylation activa- tion of Akt (also known as PKB). When Akt is inactivated, Tuberin protein (TSC2) and Hamarin protein (TSC1) make up tuberous sclerosis complex (TSC). The function of TSC on Rheb is similar to GTPase-activating protein (GAP), which is driving the loaded GTP on Rheb turn to GDP and inactivating Rheb, thus closing the mTORC1 signaling pathway [39e44]. Activated Akt phosphorylates TSC2 and inhibits GAP activity of TSC, promoting mTORC1 activa- tion [45,46]. Meanwhile, Akt phosphorylates PRAS40 subunit of mTORC1, binding it to 14-3-3 protein and inhibiting its negative regulation [26,28,47] (Fig. 2B). In addition, the inherent phos- phorylation of activated mTORC1 also phosphorylates PRAS40 subunit, forming a positive feedback loop [48,49] (Fig. 2C). In addition to the above PI3K-Akt-mTOR signaling pathway, growth factors can affect mTORC1 through other bypasses. Some growth factors, such as epidermal growth factor (EGF) can activate extra- cellular signal-regulated kinase (Erk) through activating Ras-Raf- Erk axis, then phosphorylates to inhibit TSC activity and activate mTORC1 [50] (Fig. 2D). Besides, glycogen synthase kinase 3 (GSK3) can activate TSC, and its upstream Wnt protein signal can inhibit GSK3, thus overall, signals from Wnt protein can activate mTORC1 [51,52] (Fig. 2E).

Intracellular ATP content reflects cellular energy state, mTORC1 signaling pathway can sense the intracellular low ATP state and regulate cell metabolism through AMP-activated protein kinase (AMPK) to adapt to the low energy state [53,54], assisting cell survival. AMP and ATP are both allosteric regulators of AMPK, when cells are in a low energy state, AMP/ATP ratio rises, AMPK is acti- vated, which, together with GSK3, phosphorylate TSC and activate its GAP activity, thus inhibiting downstream Rheb and mTORC1 and reducing metabolism [52,55] (Fig. 2F). In addition, AMPK can also directly phosphorylate Raptor, binding it to 14-3-3 protein and allosterically inhibit mTORC1 [56].
Stimulations from stressors can trigger stress response that drive cells to draw on the advantages and avoid disadvantages, assisting cell survival. Many stressors can change intracellular ATP level and regulate mTORC1 through AMP-AMPK axis. LKB1 protein affect cell metabolism by regulating AMPK activity (Fig. 2G), in Peutz-Jeghers syndrome (PJS) and some benign tumors, cell growth and proliferation might be related to abnormal LKB1-AMPK-TSC- mTORC1 signaling pathway caused by LKB1 gene mutation [57,58]. When hypoxia, decreased ATP level caused by mitochondrial res- piratory dysfunction can activate AMPK and down-regulate mTORC1. Meanwhile, hypoxia induces REDD1 gene expression [59], and inhibits mTORC1 through TSC related mechanism [60] (Fig. 2H). When DNA damages, p53 family protein expression is up- regulated, it has been reported that p53 can down-regulate mTORC1 activity directly by activating TSC or indirectly by acti- vating AMPK, IGF-BP3 and PTEN [61] (Fig. 2I; 2J; 2K; 2L). In addition, DNA damage can also induce the expression of REDD1 and inhibit mTORC1 [62]. In the process of tumor immunization, tumor necrosis factor-a (TNF-a) signaling pathway can inhibit TSC activity through IKKb and activate mTORC1 [63] (Fig. 2M).

2.2.1.2. Downstream signaling and effects of mTORC1. mTORC1 re- ceives upstream signals and integrates them to influence down- stream effectors through its activity changes, regulating transcription, translation, biosynthesis and autophagy etc.

In translation level, signals from upstream growth factors inhibit eIF-4E binding protein (4E-BP1) [64,65] and activate ribosomal protein S6 kinase 1 (S6K1, also called p70S6K1) [66] via mTORC1, thereby promoting mRNA translation. 4E-BP1 competitively in- hibits eIF-4E, when mTORC1 phosphorylates to inhibit 4E-BP1, 4E- BP1 dissociates with eIF-4E, which makes eIF-4E recruit initiation factor eIF-4G to 5’ terminal of mRNA and begin to translate [64,67] (Fig. 3A). When S6K1 is phosphorylation-activated by mTORC1, it will interact with downstream substrates such as eIF-4B [68,69], eEF-2 kinase (eEF-2K) [70], ribosomal protein S6 [71], CBP80 [72], SKAR [73], regulate their function and promote initiation and elongation of translation (Fig. 3B). It is reported that activated S6K1 has an inhibition to upstream insulin receptor substrate 1 (IRS1) [74,75] and forms a negative feedback loop to prevent over acti- vation (Fig. 3C).

In ribosomal biosynthesis, mTORC1 promotes rDNA transcription into rRNA through up-regulation of RNA Polymerase I (PolI) via S6K1 [76,77] (Fig. 3D). 5S rRNA is a part of ribosomal large subunit, whose transcription relies on RNA Polymerase III (Pol III), in addi- tion Pol III is also responsible to transcript tRNA. In the regulation of mTORC1 on Pol III, TFIIIC recruits mTORC1 into nucleus and binds to Pol III which is binding to the promoter, and phosphorylates to inhibit its inhibitory element Maf1, thus initiating transcription
[78] (Fig. 3E). Overall mTORC1 promotes ribosomal biosynthesis. mTORC1 also controls anabolism including nucleotides, lipids,
saccharides, proteins and even ribosome. The most important nucleotides supply in organisms is de novo synthesis, and mTORC1 can regulate both purine and pyrimidine nucleotide de novo synthesis. mTORC1 increases the expression of methylenete- trahydrofolate dehydrogenase 2 (MTHFD2) in mitochondrial tet- rahydrofolate cycle by activating ATF4, thus promoting purine nucleotide de novo synthesis on the supply of one carbon unit [79] (Fig. 3F). On the other hand, mTORC1 phosphorylation-activates S6K1, thus phosphorylation-activate CAD [carbamoyl-phosphate synthetase 2 (CPS-2), aspartate transcarbamoylase (ATC), dihy- droorotatase (DHO)] that controls the first three steps of pyrimi- dine de novo synthesis, thus mTORC1 promotes pyrimidine nucleotide de novo synthesis [80,81] (Fig. 3G). Sterol responsive element binding protein (SREBP) is a transcription factor controlling the expression of genes related to fatty acids and cholesterol biosynthesis, mTORC1 can promote lipid anabolism through SREBP activation [82]. SREBP activation by mTORC1 can through S6K1 activation [83], or through phosphorylation- inhibiton of the nuclear localization of Lipin 1 and remove its inhibition of SREBP [84] (Fig. 3H). Duvel et al. [83] reported that mTORC1 increases the expression of key enzymes related to glucose metabolism by increasing the expression of hypoxia- inducible factor 1a (HIF 1a), thus enhancing glucose uptake and glycolysis (Fig. 3I). Besides, due to the activation of SREBP, the flux of glucose metabolism through pentose phosphate pathway (PPP) increases, therefore the intermediate metabolites needed for cell growth such as NADPH also increases [83].

Fig. 1. Structure of mTOR. (A) HEAT repeat forms a-helix. (B) FAT, FATC, FRB and kinase domain, FAT domain and FATC domain get close when folded. (C) ATP binding site of mTOR (in circle). (D) Primary structure and domain distribution of mTOR. (E) Tertiary structure of mTOR. (F) Molecular Composition of Two Types of mTOR Complex. (G) Cryo-EM structure of mTORC1(PDB ID: 5H64). (H) Cryo-EM structure of mTORC2(PDB ID: 5ZCS). Cryo-EM data was visualized by PyMOL. NR refers to Negative Regulatory domain.

Fig. 2. Upstream Regulations of mTOR Signaling. (A) Amino Acid-Rag-mTORC1 axis activates mTORC1. (B) Insulin-PI3K-Akt-TSC axis activates mTORC1. (C) mTORC1-PRAS40 positive feedback loop. (D) EGFR-Ras-Raf-Erk-TSC axis activates mTORC1. (E) Wnt-GSK3-TSC axis activates mTORC1. (F) Energy-AMPK-mTORC1 axis activates mTORC1. (G) LKB1-AMPK- mTORC1 axis inhibits mTORC1. (H) REDD1-TSC axis inhibits mTORC1. (I) p53-AMPK-mTORC1 axis inhibits mTORC1. (J) p53-TSC axis inhibits mTORC1. (K) p53-PTEN-PIP3 axis inhibits mTORC1. (L) p53-IGF BP3-IGF1 axis inhibits mTORC1. (M) TNF a-TNFR-IKKb-TSC axis activates mTORC1.

When overfeed, our bodies will convert the extra energy into triglyceride (TG) and store in adipocytes, forming white adipose tissue (WAT). It is proved that specific absence of Raptor in mice adipocytes will lead to increase of fatty acid oxidation and decrease of WAT [85]. And the up-regulation of peroxisome proliferator- activated receptor-g (PPAR-g) by mTORC1 mediates preadipocyte differentiation and lipid accumulation [86e89]. Some think the mechanism of up-regulation of PPAR-g might be increase of expression [90], or activated SREBP-1c enhances PPAR-g activity [91,92].

Autophagy is the process of cell controlled self-degradation of some contents, generally damaged, redundant or even harmful contents, which belongs to the mechanism of material recycling and self-protection. mTORC1 inhibits autophagy, which means that inhibition of mTORC1 will induce autophagy [93]. Formation of autophagosome relies on the ULK-Atg13-FIP200 complex, mTORC1 phosphorylates ULK and Atg13, inhibits their activity and thus in- hibits autophagy [94,95] (Fig. 3J). Besides, Kim et al. [96] reported that AMPK, an upstream inhibitor of mTORC1, can activate ULK (Fig. 3K), thus activating autophagy and enhancing energy supply while inhibiting mTORC1 activity. When cells are hungry, tran- scription factor EB (TFEB), which responds from the transcription level, activates lysosomal biosynthesis and enhances autophagy, while at normal state, mTORC1 phosphorylation-inhibits TFEB, thus inhibiting autophagy from the transcription level [97] (Fig. 3L). In 2016, Rousseau and Bertolotti [98] reported that mTORC1 can also regulate autophagy at intracellular proteasome abundance level by controlling the activity of Erk 5, a member of mitogen activated protein kinase (MAPK) family (Fig. 3M). In addition, since auto- phagy is a degradation metabolism and its degradation product amino acids have a positive regulatory effect on mTORC1, so it is not difficult to speculate that a negative feedback loop formed, which can prevent autophagy from over activation and maintain cell stability.

2.2.2. mTORC2 signaling, regulation and effect

At present, there are few reports about the upstream signaling pathway of mTORC2, and the related pathway is still under study. Early studies have shown that Ras and ribosome can interact with and activate mTORC2 [99,100] (Fig. 4A; 4B). In 2015, Liu et al. [101] reported for the first time that 3,4,5-phosphatidylinositol-3- phosphate (PIP3), a non-protein mTORC2 pathway regulator, in- teracts with the PH domain of msin1 subunit to release the barrier of PH domain on mTORC2 kinase domain and triggers the activa- tion of mTORC2 (Fig. 4C).
The downstream effects of mTORC2 are mainly related to cell survival. mTORC2 activates several subtypes of protein kinase C (PKC) of AGC protein kinase family to control cytoskeleton rear- rangement and cell migration [14,15,102e104] (Fig. 4D). In addition,mTORC2 activates Akt [105], thus regulating cell metabolism and growth via mTORC1, or inhibiting apoptosis and cell cycle arrest and regulating glycolysis/gluconeogenesis ratio through phos- phorylation inhibition of Forkhead box protein O 1/3 (FoxO 1/3) [106e113] (Fig. 4E). Besides, Akt also phosphorylation-activates upstream mTORC2, forming a positive feedback loop and continu- ously amplifying signals [114] (Fig. 4F). mTORC2 activates the serum/glucocorticoid regulated kinase 1 (SGK1) of the AGC protein kinase family, which promotes ion transportation and cell growth [115] (Fig. 4G). Hippo signaling pathway is a restrictive regulatory pathway of cell growth, when activated, it inhibits cell growth and promotes apoptosis. YAP is a transcription adjuvant, which is located in the nucleus and combined with the transcription factor TEAD to enhance the expression of metabolism-related proteins. When Hippo pathway is activated, LATS 1/2 kinase mediates the cytoplasmic localization and phosphorylation of YAP, and mediates the ubiquitination degradation of phosphorylated YAP [116e118]. Mammalian Sterile 20-like kinase (MST 1/2) is a controlling element of Hippo signaling pathway which can activate down- stream LATS 1/2 kinase, promoting YAP degradation and mediating apoptosis of abnormally growing cell [119]. And mTORC2 phos- phorylates and inhibits MST 1/2, promoting cell growth and inhibiting apoptosis [120] (Fig. 4H).

Fig. 3. Downstream Effects of mTORC1 Signaling. (A) mTORC1-4E BP1 axis promotes translation. (B) mTORC1-S6K1 axis promotes translation. (C) S6K1-IRS1 negative feedback loop. (D) mTORC1-S6K1-PolI axis promotes ribosome biosynthesis. (E) mTORC1-S6K1-Pol III axis promotes ribosome biosynthesis. (F) mTORC1-ATF4-MTHFD2 axis promotes de novo purine synthesis. (G) mTORC1-S6K1-CAD axis promotes de novo pyrimidine synthesis. (H) mTORC1-SREBP axis promotes lipid synthesis and pentose phosphate pathway. (I) mTORC1-HIF 1a axis promotes carbohydrate metabolism. (J) mTORC1-ULK Atg13 FIP200 complex axis inhibits autophagosome formation. (K) AMPK promotes autophagosome formation. (L) mTORC1-TFEB axis inhibits lysosome biosynthesis. (M) mTORC1-Erk 5-Adc17 axis controls proteasome abundance.

In recent years, it has been reported that mTORC2 can sense intracellular glucose and glutamine levels, and regulate each other through the tricarboxylic acid cycle (TAC), suggesting that mTORC2 signaling is closely related to cell energy status [121]. mTORC2 promotes the decomposition of glutamine to a-ketoglutarate to supplement into TAC [122], while glucose, glutamine and a-keto- glutarate all have negative regulatory effects on mTORC2 [122] (Fig. 4I). At present, the direct regulatory metabolites and their mechanisms are still under study.

mTORC1 signaling pathway is closely related to mTORC2 signaling pathway, influencing and regulating each other. Long term mTORC1 inhibition will be fed back to mTORC2 through signaling network, which is the reason why prolonged Rapamycin treatment will inhibit mTORC2 at the same time, although it is not sensitive to Rapamycin.

2.3. mTOR related diseases

The occurrence and development of many diseases are related to the abnormal mTOR signal, and most of them show the over- activation or over-expression of mTOR (Table 1). The over activa- tion of mTOR may be due to the over expression of PI3K and Akt, or the dysfunction of PTEN, TSC1/2 and LKB1. Since mTOR signaling pathway controls metabolism, growth, proliferation and survival of the cell, the continuous over-activated mTOR signal will lead to the improvement of cell metabolism level, continuous growth and proliferation, cell life extension or even cell immortalization, which can directly or indirectly induce metabolic diseases, cancer and aging diseases [123]. And inhibition of this state can effectively delay or treat cancer, cardiovascular damage and other diseases caused by over-activation of mTOR. Therefore, mTOR inhibitors are hot areas at present and many mTOR inhibitors were developed. Next, we will introduce mTOR inhibitors that have been reported.

3. mTOR inhibitors

At present, mTOR inhibitors are mainly divided into four cate- gories: antibiotic allosteric mTOR inhibitors (first generation), ATP- competitive mTOR inhibitors (second generation), mTOR/PI3K dual inhibitors (second generation) and other new mTOR inhibitors (third generation). In this article, these four categories of mTOR inhibitors and their structures, properties and some clinical re- searches will be introduced sequentially (Table 2).

3.1. The first generation: antibiotic allosteric mTOR inhibitors

The first generation of mTOR inhibitors are mainly Rapamycin and its derivatives (also known as Rapalogs), targeting mTOR and FKBP12 (Table 2). The mechanism of action is basically the same, Rapalogs bind with FKBP12 to form a complex, which then binds to FRB domain of mTOR and changes the conformation of mTOR, thus inhibiting the kinase activity of mTORC1 [12] (Fig. 5).

3.1.1. Rapamycin

Rapamycin (1), also called Sirolimus or Rapamune, is the first inhibitor of mTOR, which belongs to macrolides in structure. Rapamycin is an allosteric inhibitor of mTOR, which can directly inhibit mTORC1 and simultaneously cause the inhibition of mTORC2 through the signaling network during long-term admin- istration. Rapamycin was initially used as an immunosuppressant in kidney transplantation because it can reduce the activation of T cells and B cells by Interleukin-2 (IL-2) [131]. Later, Rapamycin was used in anti-tumor, anti-cardiovascular reconstruction, anti- coronary restenosis and other aspects. In recent years, Rapamycin was also used in the treatment of infants with cardiac rhabdo- myoma [132], and the combination of Rapamycin and Vorinostat, an HDAC inhibitor, was used to treat malignant tumors in a Phase I study [133]. At present, Rapamycin is approved by FDA to be mar- keted as oral solution and tablet. Although the biological half-life of Rapamycin is more than 50 h, its biological utilization is low due to its poor water solubility and stability, and its clinical application is greatly limited. Therefore, many pharmaceutical companies have carried out structural modification of Rapamycin and developed a series of derivatives with more optimized pharmacokinetic prop- erties, such as Temsirolimus (2), Everolimus (3), Ridaforolimus (4), Umirolimus (5), Zotarolimus (6) etc.

Combining Rapamycin with nanoparticle albumin to form nanoparticle albumin-bound Rapamycin (nab-Rapamycin), which has immunosuppressive and potential anti-angiogenic and anti- tumor activities, and can enter tumor cells through albumin mediated process. Preclinical study showed that nab-Rapamycin can reduce the viability of tumor cells [134]. Besides, in xenograft models of human breast cancer, the antitumor activity of nab- Rapamycin will be enhanced in combination with Doxorubicin (Adriamycin), Vorinostat, Erlotinib or Perifosine [135].
When FKBP12-Rapamycin complex binds to mTOR, Rapamycin is embedded in the cavity formed by FKBP12 and FRB domain of mTOR (Fig. 6A). Since the C-9 to O-24 part of Rapamycin is embedded in the cavity of FKBP12, modification on this part might cause inactive compounds due to inability of binding to FKBP12 (Fig. 6B; blue part of Fig. 6F). Besides, the methoxy group at C-7 of Rapamycin faces outward relative to FKBP12, therefore the ReS configuration of C-7 substituent might have little effect on the af- finity between the derivative and FKBP12 (Fig. 6C). However, since the orientation of C-7 substituent is also the binding direction to FRB, the size and shape of the substituent have a great influence on the activity and too large substituent will hinder the binding of FKBP12-Rapamycin complex to mTOR (Fig. 6D). The interaction sites of Rapamycin with FRB are mainly C-1 to C-8 and C-36 lipo- philic part, and amino acid residues of FRB involved are mainly Tyr2038, Phe2039, Trp2101, Tyr2104, Tyr2105 and Phe2108, while Ser2035 also has some interaction with C-31 to C-33 high polarity part of Rapamycin (Green part of Fig. 6F). In addition, X-ray diffraction data visualization shows that the whole cyclohexane ring is relatively close to FKBP12 and FRB, which indicates there might be some interaction (Fig. 6E; red box of Fig. 6F). The dominant conformation of C-42 position also suggests that there are some choices for modification. Introduction of a group slightly larger than hydroxyl group or a plane group with a proper orientation can enhance the interaction, but introduction of a much larger group will hinder the interaction.

Overall, the structural modification on the interacting region can change the interaction between the compound and FKBP12 and mTOR, thus change its pharmacological activity. Among them, C-7 and C-42 position are relatively easy for modification. In addition, modification near the lactone group can affect the hydrolysis pro- cess through steric hindrance, thus prolonging the half-life of the compound.

3.1.2. Temsirolimus

Temsirolimus (CCI-779, 2), also known as Torisel, is the first Rapamycin derivative developed by Wyeth. Temsirolimus is a prodrug prepared by esterification of Rapamycin 42-OH and 2,2- dihydroxymethylpropionic acid, whose water solubility and sta- bility are both better than Rapamycin [136]. However, esters are easily degraded by oral administration, so Temsirolimus is only used for intravenous administration. After entering into the body,Temsirolimus was quickly removed from the plasma, and trans- formed into Rapamycin via CYP3A4, then maintained a relatively high concentration in the body for several days [137]. This also suggests that patients with high CYP3A4 activity or high expression in vivo need to increase the dosage properly when taking Temsir- olimus. In vitro and in vivo experiments showed that Temsirolimus had better growth inhibition effect on PTEN—/- tumor cells [138].

Fig. 4. Regulations and Effects of mTORC2 Signaling. (A) Ras activates mTORC2. (B) Ribosome activates mTORC2. (C) PIP3 activates mTORC2. (D) mTORC2 activates PKC. (E) mTORC2- Akt-Fox O1/3 axis inhibits Fox O1/3. (F) mTORC2-Akt positive feedback loop. (G) mTORC2 activates S6K1. (H) mTORC2 inhibits MST1. (I) Relations between mTORC2 and carbo- hydrate metabolism.

In late May 2007, the FDA approved Temsirolimus for the treatment of advanced renal cell carcinoma (RCC) [139,140], in November, it was approved by European Medicines Agency (EMEA). Temsirolimus monotherapy has shown excellent thera- peutic effect in mantle cell lymphoma (MCL), which drove its approvement in treatment of MCL by EMEA in 2012. A Phase III study showed that Temsirolimus can prolong the progression-free survival (PFS) of patients with relapsed or refractory MCL [141], which means Temsirolimus can slow down the progress of MCL. In a Phase II study, Temsirolimus was used to treat platinum- refractory/resistant ovarian cancer or advanced/recurrent endo- metrial carcinoma, and the result showed that Temsirolimus treatment was well tolerated but did not meet the predefined ef- ficacy criteria [142]. Besides, combination of Irinotecan/Temozolo- mide with Temsirolimus in children with refractory or relapsed neuroblastoma is under a Phase II study [143], combination of Perifosine with Temsirolimus for recurrent pediatric solid tumors is under a Phase I study [144].

3.1.3. Everolimus

Everolimus (RAD001, 3), also known as Afinitor, is an oral Rapamycin derivative developed by Novartis. Prepared by ether- ification of Rapamycin 42-OH and ethylene glycol, Everolimus with an improved water solubility and stability [136] and a half-life about 30 h.Everolimus is effective in the treatment of many tumors, and it has been approved to be used in advanced RCC and its follow-up treatment [145,146]. Everolimus shows considerable therapeutic effects in subependymal giant-cell astrocytoma (SEGA) with or without tuberous sclerosis [147,148], renal angiomyolipoma [148,149], advanced non-functional lung/gastrointestinal neuro- endocrine tumor (NET) [150] and advanced non-small-cell lung cancer (NSCLC) [151]. In a Phase III study, Evrolimus can prolong PFS and overall survival (OS) for approximately 6 months in pa- tients with advanced pancreatic NET (PNET), and was associated with a low rate of severe adverse events [152,153], making Ever- olimus to be a considerable therapy for PNET. Besides, in patients with advanced follicular-derived thyroid cancer, Everolimus has clinically relevant antitumor activity and relatively low toxicity profile in a Phase II study [154].

However, Everolimus does not show low toxicity and high efficacy for all tumors, in some types of tumor, Everolimus only sta- bilizes the disease, even shows invalid. For example, in patients with previously treated gastric cancer [155] or with advanced he- patocellular carcinoma (HCC) that sorafenib intolerant/ineffective [156], Everolimus did not improve their OS much. And there was no clinically relevant response using Everolimus in patients with relapsed or cisplatin refractory germ cell cancer (GCC) in a Phase II study [157], Another study on patients with previously treated recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) showed that Everolimus is ineffective, treatments of 3 patients were stopped due to toxicity [158].

3.1.4. Ridaforolimus

Ridaforolimus (AP23573, MK-8669, 4), also known as Defor- olimus, is an oral derivative of Rapamycin developed by Merck and ARIAD). It is prepared by esterification of Rapamycin 42-OH and dimethylphosphinic acid. Ridaforolimus is the latest Rapamycin derivative in treating sarcoma. In a Phase II study, single-agent Ridaforolimus showed a half-year overall PFS rate of 23.4% in pa- tients with advanced and pretreated bone and soft tissue sarcomas [159]. However, another international Phase III study reported that Ridaforolimus can only delayed tumor progression to a small sta- tistically significant degree in patients with metastatic sarcoma [160]. In addition, in a Phase II study in patients with advanced endometrial carcinoma, single-agent Ridaforolimus has a consid- erable anti-tumor activity and tolerance, but with toxicity at the same time [161,162]. In patients with recurrent or metastatic endometrial carcinoma, Redaforolimus was reasonably tolerated and demonstrated modest activity [163]. Besides, in a Phase I study in 2016, Ridaforolimus is orally bioavailable and well tolerated in children with advanced solid tumors, providing a promising ther- apeutic option in pediatric malignancies [164].

Fig. 5. Structure of Rapamycin and its derivatives.

3.1.5. Umirolimus

Umirolimus (TRM-986, 5), also known as Biolimus, Biolimus A9 and BA9, i.e. 42-O-(2-ethoxyethyl)-Rapamycin, is a highly lipophilic semi-synthetic derivative of Rapamycin developed by Biosensors International in order to be used in drug eluting stent. Umirolimus shows potent activity in immunosuppression and anti- proliferation. Due to high lipophilicity, Umirolimus is absorbed by vascular wall and passes through smooth muscle cell membrane faster than other Rapalogs [165,166]. Then Umirolimus induces smooth muscle cell cycle arrest to reduce progression of vascular stenosis. At present, Umirolimus is used in some drug eluting stent products as a patent drug of Biosensors International.

3.1.6. Zotarolimus

Zotarolimus (ABT-578, 6) is a semi-synthetic derivative of Rapamycin developed by Abbott Laboratories. Zotarolimus has a tetrazole substituent at C-42 position and an (S)-configuration at C- 42 which is contrary compared to other Rapalogs. Zotarolimus is extremely lipophilic, with the highest octanol-water partition co- efficient among of drugs using in drug-loading stent [167], which is favorable to design to use in drug-loading stents with sustained-release performance.

Fig. 6. Analysis of structure-activity relationship of Rapamycin. (A) Rapamycin is embedded in the cavity composed of FKBP12 and FRB. (B) The part of Rapamycin C-9 to O-24 is embedded in the cavity of FKBP12. (C) The methoxy group at C-7 of Rapamycin faces outward relative to FKBP12. (D) The methoxy group of C-7 of Rapamycin towards FRB. (E) Cyclohexane ring of Rapamycin is relatively close to FKBP12 and FRB. (F) Interaction region of rapamycin with FKBP12 and FRB. X-ray diffraction data (PDB ID: 1FKL, 1FAP) was visualized by PyMOL.

3.1.7. Other structural modifications of rapamycin

Luengo et al. [168] used trifluoroacetic acid (TFA) to catalyze the SN1 reaction between C-7 position of Rapamycin and nucleophilic reagent at —40 ◦C to introduce various groups (Formula 1) and obtained a series of derivatives. The results showed that C-7 posi- tion of Rapamycin is a part of its active site. The ReS configuration of the C-7 substituent has little effect on the affinity between the derivative and FKBP12, but the size and shape of the substituent have great effect on the activity. If a large substituent or a rigid structure were introduced, the derivative still has affinity for FKBP12, but has almost no effect on mTOR. The C-7 benzyloxy derivative (7) still has some immunosuppressive activity (T cell IC50 = 6 nM). Besides, activity of C-7 demethoxy derivative (8) is almost unchanged (T cell IC50 = 2 nM).

Due to the lactone structure, Rapamycin is unstable in both acid and alkali. After contacting with acid and alkali or entering human body, it hydrolyzes to produce b-hydroxyketone (9) and its dehy- dration product a, b-unsaturated ketone (10) (Formula 2), both are inactive [169]. Nelson et al. [169] modified C-22 and C-27 position of Rapamycin. Stability of C-27 carbonyl reduction product (11) has little change, but its acetylation product (12) has a half-life twice that of Rapamycin. Besides, alkylation on C-22 position can also increase the stability of the ring and the steric hindrance of ester group, making lactone hard to hydrolyze and prolonging half-life. The half-life of C-22 methylation product (13) can be prolonged to about 3 times.Sedrani et al. [170] opened the cyclohexane ring at C-39 position of Rapamycin and found that the activity of product decreased sharply, but its binding ability to FKBP12 was not affected. There- fore, the cyclohexane ring is vital for the binding of FKBP12- Rapamycin complex to FRB domain of mTOR.Ruan et al. [171] heated Rapamycin with nitroso benzene for 16 h and obtained modified products on Rapamycin C-1-C-4 con- jugated polyene structure: WYE-592 (14) and its reduction product ILS-920 (15) (Formula 3). It is worth noticing that WYE-592 and ILS- 920, although not as good as Rapamycin in terms of immunosup- pression, have the activities of promoting neuron survival, stimu- lating neurite outgrowth and neuroprotection. They are with EC50 values of 420 nM and 540 nM to neurite outgrowth and 700 nM and 150 nM to cortical neuron survival respectively.

3.1.8. Discussion

Above-mentioned modification sites of Rapamycin include C-7, C-22, C-27, C-42 position and C-1 to C-4 conjugated part (Fig. 7). Among them, the earliest and relatively mature studied modifica- tion site is C-42 position, which was also considered as the best site for modification without weakening the biological activity of the whole compound [167]. Previously we had an inference on the modification of Rapamycin by visualization using the X-ray diffraction data from Wilson et al. [172] (PBD ID: 1FKL) and Choi et al. [12] (PBD ID: 1FAP). And the results of modification on C-7, C- 22 and C-27 position are consistent with our inference.

Rapamycin as the first mTOR inhibitor, has been successfully synthesized. However, due to its complicated structure and large molecular weight, there are still many problems hindering its development, such as long synthesis route, low productivity and difficulties on enantiomer separation and chiral controlling. Be- sides, since the modification sites of Rapamycin are limited, the quantity of new compounds with changed physical and chemical properties, pharmacological activities or half-life are also limited. Therefore, discovery of other effective targets and development of new generation of small molecular mTOR inhibitors are necessary. Starting from the kinase activity of mTOR, it is a shortcut to develop a new type of mTOR inhibitors by searching compounds with mTOR inhibitory activity among the existing Ser/Thr kinase inhibitors.

In addition, above-mentioned modifications are all single site modification, in the future, it is worth a try to study multiple site combined modification. For example, combination of C-22 methylation in compound 13 and C-42 modification in compound 2e6 will obtain a series of new compounds, the rest work is to test whether these compounds retain both increased stability and enhanced inhibitory activity.

3.2. The second generation: ATP-Competitive mTOR inhibitors and mTOR/PI3K dual inhibitors

The second generation of mTOR inhibitors are small molecular ATP analogues, including ATP-competitive mTOR inhibitors and mTOR/PI3K dual inhibitors. The second generation of mTOR in- hibitors have different structures, but all of them can directly bind or act on the ATP binding sites of mTOR or PI3K to produce competitive inhibitory effects (Table 2). ATP-competitive mTOR inhibitors are highly selective to mTOR, or their binding sites are mainly located in mTOR. Thus ATP-competitive mTOR inhibitors can inhibit mTORC1 and mTORC2 simultaneously, therefore are also called mTORC1/mTORC2 dual inhibitors (TORCdIs) or selective mTOR kinase inhibitors (TORKIs). Researches showed that in addition to dysregulations of mTOR, there are also dysregulations in the occurrence and development of many diseases. Previously we have mentioned that mTOR shows a high homology with the cat- alytic domain of PI3K in C-terminal. Therefore, some compounds with mTOR kinase inhibitory activity should also have some inhibitory effect on PI3K. mTOR/PI3K dual inhibitors (TPdIs) are a type of compounds with inhibitory effect on both mTOR and PI3K. According to the above-mentioned statement, the therapeutic ef- fect of TPdIs should be better than that of TORCdIs in some diseases with simultaneous over-activation of mTOR and PI3K.

PI-103 (16), the first recognized mTOR/PI3K dual inhibitor, was obtained by Astella through high-throughput screening. It is a morpholino quinazoline derivative, with IC50 values of 3.6 nM against PI3Ka and 3.0 nM against PI3Kb [173]. Previous studies showed that PI-103 had a good inhibitory effect on some tumor cells [174], but it was stopped studying due to poor drug properties. While a series of TORCdIs and TPdIs (17e32) were developed from it as a lead compound. Up to now, there are many TORCdIs and TPdIs that has been reported and we divide them into 8 categories according to different chemical structure.

3.2.1. 1,3,5-Triazines

PKI-587 (Gedatolisib, PF-05212384, 17) is an mTOR/PI3K dual inhibitor developed by Pfizer, with a 2,4-dimorpholinyl-1,3,5- triazine structure. It has IC50 values of 1.6 nM against mTOR and 0.4 nM against PI3Ka, and a half-life of 14.4 h [175]. PKI-587 inhibited cell growth in a dose-dependent manner in cell lines of BON, QGP-1, KRJI and LCC-18 of gastroenteropancreatic NET [176]. In a Phase II study, PKI-587 demonstrated acceptable tolerability and moderate activity in patients with recurrent endometrial cancer [177] (Fig. 8).
PKI-179 (18) is a modification product of PKI-587. Fixed by a two-carbon bridge on R2 morpholine and replaced by pyridine-4-yl on benzamide part at the end of R3, PKI-179 has a lower molecular weight compared to PKI-587. PKI-179 has IC50 values of 0.42 nM against mTOR and 8 nM against PI3Ka, and shows excellent PAMPA permeability [178]. Its main metabolite by liver microsomes (19) is also active, with IC50 values of 0.8 nM against mTOR and 4 nM against PI3Ka, and is under development [178,179].

The R3 substituent of KU-BMCL-200908069-1 (20) is aromatic hydrazone derivative, KU-BMCL-200908069-1 only shows inhibi- tory effect on mTOR, with an IC50 of 270 nM [180]. KU-BMCL- 200908069-5 (21) is a derivative with aromatic hydrazone replaced by imidazole, which improved both stability and mTOR inhibitory activity (IC50 of 6 nM in enzyme level and 21 nM in cell level) [180].

PQR309 (Bimiralisib, 22) is an mTOR/PI3K dual inhibitor with a pyridine derivative substituent and IC50 values of 89 nM against mTOR and 33 nM against PI3Ka [181]. In 2019, Borsari et al. [182] reported an mTOR/PI3K dual inhibitor PQR514 (23), which is a modification product of PQR309, and with a pyrimidine derivative substituent. PQR514 has Ki values of 33 nM against mTOR and 2.2 nM against PI3Ka and a much potent inhibitory effect compared to PQR309 (PQR309 has Ki values of 62 nM against mTOR and 17 nM against PI3Ka). At present, PQR309 is in Phase I/II studies. Dehnhardt et al. [183] started from PKI-587, kept 2- morpholinyl-1,3,5-triazine skeleton and 4- [4-(N, N-disubstituted carbamyl) phenyl ureido]phenyl of R2 unchanged, and optimized on another morpholine ring and the N-substituents of R2 terminal amide. Then they obtained a series potent mTOR/PI3K dual in- hibitors with the structure of 2-(morpholin-4-yl)-4-substituted oxy-6-(4-(4-(N,N-disubstituted carbamyl) phenyl ureido)phenyl)- 1,3,5-triazines. Among them, many are with IC50 values of nM level against mTOR and PI3K, here are the four most potent compounds (Fig. 9).

On the other hand, the chemical structure of 1,3,5-triazines usually contains two morpholine or its derivative, while R2 conju- gates with triazine to form a planar structure. In modifications, conjugated planar structure can often build a condensed nucleus structure, therefore a series of TORCdIs and TPdIs with condensed nucleus structure were developed.

3.2.2. Pyrido[2,3-d] pyrimidines

Ku-0063,794 (24) is an ATP-competitive mTOR inhibitor devel- oped by AstraZeneca, with a structure of 2-[(2R,6S)-2,6- dimethylmorpholin-4-yl]-4-(morpholin-4-yl) pyrido [2,3-d] py- rimidine. It is reported that Ku-0063,794 can inhibit activation of mTORC1 and mTORC2 and with an IC50 value of 10 nM, but has no PI3K inhibitory activity [184]. Besides, Ku-0063,794 shows a stronger dephosphorylation to 4E-BP1 than Rapamycin [184].

AZD8055 (25) is also developed by AstraZeneca, with a 2,4-bis [(3S)-3-methylmorpholin-4-yl] pyrido [2,3-d] pyrimidine struc- ture and an IC50 value of 0.8 nM [185]. A study in HCC cells showed that AZD8055 can induce AMPK activation and autophagy induc- tion, leading to cell death [186]. Another study in human laryngeal cancer Hep-2 cell line showed that the expression of mTOR in AZD8055 treated cells was down-regulated, and pro-apoptotic factors Bax and Caspase 3 were up-regulated and anti-apoptotic factor Bcl-2 was down-regulated, suggesting that AZD8055 has anti-proliferation and apoptosis induction activity for Hep-2 cell line [187]. However, AZD8055 showed mixed results in early clin- ical studies despite of its good performance in preclinical studies. In a phase I study in patients with advanced solid tumor and lymphoma, AZD8055 caused transaminases increases, suggesting the liver toxicity of AZD8055 [188,189].

Formula 1. Structural modification of Rapamycin on C-7 position.

Formula 2. Hydrolysis of rapamycin.

Formula 3. Structural modification of Rapamycin on C-1 to C-4 position.

Fig. 7. Structural modification of Rapamycin.

AZD2014 (Vistusertib, 26) is a derivative of AZD8055, the methoxy group on benzene ring of AZD8055 was removed and the hydroxymethyl group was replaced by methylcarbamyl group during the modification, and therefore eliminate the phenomenon of transaminase increases caused by AZD8055. In a Phase I study, single-agent AZD2014 is effective in highly pretreated solid tumors [190]. However, another Phase II study showed that the therapeutic effect of AZD2014 in patients with VEGF-refractory metastatic clear cell renal cancer is inferior to Everolimus [191]. Besides, AZD2014 is relative to Rictor amplification [192,193]. Although encouraging clinical data were reported, AstraZeneca terminated AZD2014 in 2018, which was in Phase II study at that time.Structurally, these compounds show a common structural feature that the two substituents on the pyrimidine ring are mor- pholine or methyl morpholine, and a 3-/4-substituted phenyl is on the other pyridine ring (Fig. 10).

3.2.3. Pyrazolo[3,4-d] pyrimidines

Wyeth first reported a series of selective mTOR inhibitors with morpholino pyrazolo [3,4-d] pyrimidine structure, which are related to the structure of PI-103 [194]. These compounds have a morpholine or its derivative substituent on R2 at 4 position of pyrazolo [3,4-d] pyrimidine. Except the lead compound WAY-001 (27), they also found WAY-600 (28), WYE-687 (29) and WYE-354 (30) showed different mTOR inhibitory activity, with IC50 values against mTOR of 220 nM, 9 nM, 7 nM and 5 nM respectively [194]. The followed compound WYE-132 (WYE-125132, 31) has a more potent effect on mTOR, and with an IC50 value of 0.19 ± 0.07 nM [195]. In addition, Wyeth-BMCL-200910096-27 (32) with the similar chemical structure also showed a strong selectivity to mTOR, with IC50 values of 0.6 nM against mTOR and 150 nM against PI3K (250 times) [196] (Fig. 11).

Fig. 8. 1,3,5-Triazines mTOR inhibitors.

3.2.4. 4-Aminopyrazolo[3,4-d] pyrimidines

A new type of mTOR inhibitors, 4-aminopyrazolo [3,4-d] py- rimidines, are obtained by replacement of morpholine ring with amino group at R2 and removement of R3 on morpholino pyrazolo [3,4-d] pyrimidines. Shokat et al. [197,198] reported this type of mTOR inhibitors including PP242 (33), PP30 (34), PP487 (35) and PP121 (36), with IC50 values against mTOR of 8 nM, 80 nM, 72 nM and 10 nM respectively. Among them, PP242 and PP30 have a relatively strong selectivity to mTOR. In addition, PP242 can inhibit the kinase activity of PKC, RET and JAK2 to some extent, which may be due to its structural similarity to adenine of ATP [198]. PP487 and PP121 have some tyrosine kinase inhibitory activity and can inhibit Abl, Hck, Src, VEGFR2 and PDGFR, providing a direction for the research of tyrosine kinase inhibitors [197].

INK-128 (Sapanisertib, TAK-228, MLN0128, 37) is an mTOR/PI3K dual inhibitor developed by Millennium Pharmaceuticals. In vitro experiments, INK-128 can not only inhibit mTOR and PI3K, but also inhibit mTOR downstream effectors such as S6, 4E-BP1 and Akt. In a Phase I study in patients with advanced solid malignancies, no meaningful difference was noted in the pharmacokinetics of INK-128 when administered with or 24 h after Paclitaxel INK-128, suggesting further investigation of INK-128 in combination with other agents including Paclitaxel [199] (Fig. 12).

3.2.5. Thieno[3,2-d] pyrimidines

2-aryl-4-(morpholin-4-yl) thieno [3,2-d] pyrimidines are known PI3K inhibitors, and they also have inhibitory effect on mTOR. Although GDC-0941 (Pictilisib, 38) has some inhibitory ef- fect on mTOR, the inhibition of PI3K is stronger than mTOR, with IC50 values of 580 nM against mTOR and 3 nM against PI3Ka [200]. Modification on GDC-0941 obtains a compound with the similar chemical structure, GDC-0980 (Apitolisib, RG7422, 39), which has a Ki value of 17 nM against mTOR and IC50 values of 5 nM, 27 nM, 7 nM and 14 nM against PI3Ka, b, d and g [201]. GDC-0980 demonstrated excellent inhibitory effect on breast cancer, pancre- atic cancer, NSCLC and colon cancer cell lines, and showed a slightly poor inhibitory effect on prostate cancer and melanoma cell lines, but still better than GDC-0491 [202]. Modest but durable anti- tumor activity was demonstrated by GDC-0980 in a Phase I study [203]. However, a Phase II study showed that inhibition by GDC- 0980 was less effective than was Everolimus in metastatic RCC,likely due to full blockade of PI3K/mTOR signaling resulted in multiple on target adverse events [204].Verheijen et al. [205] replaced morpholinyl with 8-oxo-3- azabicyclo [3.2.1] octan-3-yl and obtained compounds with higher selectivity to mTOR than to PI3K. Further study demon- strated that when 2-aryl is 4-ureidophenyl, the inhibitory effect of compound on mTOR will be greatly enhanced. And a series of candidate compounds code Wyeth-BMCL-200910075-9a-h (40e47) were obtained, all of them have IC50 values of less than 2 nM. Wyeth-BMCL-200910075-9b (41), Wyeth-BMCL-200910075-9c (42) and Wyeth-BMCL-200910075-9e (44) demonstrated a selectivity to mTOR of over 1000 [Selectivity = IC50 (PI3Ka)/IC50 (mTOR)].

Fig. 9. Examples of 2-(morpholin-4-yl)-4-substituted oxy-6-(4-(4-(N, N-disubstituted carbamyl) phenyl ureido) phenyl)-1,3,5-triazines mTOR inhibitors.

On the other hand, from GDC-0941, modified substituent on 2- position and 4-position and obtained GNE-493 (48) and GNE-477
(49) respectively, both are TPdIs. GNE-493 has IC50 values of 30 nM against mTOR and 3.4 nM against PI3Ka [206], and GNE-477 has a Ki value of 21 nM against mTOR and an IC50 value of 4 nM against PI3Ka [207].

These compounds are all have common thieno [3,2-d] pyrimi- dine and a morpholine or its derivative substituted on pyrimidine ring. Besides, there is no substituent or only a methyl group on the 7 position of thiophene ring, which inferred that a large group on this site will affect the interaction and pharmacological activity (Fig. 13).

3.2.6. Sulfonamides

GSK2126458 (Omipalisib, GSK458, 50) is an mTOR/PI3K dual inhibitor with a structure of pyridine benzene sulfonamide and it has Ki values of 0.18 nM, 0.3 nM and 0.019e0.13 nM against mTORC1, mTORC2 and PI3K respectively [208]. Cell experiments showed that GSK2126458 can inhibit the progression of nasopha- ryngeal carcinoma (NPC) and improve the sensitivity of radio- therapy [209]. In a randomised and double-blind experiment in patients with idiopathic pulmonary fibrosis (IPF), GSK2126458 exposure dependent inhibition confirmed target engagement in blood and lungs. Besides, the most common adverse event was diarrhea, dose-dependent increases in insulin and glucose were also observed [210].

A similar compound, CMG002 (51), is a new mTOR/PI3K dual inhibitor developed by CMG Pharmaceutical. It is reported that combination of CMG002 and autophagy inhibitor, Chloroquine, can enhance Epstein-Barr virus-associated gastric cancer (EBVaGC) cell apoptosis, suggesting a synergistic effect [211]. Besides, single- agent CMG002 is cytotoxic in chemoresistant ovarian cancer cells, and can inhibit HCC cell proliferation and tumorigenesis in com- bination with Sorafenib, suggesting that this novel inhibitor might be a new therapeutic strategy for ovarian cancer and HCC [212,213].

XL765 (Voxtalisib, 52) is an mTOR/PI3K dual inhibitor developed by Exelixis, which is modified from single target PI3K inhibitor XL- 147 (Pilaralisib, 53) and has a structure of quinoxaline benzene sulfonamide [214]. XL765 demonstrated a better therapeutic effect on prostate cancer cell models than XL147 and Rapamycin [215]. Besides, XL765 suppresses glioblastoma (GBM) growth by inducing ER stress-dependent apoptosis, making it a promising therapeutic strategy to relieve tumor burden in GBM patients [216].

The sulfonyl group of these compounds are all connected to benzene ring; therefore, these compounds can also be further subdivided into benzene sulfonamides. Meanwhile, the nitrogen atom of sulfonyl group is connected with pyridine or other nitrogen-containing heterocycles (Fig. 14).

Fig. 10. Pyrido [2,3-d] pyrimidines mTOR inhibitors.

Fig. 11. Pyrazolo [3,4-d] pyrimidines mTOR inhibitors.

3.2.7. 3-Methylimidazo[4,5-c] quinolin-2-ones

NVP-BEZ235 (Dactolisib, BEZ235, 54) is an oral mTOR/PI3K dual inhibitor developed by Novartis, with IC50 values of 20.7 nM against mTOR and 4 nM, 75 nM, 7 nM and 5 nM against PI3Ka, b, d and g respectively and can inhibit tumor cells with both wild-type and mutated PI3Ka [217,218]. In breast cancer cell lines, NVP-BEZ235 is much more potent in inducing apoptosis than other PI3K inhibitors [219]. However, NVP-BEZ235 demonstrated significant toxicity in patients with advanced RCC [220], and a therapeutic effect that is not as good as Everolimus in treating PNET [221]. Therefore, the follow-up study on RCC and PNET did not continue. In a Phase II study in patients with locally advanced or metastatic transitional cell carcinoma, NVP-BEZ235 showed modest clinical activity and an unfavorable toxicity profile, but a minority of patients experienced a clinical benefit, suggesting that it could improve outcome in some specific patients [222].

Fig. 12. 4-Aminopyrazolo [3,4-d] pyrimidines mTOR inhibitors.

NVP-BGT226 (BGT226, 55) is another oral mTOR/PI3K dual in- hibitor developed by Novartis. It is reported that NVP-BGT226 displays cytotoxic activity in both normoxic and hypoxic HCC cells, suggesting it as a potential candidate for cancer treatment in HCC targeted therapy [223]. NVP-BGT226 was also studied in head and neck cancer, pancreatic cancer and NSCLC [224e226]. In addition, some studies showed that NVP-BEZ235 and NVP-BGT226 can improve the radiosensitivity of tumor cells and endothelial cells, thus can be used in combination with radiotherapy [227].

LY3023414 (56) is a highly water soluble oral mTOR/PI3K dual inhibitor developed by Lilly, with IC50 values of 165 nM against mTOR and 6.07 nM, 77.6 nM, 38 nM and 23.8 nM against PI3Ka, b, d and g respectively [228]. Preclinical studies showed that LY3023414 has anti-tumor activity and demonstrated good per- formance in glioma cells [229], skin squamous cell carcinoma cells [230] and esophageal adenocarcinoma rat models [231]. In a Phase I study, LY3023414 has a tolerable safety profile and single-agent activity in patients with advanced cancers [232]. And a Phase II study just finished showed that, in patients with heavily pretreated advanced endometrial cancer, LY3023414 demonstrated modest single-agent activity and a manageable safety profile [233] (Fig. 15).

3.2.8. Benzo[h]1,6-naphthyridin-2-ones

Torin 1 (57) is a selective mTOR inhibitor discovered by Liu et al. [234], with IC50 values of 0.29 nM against mTORC1 and 2 nM against mTOR at enzyme and cell level respectively. The half-life of Torin 1 is about 4min (human/mouse microsome). Recently it is reported that Torin 1 can intervene mTOR signaling in insular cortex to alleviate neuropathic pain [235]. However, although Torin 1 has a strong inhibitory effect on mTOR, its poor stability and low oral bioavailability limit its in vivo study.

Torin 2 (58), a selective mTOR inhibitor, is obtained by structural simplification of Torin 1, with IC50 values of 37.1 nM against mTORC1 and 25 nM against mTOR. Although the inhibitory effect on mTOR was not as good as Torin 1, the stability was improved and the half-life of Torin 2 in mouse microsome was prolonged to 17.7min [236]. Besides, Torin 2 also has inhibitory effect on ATM, ATR and DNAPK, IC50 values are less than 10 nM [237]. At present, preclinical studies of Torin 2 have been carried out on various of cancers including thyroid cancer, liver cancer, colorectal cancer, breast cancer and leukemia [238e246]. In the study, Torin 2 was also found to be able to inhibit the repair process of DNA damage caused by ionizing radiation [247]. It is even an anti-malarial that can block replenishment of Plasmodium liver stage parasitophorous vacuole membrane proteins [248]. Now, Torin 2 is also studied as a lead compound, and its derivatives are under development [249].

Fig. 13. Thieno [3,2-d] pyrimidines mTOR inhibitors.

These compounds show similar structures with 3- methylimidazo [4,5-c] quinolin-2-ones. Therefore, these two cate- gories of mTOR inhibitors can be considered as quinoline condense with pyrrolidone or pyridone respectively, and alkaline heterocyclic groups such as pyridine or quinoline are in the specific positions of quinoline ring (Fig. 16).

3.2.9. Other TORCdIs and TPdIs

In addition to above-mentioned pyrido-, pyrazolo- and thieno- pyrimidines, there are some mTOR inhibitors with other hetero- cyclic pyrimidine such as triazolo- and imidazolo-. They have no obvious structural similarity, but most contain heterocyclic py- rimidine ring structure (Fig. 17). PKI-402 (59) is an mTOR/PI3K dual inhibitor with triazolo [4,5-d] pyrimidine structure, and it has IC50 values of 1.7 nM, 1.4 nM and 9.2 nM against mTOR, PI3Ka and PI3Kg respectively [250]. PF-04691502 (60) is an mTOR/PI3K dual inhib- itor with pyrido [2,3-d] pyrimidine structure and has Ki values of 16 ± 4.9 nM against mTOR and 1.6e2.1 nM against PI3Ka-g [251]. VS-5584 (SB2343, 61) is an mTOR/PI3K dual inhibitor with purine structure and has IC50 values of 37±7 nM against mTOR and 16e68 nM against PI3K [252]. GDC-0084 (62) is also an mTOR/PI3K dual inhibitor with purine structure, it has Ki values of 70 nM against mTOR and 2 nM against PI3Ka [253].
OSI-027 (ASP7486, 63) is an oral selective mTOR inhibitor with imidazo [5,1-f] triazine structure and an IC50 value of 4 nM [254]. It is reported that OSI-027 inhibits pancreatic ductal adenocarcinoma cell proliferation and enhances the therapeutic effect of gemcita- bine both in vitro and in vivo [255], and demonstrated dose- dependent manner in patients with advanced solid tumors [256]. Besides, in molecular docking, a compound with the similar chemical structure with OSI-027 and a CID of 73294902 (No name and code, here called CID-73294902, 64) demonstrated a stronger inhibition on mTOR than OSI-027 and PI-103. The interaction has a pKd of 6.69 and binding energy of —9.13 kcal/mol [257].

CC-223 (Onatasertib, 65) is a selective mTOR inhibitor with 3,4- dihydropyrazino [2,3-b] pyrazin-2-one structure and an IC50 value of 16 nM [258,259]. The main metabolite of CC-223 is O- demethyleCCe223, which is catalyzed by CYP2C9 and CYP3A in liver microsome. Drug-drug interactions (DDI) study showed that, CC-223 and O-demethyleCCe223 are inhibitors of CYP2C9 and CYP2C19 and moderate inducers of CYP3A [260]. It is reported that CC-223 inhibits HCC and HNSCC cell growth [261,262]. A study in patients with non-pancreatic NET has entered into Phase II [263].

Compound 18i (No name and code, 66) is a potent 5- ureidobenzofuranone indoles mTOR/PI3K dual inhibitor discov- ered by Zhang et al. [264], with IC50 values of 0.3 nM against mTOR and 0.2 nM against PI3K. XL388 (67) is a benzoxazepines selective mTOR inhibitor discovered by Takeuchi et al. [265], with an IC50 value of 9.9 nM. Preclinical studies in models suggested XL388 has therapeutic value on osteosarcoma and RCC [266,267]. Recently it is reported that XL388 behaves like Torin 1, can also intervene mTOR signaling in insular cortex to alleviate neuropathic pain [235,268]. Wortmannin (68) is a lactone antibiotic isolated from culture filtrates of Penicillium wortmanni by Brian et al. [269] in 1957. MacMillan et al. [270] proposed a steroid structure for Wortmannin in 1968, in 1972 Petcher et al. [271] confirmed it through crystal X- ray diffraction. Wortmannin was first a PI3K inhibitor, in 1996 Brunn et al. [272] discovered that Wortmannin has mTOR inhibi- tion activity, with an IC50 value of about 300 nM. Recent study found that, Wortmannin attenuates seizure-induced hyperactive PI3K/Akt/mTOR signaling, impaired memory and spine dysmorphology in rats [273].In addition, in molecular simulation, 2,6- Dihydroxyacetophenone (DHAP) showed a good interaction with mTOR, and could produce some inhibition activity, suggesting that it had some selectivity to mTOR [244].

Fig. 14. Sulfonamides mTOR inhibitors.

Fig. 15. 3-Methylimidazo [4,5-c] quinolin-2-ones mTOR inhibitors.

3.2.10. Discussion

In 2013, Yang et al. [274] reported co-crystal structure of a complex of mTOR with ATP and some ATP-site inhibitors. In the co- crystal structure of the complex of mTOR with ATP (PDB ID: 4JSP), visualization showed that the adenine part embedded into the adenine pocket of mTOR ATP-binding site (Blue circles in Fig. 18A), while ribose, the middle part of ATP, was located in a relatively large space called ribose pocket (Red circles in Fig. 18A). Further visual- ization showed that mTOR formed hydrogen bonds with both ends of ATP, while no hydrogen bond was formed with the middle part (Fig. 18B; 18C). Through visualization, information of the structure of ATP-binding site can be obtained: an adenine pocket (also called hinge region), a hydrophobic pocket, two hindrance regions, a ribose pocket and a top hole, respectively corresponding to the blue-1, green-2, black-3/-5, red-4 and red-6 in Fig. 18D and E. Based on these information, a hypothesis about mTOR kinase inhibitor design model is proposed.

Fig. 16. Benzo [h]1,6-naphthyridin-2-ones mTOR inhibitors.

To help understand the hypothesis, a graphical overview of the ATP-binding site is presented in Fig. 18E. In the hinge region, there are some residues that can form hydrogen bonds with nitrogen or oxygen atom. Therefore, when designing compounds, groups with nitrogen or oxygen in this region will help improve the interaction and strength. Besides, designing a group that can insert into the top hole will also help improve the interaction, or even the half-life. In addition, compound designing should avoid designing groups in the steric hindrance regions (black-3/-5). The red-7 is solvent front, which represents an open region that groups here have nearly no interaction with ATP-binding site, providing a large space for modification and optimization. While the yellow-8 is an external region, which means if compounds with long enough chain to have useful interaction with this region, the inhibition and half-life might improve.

Based on the graphical overview of the ATP-binding site, there are two possible scaffolds for designing new mTOR kinase in- hibitors. Type-1 scaffold is an inverted-Y-shape scaffold consisting of 4 parts (Fig. 18F). Part-1 is corresponding to the hinge region,therefore, nitrogen or oxygen atoms in part-1 will help improve the interaction and strength. Part-2 is a central connector that connects the other three parts together. Since part-2 is located in a relatively large space, the structure of part-2 can be widely variable, providing a large space for designing. Part-3 and part-4 are for variable designing. In the second generation of mTOR inhibitors, the lead compound PI-103, 1,3,5-triazines, pyrido [2,3-d] pyrimi- dines, pyrazolo [3,4-d] pyrimidines and thieno [3,2-d] pyrimidines belong to this type of scaffold. Representative examples selected from each category are listed below (Fig. 18F). It is not hard to find out that all compounds belonging to Type-1 scaffold have a mor- pholinyl group in part-1. Previous researches showed that residues in hinge region formed hydrogen bonds with the oxygen atom of morpholinyl group [275,276]. Therefore, it is reasonable to directly use a morpholinyl group in part-1 when designing a new com- pound with Type-1 scaffold.

Fig. 17. Other TORCdIs and TPdIs.

Type-2 scaffold is a C-shape scaffold consisting of 4 parts (Fig. 18G). Part-1 is the same as Type-1 scaffold. While part-2 is corresponding to the hydrophobic pocket, thus designing nonpolar or hydrophobic group in this part will help improve the interaction. Part-3 and part-4 are changeable design site. In the second gener- ation of mTOR inhibitors, 4-aminopyrazolo [3,4-d] pyrimidines, sulfonamides, 3-methylimidazo [4,5-c] quinoline-2-ones and benzo [h]1,6-naphthyridin-2-ones belong to this type of scaffold. Representative examples selected from each category are listed below (Fig. 18G). It is worth mentioning that none of these com- pounds has morpholinyl group, instead, nitrogen atoms in het- erocycles play the role on forming hydrogen bonds with residues. Therefore, using heterocycles containing nitrogen in part-1 when designing new compounds with Type-2 scaffold is worth consid- ering. In addition, when designing a new compound, it is important to avoid designing groups in steric hindrance regions (5 and 6),whether Type-1 or Type-2 scaffold is used.

In addition, according to the shape of the ATP-binding site, relatively flat molecules are more likely to enter and bind. 3D conformations of the above-mentioned 9 representative com- pounds are constructed by Chem3D and optimized by Molecular Dynamics and Minimize Energy (Fig. 19). As it is showed, most of the representative compounds have a relatively flat conformation and show a potent inhibition on mTOR. While NVP-BEZ235 and Torin-2, with not so strong inhibitory effect as the other com- pounds, are biplane molecules with an angle. It might because the steric hindrance form by molecule itself does not allow the two large branches to be coplanar. And it also indicates the direction of further optimization in NVP-BEZ235 and Torin-2 that adjusting the entire molecular conformation to a flat 3D conformation.
Furthermore, there is an interesting phenomenon. In compounds with Type-1 scaffold, alkaline groups often appear at py- rimidine 2 position, while the activities of these compounds are considerable. Which suggests that the alkaline groups at pyrimi- dine 2 position might play a role in enhancing the pharmacological activity. However, the relationship between the intensity and structure of the alkaline group at pyrimidine 2 position and the inhibitory activity remains to be studied.

3.3. The third generation: other new synthetic mTOR inhibitors

P529 (Palomid 529, RES-529, 69), a benzo [c]chromen-6 ones oral mTOR inhibitor, is modified from dibenzo [c]chromen-6-one scaffold that possesses anti-estrogenic activity. P529 can inhibit mTORC1 and mTORC2 simultaneously [277]. In the case of P529, a comparable blockade of mTOR association with both Rictor and Raptor was observed, suggesting some possible mechanisms include binding to mTOR to inhibit complex assembly or a disruption of previously assembled complexes [277]. P529 demonstrated good performance in prostate cancer and can pro- duce sensitization effect on radiotherapy and some drugs [278e280]. A small short-term clinical trial showed that,subconjunctival injections of P529 in the treatment of neovascular age-related macular degeneration had no concerns for any ocular or systemic toxicity [281]. Besides, P529 can penetrate the BloodeBrain Barrier without restriction by ABCB1 and ABCG2, providing a candidate compound for treatment of brain tumors [282].

Fig. 18. Analysis of ATP-binding Site, Graphical Overview of ATP-binding Site and Hypothetic Compound Scaffolds of the Second Generation of mTOR Inhibitors. (A) Visualization of ATP binds to the ATP-binding site of mTOR. (B), (C) Residues form hydrogen bonds with ATP. (D) Surface visualization of ATP-binding site. (E) Graphical overview of ATP-binding site, including hinge region (adenine pocket, blue-1), hydrophobic pocket (green-2), hindrance region (black-3 and -5), ribose pocket (red-4), top hole (red-6, on the top surface of the ATP-binding site), solvent front (red-7) and external region (yellow-8). View looking down the vertical axis of the first figure of (D). (F) Type-1 scaffold of the second generation of mTOR inhibitors and representative examples, including PI-103 (16), PKI-179 (18), AZD8055 (25), WYE-132 (31) and GDC-0980 (39). (G) Type-2 scaffold of the second generation of mTOR inhibitors and representative examples, including INK-128 (37), GSK2126458 (50), NVP-BEZ235 (54) and Torin-2 (58). X-ray diffraction data (PDB ID: 4JSP) was visualized by PyMOL. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

JR-AB2-011 (70) is a 2-iminothiazolidines mTORC2 inhibitor reported by Benavides-Serrato et al. [283,284] in 2017. JR-AB2-011 binds to Rictor and block mTOR-Rictor association, therefore in- hibits mTORC2. JR-AB2-011 has an IC50 value of about 310 nM against mTORC2 which is 4 times that of its lead compound JR-AB2- 000 (71), and 6e7 times of affinity for Rictor that of JR-AB2-000. At present, JR-AB2-011 is under further study.

RapaLinks are the third generation of mTOR inhibitors different from Rapalogs, TORCdIs and TPdIs. RapaLinks consist of ATP-site inhibitor, linker and Rapalogs. The basic principle and design idea of RapaLinks is that ATP-site inhibitors and Rapamycin derivatives are connected with a certain length of carbon chain, due to mTOR targeting property of Rapamycin derivatives, the drug can be enriched in mTOR and reach to a locally high concentration state. The specific length of the carbon chain makes the whole drug present a specific conformation. When Rapamycin derivative part binds to mTOR, the ATP-site inhibitor part can appropriately interact with the ATP binding site. Therefore, Rapamycin derivative part and the specific conformation presented by the specific length of carbon chain makes the whole drug highly targeted and effective.

RapaLink-1 (72), RapaLink-2 (73) and RapaLink-3 (74) are the first RapaLinks, all of which take INK-128 as ATP binding site and Rapamycin as FRB/FKBP12 binding site, and use different Linker to connect 1-N atom of INK-128 and 42-O atom of Rapamycin [285]. Simulation results showed that, Rapalink-3 with only 11 atom chains in Linker is too short to combine two sites optimally at the same time, while Rapalink-1 and Rapalink-2 with 39 and 36 atom chain in Linker can simultaneously bind to ATP binding site and FRB [285]. Research showed that, RapaLink-1 demonstrated a better effect on GBM than Rapamycin and TORCdIs [286]. And RapaLink-1 plays an antithrombotic role in antiphospholipid syndrome by improving autophagy [287]. Besides, the latest result of RapaLink-1 against Sunitinib-resistant RCC has just published, suggesting that RapaLink-1 might be a brand-new therapy against RCC [288] (Fig. 20).

The FRB domain and the ATP-binding site are spatially close, which provides the possibility to design double-target (or “biva- lent”) mTOR inhibitors (Fig. 21A; 21B; 21C). The basic design idea of RapaLinks is to link Rapalogs and ATP-site inhibitors with a linker. The length of the linker has to be appropriate enough to make the entire molecule conformation favorable for binding to both targets. In the case of RapaLink-1 and RapaLink-2, the linkers are with 8 and 7 ethoxy repeats respectively (Fig. 21D). Future development of RapaLinks will not be limited to link Rapamycin and INK-128 like RapaLink-1 and RapaLink-2 do. Both Rapalogs and ATP-site in- hibitors are alternative and there are many combinations to be studied. Besides, whether there is an influence of the shape, conformation and physical-chemical properties of linker on the binding result remains to be studied.

In addition, since mTOR exist as the form of mTORC1 and mTORC2, it is feasible to inhibit mTOR activity through affecting subunit interaction. Theoretically, enhancing negative regulatory subunit activity, inhibiting positive regulatory subunit activity, inhibiting complex assembling and accelerating complex decom- position are all possible to inhibit mTOR activity. As it is mentioned above, Raptor and Rictor work as scaffolds, mSin1 helps locating, Protor assists complex assembling, PRAS40 and Deptor are negative regulatory subunits and mLST8 is positive regulatory subunit. Therefore, there are many aspects remain to be studied, including how these subunits interact with each other and regulate mTOR activity, whether there is a high-selectivity target being suitable for designing drugs, and what the binding mode between drug and target is. The above-mentioned P529 (69) and JR-AB2-011 (70) are drugs inhibiting mTOR through this mechanism, which could be considered as lead compounds for designing a new generation of mTOR inhibitor in the future, which we here called mTOR subunit interaction interfering agents (TSIIAs).

3.4. Natural products with mTOR inhibitory activity

In addition to the above synthetic compounds, some natural products can also directly or indirectly regulate mTOR and mTOR signaling pathway (Fig. 22). Curcumin (75), i.e. diferuloylmethane, is a highly symmetric molecule, mainly isolated from Curcuma rcenyujin, Curcuma longa, Curcuma phaeocaulis and Acorus calamus. Many cultured cell models and animal models showed that Curcumin has the activity of anti-inflammation, anti-proliferation, anti-invasion, anti-angio- genesis radiosensitization and chemosensitization, and is a po- tential candidate as a therapeutic agent in wound healing, diabetes, AD, PD, cardiovascular disease, pulmonary disease and arthritis [289]. In 2007, Li et al. [290] reported that Curcumin shows anti- cancer activity by inhibiting the expression of MDM2 through PI3K/mTOR/ETS2 pathway. In 2009, Johnson et al. [291] reported a direct evidence that Curcumin can dose-dependently decrease mTOR, Rictor and Raptor, suggesting that the anti-proliferation activity of Curcumin might related to mTOR as well as Curcumin is an mTOR inhibitor which inhibits mTORC1 and mTORC2 simultaneously.(—)-Epigallocatechin gallate (EGCG, 76) is a polyphenol isolated from tea. In 2011, EGCG was proven to be an ATP-competitive mTOR/PI3K dual inhibitor, with Ki values of 320 nM against mTOR and 380 nM against PI3K [292]. Besides, EGCG can also regulate AMPK, Akt and PTEN, thus playing a role in hep- atocarcinoma, colorectal carcinoma, pancreatic cancer, obesity and obesity-related diseases [293e296]. The newest study showed that, EGCG can protect vascular endothelial cell from oxidative stress injury by inducing autophagy through mTOR signaling targeting [297].

Resveratrol (RSV, 77) is a non-flavonoid polyphenol, mainly isolated from Vitis vinifera and Reynoutria japonica. Currently known that Resveratrol has anti-inflammatory, anti-cancer, anti- aging, anti-oxidant and cardiovascular protection effects [298]. It is reported that Resveratrol inhibits mTORC1 activity by enhancing the interaction between mTOR and negative regulatory subunit Deptor [299].
Sulforaphane (SFN, 78), i.e. 4-methylsulfonyl-1-isothiocyanate, is a common natural product in plants of Cruciferae, has a dose- and time-dependent inhibitory effect on mTOR [300]. Studies show that Sulforaphane inhibits protein synthesis, proliferation and invasion [301e303], and can induce cell cycle arrest and apoptosis and inhibit cell growth [304,305].

In addition to the natural products that directly inhibit mTOR, there are also some natural products that can regulate the signaling pathway of mTOR, thus producing mTOR inhibitory effect. Cryp- totanshinone (CPT, 79) is a monomer isolated from Salvia miltior- rhiza, can inhibit mTORC1 signaling and produce anti-proliferation effect [306]. Genistein (80) is an iso-flavonoid natural product, mainly exist in plants of Leguminosae, can down-regulate mTOR signaling and produce autophagy-induction and anti-proliferation effects [307e309]. Caffeine (81), i.e. 1,3,7-trimethylxanthine, is a xanthine alkaloid isolated from tea and coffee bean, can induce autophagy and apoptosis through down-regulation of mTOR signaling pathway [310,311].Tewari et al. [312] summarized a series of natural products with direct or potential regulatory effect on PI3K/Akt/mTOR signaling pathway, including monomeric compounds such as Salidroside (Rhodioloside, 82), Oridonin (Rubescensin A, 83), Capsaicin (84),Arctigenin (ATG, 85), Apigenin (86), Vitexin (87), Andrographolide (88), Rotundic acid (89), Gingkolic acid (90), Afrocyclamin A (91), and semi-synthetic natural product Docetaxel (92) as well as extras from Celastrus orbiculatus and Rhizoma Amorphophalli, pointing out the research direction for the development of new mTOR inhibitors and anticancer drugs in the future.

Fig. 19. 3D Conformations of 9 Representative Compounds. (A) PI-103. (B) PKI-179. (C) AZD8055. (D) WYE-132. (E) GDC-0980. (F) INK-128. (G) GSK2126458. (H) NVP-BEZ235. (I)Torin-2.3D conformations were constructed by Chem3D and optimized by Molecular Dynamics and Minimize Energy.

Fig. 20. Other new synthetic mTOR inhibitors.

Fig. 21. RapaLinks Are Double-target mTOR Inhibitors. (A), (B), (C) FRB/FKBP12-binding site and ATP-binding site are spatially close (different view direction). Molecular model constructed by X-ray diffraction data of ATP binding to mTOR catalytic domain (PDB ID: 4JSP) and FKBP12-Rapamycin complex interacting with mTOR FRB domain (PDB ID: 1FAP).(D) Structure of RapaLink-1 and RapaLink-2. Blue shading corresponds to Rapalogs and red shading corresponds to ATP-site inhibitors. Visualization and alignment and were accomplished by PyMOL. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

4. Summary and prospect

mTOR as the total switch of cell metabolism, is a very effective target protein of anticancer drugs. Through inhibiting the over- activated mTOR signal in tumors, a series of strong antitumor ef- fects can be produced. Therefore, many mTOR inhibitors are used clinically as anticancer drugs. However, long-term use of the same mTOR inhibitor will lead to the establishment of tolerance, and thus resistant to drugs, so it is necessary to keep developing new mTOR inhibitors.

Although the discovery of allosteric mTOR inhibitors is early and the studies on Rapalogs are mature, it is still difficult to synthesize Rapalogs due to high molecular weight and many chiral carbons. Besides, the modification sites are limited. Therefore, the future development direction of this category of mTOR inhibitors will be the treatment to other disease based on the existing drugs and their preparations. As for those compounds with considerable effect but poor drug properties can start from structural modification to improve their drug properties or develop new drug formulations. In addition, above-mentioned modifications are all single site modi- fication, future studies can start with modifications of multiple site combination.

Synthesis of small molecular mTOR inhibitors targeting ATP- binding sites (TORCdIs and TPdIs) are relatively easy, and their in- hibition to mTOR is more thorough. Therefore, small molecular mTOR inhibitors will also be a great research direction in the future. New compounds with mTOR inhibitory activity can be obtained through designing molecules with Type-1 scaffold (inverted-Y- shape scaffold) or Type-2 scaffold (C-shape scaffold). In Type-1 scaffold, nitrogen/oxygen-containing groups in part-1 are favor- able for improving drug property, while the morpholinyl group is the most common used. Part-2 has a good compatibility since there is a relatively large space. In Type-2 scaffold, nitrogen/oxygen- containing groups in part-1 and nonpolar/hydrophobic groups in part-2 are favorable for improving drug property. A possible reason of no morpholinyl group in part-1 of drugs with Type-2 scaffold is that the part-1 in Type-2 scaffold has to connect part-2 and part-3, while the morpholinyl group is not large enough to form an appropriate angle or conformation. Instead, nitrogen-containing heterocycles are favorable in part-1 of Type-2 scaffold, especially nitrogen-containing bicycles. Besides, avoiding groups in hindrance regions (part-5/6) and constructing a relatively flat molecule will also help improve drug property. Furthermore, among compounds with Type-1 scaffold, the relationship between the intensity and structure of the alkaline group at pyrimidine 2 position and the inhibitory activity remains to be studied.

RapaLinks are double-target mTOR inhibitors linking Rapalogs and ATP-site inhibitors with a linker. Future development of RapaLinks will not be limited to link Rapamycin and INK-128 like RapaLink-1 and RapaLink-2 do. Both Rapalogs and ATP-site in- hibitors are alternative and there are many combinations to be studied. Besides, whether there is an influence of the shape, conformation and physical-chemical properties of linker on the binding result remains to be studied.

Compounds affecting the interaction between mTOR and sub- units, such as P529, JR-AB2-011, Curcumin and Resveratrol, are also a starting point for the development of a new generation of mTOR inhibitors (mTOR subunit interaction interfering agents, TSIIAs). Theoretically, enhancing negative regulatory subunit activity, inhibiting positive regulatory subunit activity, inhibiting complex assembling and accelerating complex decomposition are all possible to inhibit mTOR activity. However, there are many aspects remain to be studied, including how these subunits interact with each other and regulate mTOR activity, whether there is a high- selectivity target being suitable for designing drugs, and what the binding mode between drug and target is.

Some natural products with mTOR inhibitory activity will also be the research object in the future. After clarifying the mechanism of mTOR inhibitory effect of a natural product, it could be a lead compound.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 22. Natural products with mTOR inhibitory activity.

Acknowledgement

We firstly want to thank Jilin University for providing many online databases including ACS, Elsevier, Springer and CNKI. Next, we thank National Center for Biotechnology Information (NCBI) for providing free literature and information retrieval platforms such as PubMed, PubChem and PMC. We also want to thank Protein Data Bank (PDB) for free Cryo-EM and X-ray diffraction data.

References

[1] J. Heitman, N.R. Movva, M.N. Hall, Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast, Science 253 (5022) (1991) 905e909.
[2] E. Jacinto, M.N. Hall, Tor signalling in bugs, brain and brawn, Nat. Rev. Mol. Cell Biol. 4 (2) (2003) 117e126.
[3] T. Schmelzle, M.N. Hall, TOR, a central controller of cell growth, Cell 103 (2) (2000) 253e262.
[4] E.J. Brown, M.W. Albers, T.B. Shin, et al., A mammalian protein targeted by G1-arresting rapamycin-receptor complex, Nature 369 (6483) (1994) 756e758.
[5] D.M. Sabatini, H. Erdjument-Bromage, M. Lui, P. Tempst, S.H. Snyder, RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs, Cell 78 (1) (1994) 35e43.
[6] M.I. Chiu, H. Katz, V. Berlin, RAPT1, a mammalian homolog of yeast Tor, in- teracts with the FKBP12/rapamycin complex, Proc. Natl. Acad. Sci. U. S. A. 91
(26) (1994) 12574e12578.
[7] C.J. Sabers, M.M. Martin, G.J. Brunn, et al., Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells, J. Biol. Chem. 270 (2) (1995) 815e822.
[8] A.C. Gingras, B. Raught, N. Sonenberg, Regulation of translation initiation by FRAP/mTOR, Genes Dev. 15 (7) (2001) 807e826.
[9] C.T. Keith, S.L. Schreiber, PIK-related kinases: DNA repair, recombination, and cell cycle checkpoints, Science 270 (5233) (1995) 50e51.
[10] M.A. Andrade, P. Bork, HEAT repeats in the Huntington’s disease protein, Nat. Genet. 11 (2) (1995) 115e116.
[11] R. Bosotti, A. Isacchi, E.L. Sonnhammer, FAT: a novel domain in PIK-related kinases, Trends Biochem. Sci. 25 (5) (2000) 225e227.
[12] J. Choi, J. Chen, S.L. Schreiber, J. Clardy, Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP, Science 273 (5272) (1996) 239e242.
[13] D.D. Sarbassov, S.M. Ali, D.H. Kim, et al., Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton, Curr. Biol. 14 (14) (2004) 1296e1302.
[14] E. Jacinto, R. Loewith, A. Schmidt, et al., Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive, Nat. Cell Biol. 6 (11) (2004) 1122e1128.
[15] X.F. Zheng, D. Florentino, J. Chen, G.R. Crabtree, S.L. Schreiber, TOR kinase domains are required for two distinct functions, only one of which is inhibited by rapamycin, Cell 82 (1) (1995) 121e130.
[16] D.D. Sarbassov, S.M. Ali, S. Sengupta, et al., Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB, Mol. Cell. 22 (2) (2006) 159e168.
[17] C.K. Yip, K. Murata, T. Walz, D.M. Sabatini, S.A. Kang, Structure of the human mTOR complex I and its implications for rapamycin inhibition, Mol. Cell. 38
(5) (2010) 768e774.
[18] S. Wullschleger, R. Loewith, W. Oppliger, M.N. Hall, Molecular organization of target of rapamycin complex 2, J. Biol. Chem. 280 (35) (2005) 30697e30704.
[19] H. Yang, J. Wang, M. Liu, et al., 4.4 Å Resolution Cryo-EM structure of human mTOR Complex 1, Protein Cell 7 (12) (2016) 878-887.
[20] X. Chen, M. Liu, Y. Tian, et al., Cryo-EM structure of human mTOR complex 2, Cell Res. 28 (5) (2018) 518e528.
[21] K. Hara, Y. Maruki, X. Long, et al., Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action, Cell 110 (2) (2002) 177e189.
[22] D.H. Kim, D.D. Sarbassov, S.M. Ali, et al., mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery, Cell 110 (2) (2002) 163e175.
[23] H. Nojima, C. Tokunaga, S. Eguchi, et al., The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E- BP1 through their TOR signaling (TOS) motif, J. Biol. Chem. 278 (18) (2003) 15461e15464.
[24] S.S. Schalm, D.C. Fingar, D.M. Sabatini, J. Blenis, TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function, Curr. Biol. 13 (10) (2003) 797e806.
[25] Y. Sancak, T.R. Peterson, Y.D. Shaul, et al., The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1, Science 320 (5882) (2008) 1496e1501.
[26] Y. Sancak, C.C. Thoreen, T.R. Peterson, et al., PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase, Mol. Cell. 25 (6) (2007) 903e915.
[27] L.R. Pearce, X. Huang, J. Boudeau, et al., Identification of Protor as a novel Rictor-binding component of mTOR complex-2, Biochem. J. 405 (3) (2007) 513e522.
[28] E. Vander Haar, S.I. Lee, S. Bandhakavi, T.J. Griffin, D.H. Kim, Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40, Nat. Cell Biol. 9 (3) (2007) 316e323.
[29] T.R. Peterson, M. Laplante, C.C. Thoreen, et al., DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival, Cell 137 (5) (2009) 873e886.
[30] S.Y. Woo, D.H. Kim, C.B. Jun, et al., PRR5, a novel component of mTOR complex 2, regulates platelet-derived growth factor receptor beta expression and signaling, J. Biol. Chem. 282 (35) (2007) 25604e25612.
[31] M.A. Frias, C.C. Thoreen, J.D. Jaffe, et al., mSin1 is necessary for Akt/PKB phosphorylation, and its isoforms define three distinct mTORC2s, Curr. Biol. 16 (18) (2006) 1865e1870.
[32] Q. Yang, K. Inoki, T. Ikenoue, K.L. Guan, Identification of Sin 1 as an essential TORC2 component required for complex formation and kinase activity, Genes Dev. 20 (20) (2006) 2820e2832.
[33] K. Hara, K. Yonezawa, Q.P. Weng, M.T. Kozlowski, C. Belham, J. Avruch, Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism, J. Biol. Chem. 273 (23) (1998) 14484e14494.
[34] X. Wang, L.E. Campbell, C.M. Miller, C.G. Proud, Amino acid availability regulates p70 S6 kinase and multiple translation factors, Biochem. J. 334 (Pt 1) (1998) 261e267.
[35] E. Kim, P. Goraksha-Hicks, L. Li, T.P. Neufeld, K.L. Guan, Regulation of TORC1 by Rag GTPases in nutrient response, Nat. Cell Biol. 10 (8) (2008) 935e945.
[36] Y. Sancak, L. Bar-Peled, R. Zoncu, A.L. Markhard, S. Nada, D.M. Sabatini, Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids, Cell 141 (2) (2010) 290e303.
[37] L.J. Saucedo, X. Gao, D.A. Chiarelli, L. Li, D. Pan, B.A. Edgar, Rheb promotes cell growth as a component of the insulin/TOR signalling network, Nat. Cell Biol. 5 (6) (2003) 566e571.
[38] H. Stocker, T. Radimerski, B. Schindelholz, et al., Rheb is an essential regu- lator of S6K in controlling cell growth in Drosophila, Nat. Cell Biol. 5 (6) (2003) 559e565.
[39] K. Inoki, Y. Li, T. Xu, K.L. Guan, Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling, Genes Dev. 17 (15) (2003) 1829e1834.
[40] Y. Zhang, X. Gao, L.J. Saucedo, B. Ru, B.A. Edgar, D. Pan, Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins, Nat. Cell Biol. 5 (6) (2003) 578e581.
[41] A. Garami, F.J. Zwartkruis, T. Nobukuni, et al., Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2, Mol. Cell. 11 (6) (2003) 1457e1466.
[42] A.R. Tee, B.D. Manning, P.P. Roux, L.C. Cantley, J. Blenis, Tuberous sclerosis complex gene products, Tuberin and Hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb, Curr. Biol. 13
(15) (2003) 1259e1268.
[43] X. Gao, D. Pan, TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth, Genes Dev. 15 (11) (2001) 1383e1392.
[44] N. Tapon, N. Ito, B.J. Dickson, J.E. Treisman, I.K. Hariharan, The Drosophila tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation, Cell 105 (3) (2001) 345e355.
[45] S.L. Cai, A.R. Tee, J.D. Short, et al., Activity of TSC2 is inhibited by AKT- mediated phosphorylation and membrane partitioning, J. Cell Biol. 173 (2) (2006) 279e289.
[46] Y. Li, K. Inoki, R. Yeung, K.L. Guan, Regulation of TSC2 by 14-3-3 binding,
J. Biol. Chem. 277 (47) (2002) 44593e44596.
[47] K.S. Kovacina, G.Y. Park, S.S. Bae, et al., Identification of a proline-rich Akt substrate as a 14-3-3 binding partner, J. Biol. Chem. 278 (12) (2003) 10189e10194.
[48] N. Oshiro, R. Takahashi, K. Yoshino, et al., The proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target of rapamycin complex 1, J. Biol. Chem. 282 (28) (2007) 20329e20339.
[49] B.D. Fonseca, E.M. Smith, V.H. Lee, C. MacKintosh, C.G. Proud, PRAS40 is a target for mammalian target of rapamycin complex 1 and is required for signaling downstream of this complex, J. Biol. Chem. 282 (34) (2007) 24514e24524.
[50] L. Ma, Z. Chen, H. Erdjument-Bromage, P. Tempst, P.P. Pandolfi, Phosphory- lation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis, Cell 121 (2) (2005) 179e193.
[51] R.M. Castilho, C.H. Squarize, L.A. Chodosh, B.O. Williams, J.S. Gutkind, mTOR mediates Wnt-induced epidermal stem cell exhaustion and aging, Cell Stem Cell 5 (3) (2009) 279e289.
[52] K. Inoki, H. Ouyang, T. Zhu, et al., TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth, Cell 126 (5) (2006) 955e968.
[53] P.B. Dennis, A. Jaeschke, M. Saitoh, B. Fowler, S.C. Kozma, G. Thomas, Mammalian TOR: a homeostatic ATP sensor, Science 294 (5544) (2001) 1102e1105.
[54] D.G. Hardie, AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy, Nat. Rev. Mol. Cell Biol. 8 (10) (2007) 774e785.
[55] K. Inoki, T. Zhu, K.L. Guan, TSC2 mediates cellular energy response to control cell growth and survival, Cell 115 (5) (2003) 577e590.
[56] D.M. Gwinn, D.B. Shackelford, D.F. Egan, et al., AMPK phosphorylation of raptor mediates a metabolic checkpoint, Mol. Cell. 30 (2) (2008) 214e226.
[57] M.N. Corradetti, K. Inoki, N. Bardeesy, R.A. DePinho, K.L. Guan, Regulation of the TSC pathway by LKB1: evidence of a molecular link between tuberous sclerosis complex and Peutz-Jeghers syndrome, Genes Dev. 18 (13) (2004) 1533e1538.
[58] R.J. Shaw, N. Bardeesy, B.D. Manning, et al., The LKB1 tumor suppressor negatively regulates mTOR signaling, Canc. Cell 6 (1) (2004) 91e99.
[59] T. Shoshani, A. Faerman, I. Mett, et al., Identification of a novel hypoxia- inducible factor 1-responsive gene, RTP801, involved in apoptosis, Mol. Cell Biol. 22 (7) (2002) 2283e2293.
[60] J. Brugarolas, K. Lei, R.L. Hurley, et al., Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor com- plex, Genes Dev. 18 (23) (2004) 2893e2904.
[61] Z. Feng, W. Hu, E. de Stanchina, et al., The regulation of AMPK beta 1, TSC2, and PTEN expression by p53: stress, cell and tissue specificity, and the role of these gene products in modulating the IGF-1-AKT-mTOR pathways, Canc. Res. 67 (7) (2007) 3043e3053.
[62] L.W. Ellisen, K.D. Ramsayer, C.M. Johannessen, et al., REDD1, a develop- mentally regulated transcriptional target of p63 and p53, links p63 to regulation of reactive oxygen species, Mol. Cell. 10 (5) (2002) 995e1005.
[63] D.F. Lee, H.P. Kuo, C.T. Chen, et al., IKK beta suppression of TSC1 links inflammation and tumor angiogenesis via the mTOR pathway, Cell 130 (3) (2007) 440e455.
[64] K. Hara, K. Yonezawa, M.T. Kozlowski, et al., Regulation of eIF-4E BP1 phosphorylation by mTOR, J. Biol. Chem. 272 (42) (1997) 26457e26463.
[65] S.R. von Manteuffel, A.C. Gingras, X.F. Ming, N. Sonenberg, G. Thomas, 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is indepen- dent of mitogen-activated protein kinase, Proc. Natl. Acad. Sci. U. S. A. 93 (9) (1996) 4076e4080.
[66] E.J. Brown, P.A. Beal, C.T. Keith, J. Chen, T.B. Shin, S.L. Schreiber, Control of p70 s6 kinase by kinase activity of FRAP in vivo, Nature 377 (6548) (1995) 441e446.
[67] A. Haghighat, S. Mader, A. Pause, N. Sonenberg, Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E, EMBO J. 14 (22) (1995) 5701e5709.
[68] M.K. Holz, B.A. Ballif, S.P. Gygi, J. Blenis, mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events, Cell 123 (4) (2005) 569e580.
[69] D. Shahbazian, P.P. Roux, V. Mieulet, et al., The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity, EMBO J. 25 (12) (2006) 2781e2791.
[70] G. Lenz, J. Avruch, Glutamatergic regulation of the p70S6 kinase in primary mouse neurons, J. Biol. Chem. 280 (46) (2005) 38121e38124.
[71] P. Jeno, L.M. Ballou, I. Novak-Hofer, G. Thomas, Identification and characterization of a mitogen-activated S6 kinase, Proc. Natl. Acad. Sci. U. S.
A. 85 (2) (1988) 406e410.
[72] K.F. Wilson, W.J. Wu, R.A. Cerione, Cdc 42 stimulates RNA splicing via the S6 kinase and a novel S6 kinase target, the nuclear cap-binding complex, J. Biol. Chem. 275 (48) (2000) 37307e37310.
[73] X.M. Ma, S.O. Yoon, C.J. Richardson, K. Julich, J. Blenis, SKAR links pre-mRNA splicing to mTOR/S6K1-mediated enhanced translation efficiency of spliced mRNAs, Cell 133 (2) (2008) 303e313.
[74] T. Haruta, T. Uno, J. Kawahara, et al., A rapamycin-sensitive pathway down- regulates insulin signaling via phosphorylation and proteasomal degradation of insulin receptor substrate-1, Mol. Endocrinol. 14 (6) (2000) 783e794.
[75] O.J. Shah, Z. Wang, T. Hunter, Inappropriate activation of the TSC/Rheb/ mTOR/S6K cassette induces IRS1/2 depletion, insulin resistance, and cell survival deficiencies, Curr. Biol. 14 (18) (2004) 1650e1656.
[76] K.M. Hannan, Y. Brandenburger, A. Jenkins, et al., mTOR-dependent regula- tion of ribosomal gene transcription requires S6K1 and is mediated by phosphorylation of the carboxy-terminal activation domain of the nucleolar transcription factor UBF, Mol. Cell Biol. 23 (23) (2003) 8862e8877.
[77] C. Mayer, J. Zhao, X. Yuan, I. Grummt, mTOR-dependent activation of the transcription factor TIF-IA links rRNA synthesis to nutrient availability, Genes Dev. 18 (4) (2004) 423e434.
[78] T. Kantidakis, B.A. Ramsbottom, J.L. Birch, S.N. Dowding, R.J. White, mTOR associates with TFIIIC, is found at tRNA and 5S rRNA genes, and targets their repressor Maf1, Proc. Natl. Acad. Sci. U. S. A. 107 (26) (2010) 11823e11828.
[79] I. Ben-Sahra, G. Hoxhaj, S.J.H. Ricoult, J.M. Asara, B.D. Manning, mTORC1 induces purine synthesis through control of the mitochondrial tetrahy- drofolate cycle, Science 351 (6274) (2016) 728e733.
[80] I. Ben-Sahra, J.J. Howell, J.M. Asara, B.D. Manning, Stimulation of de novo pyrimidine synthesis by growth signaling through mTOR and S6K1, Science 339 (6125) (2013) 1323e1328.
[81] A.M. Robitaille, S. Christen, M. Shimobayashi, et al., Quantitative phospho- proteomics reveal mTORC1 activates de novo pyrimidine synthesis, Science 339 (6125) (2013) 1320e1323.
[82] T. Porstmann, C.R. Santos, B. Griffiths, et al., SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth, Cell Metabol. 8 (3) (2008) 224e236.
[83] K. Duvel, J.L. Yecies, S. Menon, et al., Activation of a metabolic gene regula- tory network downstream of mTOR complex 1, Mol. Cell. 39 (2) (2010) 171e183.
[84] T.R. Peterson, S.S. Sengupta, T.E. Harris, et al., mTOR complex 1 regulates lipin 1 localization to control the SREBP pathway, Cell 146 (3) (2011) 408e420.
[85] P. Polak, N. Cybulski, J.N. Feige, J. Auwerx, M.A. Rüegg, M.N. Hall, Adipose- specific knockout of raptor results in lean mice with enhanced mitochondrial respiration, Cell Metabol. 8 (5) (2008) 399e410.
[86] J.E. Kim, J. Chen, Regulation of peroxisome proliferator-activated receptor- gamma activity by mammalian target of rapamycin and amino acids in adipogenesis, Diabetes 53 (11) (2004) 2748e2756.
[87] W.C. Yeh, B.E. Bierer, S.L. McKnight, Rapamycin inhibits clonal expansion and adipogenic differentiation of 3T3-L1 cells, Proc. Natl. Acad. Sci. U. S. A. 92 (24) (1995) 11086e11090.
[88] A. Gagnon, S. Lau, A. Sorisky, Rapamycin-sensitive phase of 3T3-L1 pre- adipocyte differentiation after clonal expansion, J. Cell. Physiol. 189 (1) (2001) 14e22.
[89] H.H. Zhang, J. Huang, K. Düvel, et al., Insulin stimulates adipogenesis through the Akt-TSC2-mTORC1 pathway, PLoS One 4 (7) (2009), e6189.
[90] O. Le Bacquer, E. Petroulakis, S. Paglialunga, et al., Elevated sensitivity to diet- induced obesity and insulin resistance in mice lacking 4E-BP1 and 4E-BP2,
J. Clin. Invest. 117 (2) (2007) 387e396.
[91] J.B. Kim, B.M. Spiegelman, ADD1/SREBP1 promotes adipocyte differentiation and gene expression linked to fatty acid metabolism, Genes Dev. 10 (9) (1996) 1096e1107.
[92] J.B. Kim, H.M. Wright, M. Wright, B.M. Spiegelman, ADD1/SREBP1 activates PPARgamma through the production of endogenous ligand, Proc. Natl. Acad. Sci. U. S. A. 95 (8) (1998) 4333e4337.
[93] T. Noda, Y. Ohsumi, Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast, J. Biol. Chem. 273 (7) (1998) 3963e3966.
[94] N. Hosokawa, T. Hara, T. Kaizuka, et al., Nutrient-dependent mTORC1 asso- ciation with the ULK1-Atg13-FIP200 complex required for autophagy, Mol. Biol. Cell 20 (7) (2009) 1981e1991.
[95] C.H. Jung, C.B. Jun, S.H. Ro, et al., ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery, Mol. Biol. Cell 20 (7) (2009) 1992e2003.
[96] J. Kim, M. Kundu, B. Viollet, K.L. Guan, AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1, Nat. Cell Biol. 13 (2) (2011) 132e141.
[97] C. Settembre, R. Zoncu, D.L. Medina, et al., A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB, EMBO J. 31 (5) (2012) 1095e1108.
[98] A. Rousseau, A. Bertolotti, An evolutionarily conserved pathway controls proteasome homeostasis, Nature 536 (7615) (2016) 184e189.
[99] P.G. Charest, Z. Shen, A. Lakoduk, A.T. Sasaki, S.P. Briggs, R.A. Firtel, A Ras signaling complex controls the RasC-TORC2 pathway and directed cell migration, Dev. Cell 18 (5) (2010) 737e749.
[100] V. Zinzalla, D. Stracka, W. Oppliger, M.N. Hall, Activation of mTORC2 by as- sociation with the ribosome, Cell 144 (5) (2011) 757e768.
[101] P. Liu, W. Gan, Y.R. Chin, et al., PtdIns(3,4,5)P3-Dependent activation of the mTORC2 kinase complex, Canc. Discov. 5 (11) (2015) 1194e1209.
[102] X. Gan, J. Wang, C. Wang, et al., PRR5L degradation promotes mTORC2- mediated PKC-d phosphorylation and cell migration downstream of Ga12, Nat. Cell Biol. 14 (7) (2012) 686e696.
[103] V. Thomanetz, N. Angliker, D. Cloe€tta, et al., Ablation of the mTORC2 component rictor in brain or Purkinje cells affects size and neuron morphology, J. Cell Biol. 201 (2) (2013) 293e308.
[104] X. Li, T. Gao, mTORC2 phosphorylates protein kinase Cz to regulate its sta-
bility and activity, EMBO Rep. 15 (2) (2014) 191e198.
[105] D.D. Sarbassov, D.A. Guertin, S.M. Ali, D.M. Sabatini, Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex, Science 307 (5712) (2005) 1098e1101.
[106] G.J. Kops, N.D. de Ruiter, A.M. De Vries-Smits, D.R. Powell, J.L. Bos,
B.M. Burgering, Direct control of the Forkhead transcription factor AFX by protein kinase B, Nature 398 (6728) (1999) 630e634.
[107] A. Brunet, A. Bonni, M.J. Zigmond, et al., Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor, Cell 96 (6) (1999) 857e868.
[108] W.H. Biggs 3rd, J. Meisenhelder, T. Hunter, W.K. Cavenee, K.C. Arden, Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1, Proc. Natl. Acad. Sci. U. S. A. 96 (13) (1999) 7421e7426.
[109] E.D. Tang, G. Nun~ez, F.G. Barr, K.L. Guan, Negative regulation of the forkhead
transcription factor FKHR by Akt, J. Biol. Chem. 274 (24) (1999) 16741e16746.
[110] H. Takaishi, H. Konishi, H. Matsuzaki, et al., Regulation of nuclear trans- location of forkhead transcription factor AFX by protein kinase B, Proc. Natl. Acad. Sci. U. S. A. 96 (21) (1999) 11836e11841.
[111] A.M. Brownawell, G.J. Kops, I.G. Macara, B.M. Burgering, Inhibition of nuclear import by protein kinase B (Akt) regulates the subcellular distribution and activity of the forkhead transcription factor AFX, Mol. Cell Biol. 21 (10) (2001) 3534e3546.
[112] P.F. Dijkers, R.H. Medema, J.W. Lammers, L. Koenderman, P.J. Coffer, Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1, Curr. Biol. 10 (19) (2000) 1201e1204.
[113] R.H. Medema, G.J. Kops, J.L. Bos, B.M. Burgering, AFX-like Forkhead tran- scription factors mediate cell-cycle regulation by Ras and PKB through p27kip1, Nature 404 (6779) (2000) 782e787.
[114] G. Yang, D.S. Murashige, S.J. Humphrey, D.E. James, A positive feedback loop between akt and mTORC2 via SIN1 phosphorylation, Cell Rep. 12 (6) (2015) 937e943.
[115] J.M. Garcia-Martinez, D.R. Alessi, mTOR complex 2 (mTORC2) controls hy- drophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1), Biochem. J. 416 (3) (2008) 375e385.
[116] B. Zhao, X. Wei, W. Li, et al., Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control, Genes Dev. 21 (21) (2007) 2747e2761.
[117] Y. Hao, A. Chun, K. Cheung, B. Rashidi, X. Yang, Tumor suppressor LATS1 is a negative regulator of oncogene YAP, J. Biol. Chem. 283 (9) (2008) 5496e5509.
[118] B. Zhao, L. Li, K. Tumaneng, C.Y. Wang, K.L. Guan, A coordinated phosphor- ylation by Lats and CK1 regulates YAP stability through SCF(beta-TRCP), Genes Dev. 24 (1) (2010) 72e85.
[119] E.H. Chan, M. Nousiainen, R.B. Chalamalasetty, A. Sch€afer, E.A. Nigg,
H.H. Sillje´, The Ste20-like kinase Mst2 activates the human large tumor suppressor kinase Lats1, Oncogene 24 (12) (2005) 2076e2086.
[120] S. Sciarretta, P. Zhai, Y. Maejima, et al., mTORC2 regulates cardiac response to stress by inhibiting MST1, Cell Rep. 11 (1) (2015) 125e136.
[121] S.I. Arriola Apelo, D.W. Lamming, mTORC2 puts its shoulder to Krebs’ Wheel, Mol. Cell. 63 (5) (2016) 723e725.
[122] J.G. Moloughney, P.K. Kim, N.M. Vega-Cotto, et al., mTORC2 responds to glutamine catabolite levels to modulate the hexosamine biosynthesis enzyme GFAT1, Mol. Cell. 63 (5) (2016) 811e826.
[123] F. Boutouja, C.M. Stiehm, H.W. Platta, mTOR: a cellular regulator interface in health and disease, Cells 8 (1) (2019) 18.
[124] L. Khamzina, A. Veilleux, S. Bergeron, A. Marette, Increased activation of the mammalian target of rapamycin pathway in liver and skeletal muscle of obese rats: possible involvement in obesity-linked insulin resistance, Endocrinology 146 (3) (2005) 1473e1481.
[125] M.R. Rajan, E. Nyman, C. Bra€nnmark, C.S. Olofsson, P. Strålfors, Inhibition of
FOXO1 transcription factor in primary human adipocytes mimics the insulin- resistant state of type 2 diabetes, Biochem. J. 475 (10) (2018) 1807e1820.
[126] S.J. Kim, M.A. DeStefano, W.J. Oh, et al., mTOR complex 2 regulates proper turnover of insulin receptor substrate-1 via the ubiquitin ligase subunit Fbw8, Mol. Cell. 48 (6) (2012) 875e887.
[127] D. Benjamin, M. Colombi, C. Moroni, M.N. Hall, Rapamycin passes the torch: a new generation of mTOR inhibitors, Nat. Rev. Drug Discov. 10 (11) (2011) 868e880.
[128] K. Pierzynowska, L. Gaffke, Z. Cyske, et al., Autophagy stimulation as a promising approach in treatment of neurodegenerative diseases, Metab. Brain Dis. 33 (4) (2018) 989e1008.
[129] Z. Wen, J. Zhang, P. Tang, N. Tu, K. Wang, G. Wu, Overexpression of miR-185 inhibits autophagy and apoptosis of dopaminergic neurons by regulating the AMPK/mTOR signaling pathway in Parkinson’s disease, Mol. Med. Rep. 17 (1) (2018) 131e137.
[130] S. Sciarretta, M. Forte, G. Frati, J. Sadoshima, New insights into the role of mTOR signaling in the cardiovascular system, Circ. Res. 122 (3) (2018) 489e505.
[131] S. Mukherjee, U. Mukherjee, A comprehensive review of immunosuppres- sion used for liver transplantation, J Transplant 2009 (2009) 701464.
[132] M. Lucchesi, E. Chiappa, F. Giordano, F. Mari, L. Genitori, I. Sardi, Sirolimus in infants with multiple cardiac rhabdomyomas associated with tuberous sclerosis complex, Case Rep Oncol 11 (2) (2018) 425e430.
[133] H. Park, I. Garrido-Laguna, A. Naing, et al., Phase I dose-escalation study of the mTOR inhibitor sirolimus and the HDAC inhibitor vorinostat in patients with advanced malignancy, Oncotarget 7 (41) (2016) 67521e67531.
[134] D. Cirstea, T. Hideshima, S. Rodig, et al., Dual inhibition of akt/mammalian target of rapamycin pathway by nanoparticle albumin-bound-rapamycin and perifosine induces antitumor activity in multiple myeloma, Mol. Canc. Therapeut. 9 (4) (2010) 963e975.
[135] A.M. Gonzalez-Angulo, F. Meric-Bernstam, S. Chawla, et al., Weekly nab- Rapamycin in patients with advanced nonhematologic malignancies: final results of a phase I trial, Clin. Canc. Res. 19 (19) (2013) 5474e5484.
[136] L.H. Meng, X.F. Zheng, Toward rapamycin analog (rapalog)-based precision cancer therapy, Acta Pharmacol. Sin. 36 (10) (2015) 1163e1169.
[137] E. Raymond, J. Alexandre, S. Faivre, et al., Safety and pharmacokinetics of escalated doses of weekly intravenous infusion of CCI-779, a novel mTOR inhibitor, in patients with cancer, J. Clin. Oncol. 22 (12) (2004) 2336e2347.
[138] M.S. Neshat, I.K. Mellinghoff, C. Tran, et al., Enhanced sensitivity of PTEN- deficient tumors to inhibition of FRAP/mTOR, Proc. Natl. Acad. Sci. U. S. A. 98 (18) (2001) 10314e10319.
[139] G. Hudes, M. Carducci, P. Tomczak, et al., Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma, N. Engl. J. Med. 356 (22) (2007) 2271e2281.
[140] V.E. Kwitkowski, T.M. Prowell, A. Ibrahim, et al., FDA approval summary: temsirolimus as treatment for advanced renal cell carcinoma, Oncol. 15 (4) (2010) 428e435.
[141] G. Hess, R. Herbrecht, J. Romaguera, et al., Phase III study to evaluate tem- sirolimus compared with investigator’s choice therapy for the treatment of relapsed or refractory mantle cell lymphoma, J. Clin. Oncol. 27 (23) (2009) 3822e3829.
[142] G. Emons, C. Kurzeder, B. Schmalfeldt, et al., Temsirolimus in women with platinum-refractory/resistant ovarian cancer or advanced/recurrent endo- metrial carcinoma. A phase II study of the AGO-study group (AGO-GYN8), Gynecol. Oncol. 140 (3) (2016) 450e456.
[143] R. Mody, A. Naranjo, C. Van Ryn, et al., Irinotecan-temozolomide with tem- sirolimus or dinutuximab in children with refractory or relapsed neuro- blastoma (COG ANBL1221): an open-label, randomised, phase 2 trial, Lancet Oncol. 18 (7) (2017) 946e957.
[144] O.J. Becher, S.W. Gilheeney, Y. Khakoo, et al., A phase I study of perifosine with temsirolimus for recurrent pediatric solid tumors, Pediatr. Blood Canc. 64 (7) (2017), https://doi.org/10.1002/pbc.26409.
[145] R.J. Motzer, B. Escudier, S. Oudard, et al., Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebo-controlled phase III trial, Lancet 372 (9637) (2008) 449e456.
[146] S. Buti, A. Leonetti, A. Dallatomasina, M. Bersanelli, Everolimus in the man- agement of metastatic renal cell carcinoma: an evidence-based review of its place in therapy, Core Evid. 11 (2016) 23e36.
[147] D.A. Krueger, M.M. Care, K. Holland, et al., Everolimus for subependymal giant-cell astrocytomas in tuberous sclerosis, N. Engl. J. Med. 363 (19) (2010) 1801e1811.
[148] D.N. Franz, E. Belousova, S. Sparagana, et al., Long-term use of everolimus in patients with tuberous sclerosis complex: final results from the EXIST-1 study, PLoS One 11 (6) (2016), e0158476.
[149] J.J. Bissler, J.C. Kingswood, E. Radzikowska, et al., Everolimus for angiomyo- lipoma associated with tuberous sclerosis complex or sporadic lym- phangioleiomyomatosis (EXIST-2): a multicentre, randomised, double-blind, placebo-controlled trial, Lancet 381 (9869) (2013) 817e824.
[150] J.C. Yao, N. Fazio, S. Singh, et al., Everolimus for the treatment of advanced, non-functional neuroendocrine tumours of the lung or gastrointestinal tract (RADIANT-4): a randomised, placebo-controlled, phase 3 study, Lancet 387 (10022) (2016) 968e977.
[151] J.C. Soria, F.A. Shepherd, J.Y. Douillard, et al., Efficacy of everolimus (RAD001) in patients with advanced NSCLC previously treated with chemotherapy alone or with chemotherapy and EGFR inhibitors, Ann. Oncol. 20 (10) (2009) 1674e1681.
[152] J.C. Yao, M.H. Shah, T. Ito, et al., Everolimus for advanced pancreatic neuro- endocrine tumors, N. Engl. J. Med. 364 (6) (2011) 514e523.
[153] J.C. Yao, M. Pavel, C. Lombard-Bohas, et al., Everolimus for the treatment of advanced pancreatic neuroendocrine tumors: overall survival and circu- lating biomarkers from the randomized, phase III RADIANT-3 study, J. Clin. Oncol. 34 (32) (2016) 3906e3913.
[154] T.C. Schneider, D. de Wit, T.P. Links, et al., Everolimus in patients with advanced follicular-derived thyroid cancer: results of a phase II clinical trial,
J. Clin. Endocrinol. Metab. 102 (2) (2017) 698e707.
[155] A. Ohtsu, J.A. Ajani, Y.X. Bai, et al., Everolimus for previously treated advanced gastric cancer: results of the randomized, double-blind, phase III GRANITE-1 study, J. Clin. Oncol. 31 (31) (2013) 3935e3943.
[156] A.X. Zhu, M. Kudo, E. Assenat, et al., Effect of everolimus on survival in advanced hepatocellular carcinoma after failure of sorafenib: the EVOLVE-1 randomized clinical trial, J. Am. Med. Assoc. 312 (1) (2014) 57e67.
[157] M. Fenner, C. Oing, A. Dieing, et al., Everolimus in patients with multiply relapsed or cisplatin refractory germ cell tumors: results of a phase II, single- arm, open-label multicenter trial (RADIT) of the German Testicular Cancer Study Group, J. Canc. Res. Clin. Oncol. 145 (3) (2019) 717e723.
[158] J.L. Geiger, J.E. Bauman, M.K. Gibson, et al., Phase II trial of everolimus in patients with previously treated recurrent or metastatic head and neck squamous cell carcinoma, Head Neck 38 (12) (2016) 1759e1764.
[159] S.P. Chawla, A.P. Staddon, L.H. Baker, et al., Phase II study of the mammalian target of rapamycin inhibitor ridaforolimus in patients with advanced bone and soft tissue sarcomas, J. Clin. Oncol. 30 (1) (2012) 78e84.
[160] G.D. Demetri, S.P. Chawla, I. Ray-Coquard, et al., Results of an international randomized phase III trial of the mammalian target of rapamycin inhibitor ridaforolimus versus placebo to control metastatic sarcomas in patients after benefit from prior chemotherapy, J. Clin. Oncol. 31 (19) (2013) 2485e2492.
[161] N. Colombo, D.S. McMeekin, P.E. Schwartz, et al., Ridaforolimus as a single agent in advanced endometrial cancer: results of a single-arm, phase 2 trial, Br. J. Canc. 108 (5) (2013) 1021e1026.
[162] A.M. Oza, S. Pignata, A. Poveda, et al., Randomized phase II trial of ridafor- olimus in advanced endometrial carcinoma, J. Clin. Oncol. 33 (31) (2015) 3576e3582.
[163] D. Tsoref, S. Welch, S. Lau, et al., Phase II study of oral ridaforolimus in women with recurrent or metastatic endometrial cancer, Gynecol. Oncol. 135 (2) (2014) 184e189.
[164] A.D. Pearson, S.M. Federico, I. Aerts, et al., A phase 1 study of oral ridafor- olimus in pediatric patients with advanced solid tumors, Oncotarget 7 (51) (2016) 84736e84747.
[165] M. Nakamura, Y. Otsuka, Y. Ueda, K. Mitsudo, Favorable pharmacokinetics of biolimus A9 after deployment of Nobori stent for coronary artery disease: insights from Nobori PK study in Japanese subjects, Cardiovasc Interv Ther 27 (1) (2012) 24e30.
[166] W. Steudel, C. Dingmann, Y.L. Zhang, et al., Randomized, double-blind, pla- cebo-controlled, single intravenous dose-escalation study to evaluate the safety, tolerability, and pharmacokinetics of the novel coronary smooth muscle cell proliferation inhibitor Biolimus A9 in healthy individuals, J. Clin. Pharmacol. 51 (1) (2011) 29e39.
[167] S.E. Burke, R.E. Kuntz, L.B. Schwartz, Zotarolimus (ABT-578) eluting stents, Adv. Drug Deliv. Rev. 58 (3) (2006) 437e446.
[168] J.I. Luengo, D.S. Yamashita, D. Dunnington, et al., Structure-activity studies of rapamycin analogs: evidence that the C-7 methoxy group is part of the effector domain and positioned at the FKBP12-FRAP interface, Chem. Biol. 2
(7) (1995) 471e481.
[169] F.C. Nelson, S.J. Stachel, C.P. Eng, S.N. Sehgal, Manipulation of the C(22)-C(27) region of rapamycin: stability issues and biological implications, Bioorg. Med. Chem. Lett 9 (2) (1999) 295e300.
[170] R. Sedrani, L.H. Jones, A.M. Jutzi-Eme, W. Schuler, S. Cottens, Cleavage of the cyclohexyl-subunit of rapamycin results in loss of immunosuppressive ac- tivity, Bioorg. Med. Chem. Lett 9 (3) (1999) 459e462.
[171] B. Ruan, K. Pong, F. Jow, et al., Binding of rapamycin analogs to calcium channels and FKBP52 contributes to their neuroprotective activities, Proc. Natl. Acad. Sci. U. S. A. 105 (1) (2008) 33e38.
[172] K.P. Wilson, M.M. Yamashita, M.D. Sintchak, et al., Comparative X-ray structures of the major binding protein for the immunosuppressant FK506 (tacrolimus) in unliganded form and in complex with FK506 and rapamycin, Acta Crystallogr D Biol Crystallogr 51 (Pt 4) (1995) 511-521.
[173] M. Hayakawa, H. Kaizawa, H. Moritomo, et al., Synthesis and biological evaluation of pyrido[3’,2’:4,5]furo[3,2-d]pyrimidine derivatives as novel PI3 kinase p110alpha inhibitors, Bioorg. Med. Chem. Lett 17 (9) (2007) 2438e2442.
[174] Q.W. Fan, C.K. Cheng, T.P. Nicolaides, et al., A dual phosphoinositide-3-kinase alpha/mTOR inhibitor cooperates with blockade of epidermal growth factor receptor in PTEN-mutant glioma, Canc. Res. 67 (17) (2007) 7960e7965.
[175] A.M. Venkatesan, C.M. Dehnhardt, E. Delos Santos, et al., Bis(morpholino- 1,3,5-triazine) derivatives: potent adenosine 50 -triphosphate competitive
phosphatidylinositol-3-kinase/mammalian target of rapamycin inhibitors: discovery of Compound 26(PKI-587), a highly efficacious dual inhibitor,
J. Med. Chem. 53 (6) (2010) 2636e2645.
[176] H. Freitag, F. Christen, F. Lewens, et al., Inhibition of mTOR’s catalytic site by PKI-587 is a promising therapeutic option for gastroenteropancreatic neuroendocrine tumor disease, Neuroendocrinology 105 (1) (2017) 90e104.
[177] J.M. Del Campo, M. Birrer, C. Davis, et al., A randomized phase II non- comparative study of PF-04691502 and gedatolisib (PF-05212384) in pa- tients with recurrent endometrial cancer, Gynecol. Oncol. 142 (1) (2016) 62e69.
[178] A.M. Venkatesan, Z. Chen, O. dos Santos, et al., PKI-179: an orally efficacious dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor, Bioorg. Med. Chem. Lett 20 (19) (2010) 5869e5873.
[179] Z. Chen, A.M. Venkatesan, O. Dos Santos, et al., Stereoselective synthesis of an active metabolite of the potent PI3 kinase inhibitor PKI-179, J. Org. Chem. 75
(5) (2010) 1643e1651.
[180] K.A. Menear, S. Gomez, K. Malagu, et al., Identification and optimisation of novel and selective small molecular weight kinase inhibitors of mTOR, Bio- org. Med. Chem. Lett 19 (20) (2009) 5898e5901.
[181] F. Beaufils, N. Cmiljanovic, V. Cmiljanovic, et al., 5-(4,6-Dimorpholino-1,3,5- triazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (PQR309), a potent, brain- penetrant, orally bioavailable, pan-class I PI3K/mTOR inhibitor as clinical candidate in oncology, J. Med. Chem. 60 (17) (2017) 7524e7538.
[182] C. Borsari, D. Rageot, F. Beaufils, et al., Preclinical development of PQR514, a highly potent PI3K inhibitor bearing a difluoromethyl-pyrimidine moiety, ACS Med. Chem. Lett. 10 (10) (2019) 1473e1479.
[183] C.M. Dehnhardt, A.M. Venkatesan, Z. Chen, et al., Identification of 2- oxatriazines as highly potent pan-PI3K/mTOR dual inhibitors, Bioorg. Med. Chem. Lett 21 (16) (2011) 4773e4778.
[184] J.M. García-Martínez, J. Moran, R.G. Clarke, et al., Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR), Biochem. J. 421 (1) (2009) 29e42.
[185] C.M. Chresta, B.R. Davies, I. Hickson, et al., AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity, Canc. Res. 70 (1) (2010) 288e298.
[186] M. Hu, H. Huang, R. Zhao, et al., AZD8055 induces cell death associated with autophagy and activation of AMPK in hepatocellular carcinoma, Oncol. Rep. 31 (2) (2014) 649e656.
[187] L. Zhao, B. Teng, L. Wen, et al., mTOR inhibitor AZD8055 inhibits proliferation and induces apoptosis in laryngeal carcinoma, Int. J. Clin. Exp. Med. 7 (2) (2014) 337e347.
[188] A. Naing, C. Aghajanian, E. Raymond, et al., Safety, tolerability, pharmaco- kinetics and pharmacodynamics of AZD8055 in advanced solid tumours and lymphoma, Br. J. Canc. 107 (7) (2012) 1093e1099.
[189] H. Asahina, H. Nokihara, N. Yamamoto, et al., Safety and tolerability of AZD8055 in Japanese patients with advanced solid tumors; a dose-finding phase I study, Invest. N. Drugs 31 (3) (2013) 677e684.
[190] B. Basu, E. Dean, M. Puglisi, et al., First-in-Human pharmacokinetic and pharmacodynamic study of the dual m-TORC 1/2 inhibitor AZD2014, Clin. Canc. Res. 21 (15) (2015) 3412e3419.
[191] T. Powles, M. Wheater, O. Din, et al., A randomised phase 2 study of AZD2014 versus everolimus in patients with VEGF-refractory metastatic clear cell renal cancer, Eur. Urol. 69 (3) (2016) 450e456.
[192] S.T. Kim, S.Y. Kim, S.J. Klempner, et al., Rapamycin-insensitive companion of mTOR (RICTOR) amplification defines a subset of advanced gastric cancer and is sensitive to AZD2014-mediated mTORC1/2 inhibition, Ann. Oncol. 28
(3) (2017) 547e554.
[193] N. Sakre, G. Wildey, M. Behtaj, et al., RICTOR amplification identifies a sub- group in small cell lung cancer and predicts response to drugs targeting mTOR, Oncotarget 8 (4) (2017) 5992e6002.
[194] K. Yu, L. Toral-Barza, C. Shi, et al., Biochemical, cellular, and in vivo activity of novel ATP-competitive and selective inhibitors of the mammalian target of rapamycin, Canc. Res. 69 (15) (2009) 6232e6240.
[195] K. Yu, C. Shi, L. Toral-Barza, et al., Beyond rapalog therapy: preclinical pharmacology and antitumor activity of WYE-125132, an ATP-competitive and specific inhibitor of mTORC1 and mTORC2, Canc. Res. 70 (2) (2010) 621e631.
[196] D.J. Richard, J.C. Verheijen, K. Curran, et al., Incorporation of water- solubilizing groups in pyrazolopyrimidine mTOR inhibitors: discovery of highly potent and selective analogs with improved human microsomal stability, Bioorg. Med. Chem. Lett 19 (24) (2009) 6830e6835.
[197] B. Apsel, J.A. Blair, B. Gonzalez, et al., Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases, Nat. Chem. Biol. 4 (11) (2008) 691e699.
[198] M.E. Feldman, B. Apsel, A. Uotila, et al., Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2, PLoS Biol. 7 (2) (2009) e38.
[199] H.A. Burris 3rd, C.D. Kurkjian, L. Hart, et al., TAK-228 (formerly MLN0128), an investigational dual TORC1/2 inhibitor plus paclitaxel, with/without trastu- zumab, in patients with advanced solid malignancies, Canc. Chemother. Pharmacol. 80 (2) (2017) 261e273.
[200] A.J. Folkes, K. Ahmadi, W.K. Alderton, et al., The identification of 2-(1H- indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4- yl-thieno[3,2-d]pyrimidine (GDC-0941) as a potent, selective, orally bioavailable inhibitor of class I PI3 kinase for the treatment of cancer, J. Med. Chem. 51 (18) (2008) 5522e5532.
[201] D.P. Sutherlin, L. Bao, M. Berry, et al., Discovery of a potent, selective, and orally available class I phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) kinase inhibitor (GDC-0980) for the treatment of cancer, J. Med. Chem. 54 (21) (2011) 7579e7587.
[202] J.J. Wallin, K.A. Edgar, J. Guan, et al., GDC-0980 is a novel class I PI3K/mTOR kinase inhibitor with robust activity in cancer models driven by the PI3K pathway, Mol. Canc. Therapeut. 10 (12) (2011) 2426e2436.
[203] S.O. Dolly, A.J. Wagner, J.C. Bendell, et al., Phase I study of apitolisib (GDC- 0980), dual phosphatidylinositol-3-kinase and mammalian target of rapa- mycin kinase inhibitor, in patients with advanced solid tumors, Clin. Canc. Res. 22 (12) (2016) 2874e2884.
[204] T. Powles, M.R. Lackner, S. Oudard, et al., Randomized open-label phase II trial of apitolisib (GDC-0980), a novel inhibitor of the PI3K/mammalian target of rapamycin pathway, versus everolimus in patients with metastatic renal cell carcinoma, J. Clin. Oncol. 34 (14) (2016) 1660e1668.
[205] J.C. Verheijen, K. Yu, L. Toral-Barza, I. Hollander, A. Zask, Discovery of 2- arylthieno[3,2-d]pyrimidines containing 8-oxa-3-azabi-cyclo[3.2.1]octane in the 4-position as potent inhibitors of mTOR with selectivity over PI3K, Bioorg. Med. Chem. Lett 20 (1) (2010) 375e379.
[206] D.P. Sutherlin, D. Sampath, M. Berry, et al., Discovery of (thienopyrimidin-2- yl)aminopyrimidines as potent, selective, and orally available pan-PI3-kinase and dual pan-PI3-kinase/mTOR inhibitors for the treatment of cancer, J. Med. Chem. 53 (3) (2010) 1086e1097.
[207] T.P. Heffron, M. Berry, G. Castanedo, et al., Identification of GNE-477, a potent and efficacious dual PI3K/mTOR inhibitor, Bioorg. Med. Chem. Lett 20 (8) (2010) 2408e2411.
[208] S.D. Knight, N.D. Adams, J.L. Burgess, et al., Discovery of GSK2126458, a highly potent inhibitor of PI3K and the mammalian target of rapamycin, ACS Med. Chem. Lett. 1 (1) (2010) 39e43.
[209] T. Liu, Q. Sun, Q. Li, et al., Dual PI3K/mTOR inhibitors, GSK2126458 and PKI- 587, suppress tumor progression and increase radiosensitivity in nasopha- ryngeal carcinoma, Mol. Canc. Therapeut. 14 (2) (2015) 429e439.
[210] P.T. Lukey, S.A. Harrison, S. Yang, et al., A randomised, placebo-controlled study of omipalisib (PI3K/mTOR) in idiopathic pulmonary fibrosis, Eur. Respir. J. 53 (3) (2019) 1801992.
[211] M.Y. Kim, A.J. Kruger, J.Y. Jeong, et al., Combination therapy with a PI3K/ mTOR dual inhibitor and chloroquine enhances synergistic apoptotic cell death in epstein-barr virus-infected gastric cancer cells, Mol. Cell. 42 (6) (2019) 448e459.
[212] H.J. Choi, J.H. Heo, J.Y. Park, et al., A novel PI3K/mTOR dual inhibitor, CMG002, overcomes the chemoresistance in ovarian cancer, Gynecol. Oncol. 153 (1) (2019) 135e148.
[213] M.N. Kim, S.M. Lee, J.S. Kim, S.G. Hwang, Preclinical efficacy of a novel dual PI3K/mTOR inhibitor, CMG002, alone and in combination with sorafenib in hepatocellular carcinoma, Canc. Chemother. Pharmacol. 84 (4) (2019) 809e817.
[214] R. Marone, V. Cmiljanovic, B. Giese, M.P. Wymann, Targeting phosphoino- sitide 3-kinase: moving towards therapy, Biochim. Biophys. Acta 1784 (1) (2008) 159e185.
[215] G.L. Gravina, A. Mancini, L. Scarsella, et al., Dual PI3K/mTOR inhibitor, XL765 (SAR245409), shows superior effects to sole PI3K [XL147 (SAR245408)] or mTOR [rapamycin] inhibition in prostate cancer cell models, Tumour Biol 37
(1) (2016) 341e351.
[216] H. Zhao, G. Chen, H. Liang, Dual PI3K/mTOR Inhibitor, XL765, suppresses glioblastoma growth by inducing ER stress-dependent apoptosis, Onco- Targets Ther. 12 (2019) 5415e5424.
[217] S.M. Maira, F. Stauffer, J. Brueggen, et al., Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/ mammalian target of rapamycin inhibitor with potent in vivo antitumor activity, Mol. Canc. Therapeut. 7 (7) (2008) 1851e1863.
[218] V. Serra, B. Markman, M. Scaltriti, et al., NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations, Canc. Res. 68 (19) (2008) 8022e8030.
[219] S.M. Brachmann, I. Hofmann, C. Schnell, et al., Specific apoptosis induction by the dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 amplified and PIK3CA mutant breast cancer cells, Proc. Natl. Acad. Sci. U. S. A. 106 (52) (2009) 22299e22304.
[220] M.I. Carlo, A.M. Molina, Y. Lakhman, et al., A phase ib study of BEZ235, a dual inhibitor of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR), in patients with advanced renal cell carcinoma, Oncol. 21 (7) (2016) 787e788.
[221] R. Salazar, R. Garcia-Carbonero, S.K. Libutti, et al., Phase II study of BEZ235 versus everolimus in patients with mammalian target of rapamycin inhibi- tor-Naïve advanced pancreatic neuroendocrine tumors, Oncol. 23 (7) (2018), 766ee90.
[222] E. Seront, S. Rottey, B. Filleul, et al., Phase II study of dual phosphoinositol-3- kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor BEZ235 in patients with locally advanced or metastatic transitional cell carcinoma, BJU Int. 118 (3) (2016) 408e415.
[223] C. Simioni, A. Cani, A.M. Martelli, et al., The novel dual PI3K/mTOR inhibitor NVP-BGT226 displays cytotoxic activity in both normoxic and hypoxic hepatocarcinoma cells, Oncotarget 6 (19) (2015) 17147e17160.
[224] K.Y. Chang, S.Y. Tsai, C.M. Wu, C.J. Yen, B.F. Chuang, J.Y. Chang, Novel phos- phoinositide 3-kinase/mTOR dual inhibitor, NVP-BGT226, displays potent growth-inhibitory activity against human head and neck cancer cells in vitro and in vivo, Clin. Canc. Res. 17 (22) (2011) 7116e7126.
[225] W. Glienke, L. Maute, J. Wicht, L. Bergmann, The dual PI3K/mTOR inhibitor NVP-BGT226 induces cell cycle arrest and regulates Survivin gene expression in human pancreatic cancer cell lines, Tumour Biol 33 (3) (2012) 757e765.
[226] Y. Katanasaka, Y. Kodera, M. Yunokawa, Y. Kitamura, T. Tamura, F. Koizumi, Synergistic anti-tumor effects of a novel phosphatidyl inositol-3 kinase/ mammalian target of rapamycin dual inhibitor BGT226 and gefitinib in non- small cell lung cancer cell lines, Canc. Lett. 347 (2) (2014) 196e203.
[227] E. Fokas, M. Yoshimura, R. Prevo, et al., NVP-BEZ235 and NVP-BGT226, dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitors, enhance tumor and endothelial cell radiosensitivity, Radiat. Oncol. 7 (2012) 48.
[228] M.C. Smith, M.M. Mader, J.A. Cook, et al., Characterization of LY3023414, a novel PI3K/mTOR dual inhibitor eliciting transient target modulation to impede tumor growth, Mol. Canc. Therapeut. 15 (10) (2016) 2344e2356.
[229] L. Zheng, H. Li, Y. Mo, G. Qi, B. Liu, J. Zhao, Autophagy inhibition sensitizes LY3023414-induced anti-glioma cell activity in vitro and in vivo, Oncotarget 8 (58) (2017) 98964e98973.
[230] Y. Zou, M. Ge, X. Wang, Targeting PI3K-AKT-mTOR by LY3023414 inhibits human skin squamous cell carcinoma cell growth in vitro and in vivo, Bio- chem. Biophys. Res. Commun. 490 (2) (2017) 385e392.
[231] A.H. Zaidi, J.E. Kosovec, D. Matsui, et al., PI3K/mTOR dual inhibitor, LY3023414, demonstrates potent antitumor efficacy against esophageal adenocarcinoma in a rat model, Ann. Surg. 266 (1) (2017) 91e98.
[232] J.C. Bendell, A.M. Varghese, D.M. Hyman, et al., A first-in-human phase 1 study of LY3023414, an oral PI3K/mTOR dual inhibitor, in patients with advanced cancer, Clin. Canc. Res. 24 (14) (2018) 3253e3262.
[233] M.M. Rubinstein, D.M. Hyman, I. Caird, et al., Phase 2 study of LY3023414 in patients with advanced endometrial cancer harboring activating mutations in the PI3K pathway, Cancer 126 (6) (2020) 1274e1282.
[234] Q. Liu, J.W. Chang, J. Wang, et al., Discovery of 1-(4-(4-propionylpiperazin-1- yl)-3-(trifluoromethyl)phenyl)-9-(quinolin-3-yl)benzo[h][1,6]naphthyridin- 2(1H)-one as a highly potent, selective mammalian target of rapamycin (mTOR) inhibitor for the treatment of cancer, J. Med. Chem. 53 (19) (2010) 7146e7155.
[235] S. Choi, K. Kim, M. Cha, M. Kim, B.H. Lee, mTOR signaling intervention by Torin 1 and XL388 in the insular cortex alleviates neuropathic pain, Neurosci. Lett. 718 (2020) 134742.
[236] Q. Liu, J. Wang, S.A. Kang, et al., Discovery of 9-(6-aminopyridin-3-yl)-1-(3- (trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one (Torin2) as a potent, selective, and orally available mammalian target of rapamycin (mTOR) inhibitor for treatment of cancer, J. Med. Chem. 54 (5) (2011) 1473e1480.
[237] Q. Liu, C. Xu, S. Kirubakaran, et al., Characterization of Torin2, an ATP- competitive inhibitor of mTOR, ATM, and ATR, Canc. Res. 73 (8) (2013) 2574e2586.
[238] M. Ahmed, A.R. Hussain, P. Bavi, et al., High prevalence of mTOR complex activity can be targeted using Torin2 in papillary thyroid carcinoma, Carci- nogenesis 35 (7) (2014) 1564e1572.
[239] C. Simioni, A. Cani, A.M. Martelli, et al., Activity of the novel mTOR inhibitor Torin-2 in B-precursor acute lymphoblastic leukemia and its therapeutic potential to prevent Akt reactivation, Oncotarget 5 (20) (2014) 10034e10047.
[240] S.M. Sadowski, M. Boufraqech, L. Zhang, et al., Torin2 targets dysregulated pathways in anaplastic thyroid cancer and inhibits tumor growth and metastasis, Oncotarget 6 (20) (2015) 18038e18049.
[241] A.R. Hussain, M. Al-Romaizan, M. Ahmed, et al., Dual targeting of mTOR activity with Torin2 potentiates anticancer effects of cisplatin in epithelial ovarian cancer, Mol. Med. 21 (1) (2015) 466e478.
[242] C. Wang, X. Wang, Z. Su, H. Fei, X. Liu, Q. Pan, The novel mTOR inhibitor Torin-2 induces autophagy and downregulates the expression of UHRF1 to suppress hepatocarcinoma cell growth, Oncol. Rep. 34 (4) (2015) 1708e1716.
[243] T. Watanabe, A. Sato, N. Kobayashi-Watanabe, N. Sueoka-Aragane, S. Kimura,
E. Sueoka, Torin2 potentiates anticancer effects on adult T-cell leukemia/ lymphoma by inhibiting mammalian target of rapamycin, Anticancer Res. 36
(1) (2016) 95e102.
[244] A. Awasthi, P. Kumar, C.V. Srikanth, S. Sahi, R. Puria, Invitro evaluation of Torin2 and 2, 6-dihydroxyacetophenone in colorectal cancer therapy, Pathol. Oncol. Res. 25 (1) (2019) 301e309.
[245] S.S. Chopra, A. Jenney, A. Palmer, et al., Torin2 exploits replication and checkpoint vulnerabilities to cause death of PI3K-activated triple-negative breast cancer cells, Cell Syst 10 (1) (2020) 66e81, e11.
[246] C. Wang, R. Zhang, J. Tan, et al., Effect of mesoporous silica nanoparticles co- loading with 17-AAG and Torin2 on anaplastic thyroid carcinoma by tar- geting VEGFR2, Oncol. Rep. 43 (5) (2020) 1491e1502.
[247] D. Udayakumar, R.K. Pandita, N. Horikoshi, et al., Torin2 suppresses ionizing radiation-induced DNA damage repair, Radiat. Res. 185 (5) (2016) 527e538.
[248] K.K. Hanson, A.S. Ressurreiç~ao, K. Buchholz, et al., Torins are potent anti-
malarials that block replenishment of Plasmodium liver stage para- sitophorous vacuole membrane proteins, Proc. Natl. Acad. Sci. U. S. A. 110
(30) (2013) E2838eE2847.
[249] A. Shaik, R. Bhakuni, S. Kirubakaran, Design, synthesis, and docking studies of new Torin2 analogs as potential ATR/mTOR kinase inhibitors, Molecules 23
(5) (2018) 992.
[250] C.M. Dehnhardt, A.M. Venkatesan, E. Delos Santos, et al., Lead optimization of N-3-substituted 7-morpholinotriazolopyrimidines as dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitors: discovery of PKI-402,
J. Med. Chem. 53 (2) (2010) 798e810.
[251] J. Yuan, P.P. Mehta, M.J. Yin, et al., PF-04691502, a potent and selective oral inhibitor of PI3K and mTOR kinases with antitumor activity, Mol. Canc. Therapeut. 10 (11) (2011) 2189e2199.
[252] S. Hart, V. Novotny-Diermayr, K.C. Goh, et al., VS-5584, a novel and highly selective PI3K/mTOR kinase inhibitor for the treatment of cancer, Mol. Canc. Therapeut. 12 (2) (2013) 151e161.
[253] T.P. Heffron, C.O. Ndubaku, L. Salphati, et al., Discovery of clinical develop- ment candidate GDC-0084, a brain penetrant inhibitor of PI3K and mTOR, ACS Med. Chem. Lett. 7 (4) (2016) 351e356.
[254] S.V. Bhagwat, P.C. Gokhale, A.P. Crew, et al., Preclinical characterization of OSI-027, a potent and selective inhibitor of mTORC1 and mTORC2: distinct from rapamycin, Mol. Canc. Therapeut. 10 (8) (2011) 1394e1406.
[255] X. Zhi, W. Chen, F. Xue, et al., OSI-027 inhibits pancreatic ductal adenocarcinoma cell proliferation and enhances the therapeutic effect of gemcitabine both in vitro and in vivo, Oncotarget 6 (28) (2015) 26230e26241.
[256] J. Mateo, D. Olmos, H. Dumez, et al., A first in man, dose-finding study of the mTORC1/mTORC2 inhibitor OSI-027 in patients with advanced solid malig- nancies, Br. J. Canc. 114 (8) (2016) 889e896.
[257] M. Rehan, An anti-cancer drug candidate OSI-027 and its analog as inhibitors of mTOR: computational insights into the inhibitory mechanisms, J. Cell. Biochem. 118 (12) (2017) 4558e4567.
[258] D.S. Mortensen, S.M. Perrin-Ninkovic, G. Shevlin, et al., Discovery of mammalian target of rapamycin (mTOR) kinase inhibitor CC-223, J. Med. Chem. 58 (13) (2015) 5323e5333.
[259] D.S. Mortensen, K.E. Fultz, S. Xu, et al., CC-223, a potent and selective in- hibitor of mTOR kinase: in vitro and in vivo characterization, Mol. Canc. Therapeut. 14 (6) (2015) 1295e1305.
[260] Z. Tong, R. Narayanan, C. Atsriku, et al., Assessment of drug-drug interaction potential and PBPK modeling of CC-223, a potent inhibitor of the mammalian target of rapamycin kinase, Xenobiotica 49 (1) (2019) 54e70.
[261] Z. Xie, J. Wang, M. Liu, D. Chen, C. Qiu, K. Sun, CC-223 blocks mTORC1/C2 activation and inhibits human hepatocellular carcinoma cells in vitro and in vivo, PLoS One 12 (3) (2017), e0173252.
[262] J.Y. Wang, X. Jin, X. Zhang, X.F. Li, CC-223 inhibits human head and neck squamous cell carcinoma cell growth, Biochem. Biophys. Res. Commun. 496
(4) (2018) 1191e1196.
[263] E. Wolin, A. Mita, A. Mahipal, et al., A phase 2 study of an oral mTORC1/ mTORC2 kinase inhibitor (CC-223) for non-pancreatic neuroendocrine tu- mors with or without carcinoid symptoms, PLoS One 14 (9) (2019), e0221994.
[264] N. Zhang, S. Ayral-Kaloustian, J.T. Anderson, et al., 5-ureidobenzofuranone indoles as potent and efficacious inhibitors of PI3 kinase-alpha and mTOR for the treatment of breast cancer, Bioorg. Med. Chem. Lett 20 (12) (2010) 3526e3529.
[265] C.S. Takeuchi, B.G. Kim, C.M. Blazey, et al., Discovery of a novel class of highly potent, selective, ATP-competitive, and orally bioavailable inhibitors of the mammalian target of rapamycin (mTOR), J. Med. Chem. 56 (6) (2013) 2218e2234.
[266] Y.R. Zhu, X.Z. Zhou, L.Q. Zhu, et al., The anti-cancer activity of the mTORC1/2 dual inhibitor XL388 in preclinical osteosarcoma models, Oncotarget 7 (31) (2016) 49527e49538.
[267] Z. Xiong, Y. Zang, S. Zhong, et al., The preclinical assessment of XL388, a mTOR kinase inhibitor, as a promising anti-renal cell carcinoma agent, Oncotarget 8 (18) (2017) 30151e30161.
[268] K. Kim, S. Choi, M. Cha, B.H. Lee, Effects of mTOR inhibitors on neuropathic pain revealed by optical imaging of the insular cortex in rats, Brain Res. 1733 (2020) 146720.
[269] P.W. Brian, P.J. Curtis, H.G. Hemming, G.L.F. Norris, Wortmannin, an anti- biotic produced by Penicillium wortmanni, Trans. Br. Mycol. Soc. 40 (3) (1957) 365e368.
[270] J. MacMillan, A.E. Vanstone, S.K. Yeboah, The structure of wortmannin, a steroidal fungal metabolite, Chem. Commun. (11) (1968) 613e614.
[271] T.J. Petcher, H.P. Weber, Z. Kis, Crystal structure and absolute configuration of Wortmannin and of Wortmannin p-bromobenzoate, J. Chem. Soc. Chem. Commun. 19 (1972) 1061e1062.
[272] G.J. Brunn, J. Williams, C. Sabers, G. Wiederrecht, J.C. Lawrence Jr.,
R.T. Abraham, Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortman- nin and LY294002, EMBO J. 15 (19) (1996) 5256e5267.
[273] A.N. Carter, H.A. Born, A.T. Levine, et al., Wortmannin attenuates seizure- induced hyperactive PI3K/Akt/mTOR signaling, impaired memory, and spine dysmorphology in rats, eNeuro 4 (3) (2017). ENEURO.0354-16.2017.
[274] H. Yang, D.G. Rudge, J.D. Koos, B. Vaidialingam, H.J. Yang, N.P. Pavletich, mTOR kinase structure, mechanism and regulation, Nature 497 (7448) (2013) 217e223.
[275] T.J. Sundstrom, A.C. Anderson, D.L. Wright, Inhibitors of phosphoinositide-3- kinase: a structure-based approach to understanding potency and selec- tivity, Org. Biomol. Chem. 7 (5) (2009) 840-850.
[276] E.H. Walker, M.E. Pacold, O. Perisic, et al., Structural determinants of phos- phoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine, Mol. Cell. 6 (4) (2000) 909-919.
[277] Q. Xue, B. Hopkins, C. Perruzzi, D. Udayakumar, D. Sherris, L.E. Benjamin, Palomid 529, a novel small-molecule drug, is a TORC1/TORC2 inhibitor that reduces tumor growth, tumor angiogenesis, and vascular permeability, Canc. Res. 68 (22) (2008) 9551e9557.
[278] R. Diaz, P.A. Nguewa, J.A. Diaz-Gonzalez, et al., The novel Akt inhibitor Pal- omid 529 (P529) enhances the effect of radiotherapy in prostate cancer, Br. J. Canc. 100 (6) (2009) 932e940.
[279] G.L. Gravina, F. Marampon, F. Petini, et al., The TORC1/TORC2 inhibitor, Palomid 529, reduces tumor growth and sensitizes to docetaxel and cisplatin in aggressive and hormone-refractory prostate cancer cells, Endocr. Relat. Canc. 18 (4) (2011) 385e400.
[280] G.L. Gravina, F. Marampon, D. Sherris, et al., Torc1/Torc2 inhibitor, Palomid 529, enhances radiation response modulating CRM1-mediated survivin function and delaying DNA repair in prostate cancer models, Prostate 74 (8) (2014) 852e868.
[281] M. Dalal, N. Jacobs-El, B. Nicholson, et al., Subconjunctival Palomid 529 in the treatment of neovascular age-related macular degeneration, Graefes Arch. Clin. Exp. Ophthalmol. 251 (12) (2013) 2705e2709.
[282] F. Lin, L. Buil, D. Sherris, J.H. Beijnen, O. van Tellingen, Dual mTORC1 and mTORC2 inhibitor Palomid 529 penetrates the blood-brain barrier without restriction by ABCB1 and ABCG2, Int. J. Canc. 133 (5) (2013) 1222e1233.
[283] A. Benavides-Serrato, J. Lee, B. Holmes, et al., Specific blockade of Rictor- mTOR association inhibits mTORC2 activity and is cytotoxic in glioblas- toma, PLoS One 12 (4) (2017), e0176599.
[284] A. Benavides-Serrato, J. Lee, B. Holmes, et al., Correction: specific blockade of Rictor-mTOR association inhibits mTORC2 activity and is cytotoxic in glio- blastoma, PLoS One 14 (2) (2019), e0212160.
[285] V.S. Rodrik-Outmezguine, M. Okaniwa, Z. Yao, et al., Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor, Nature 534 (7606) (2016) 272e276.
[286] Q. Fan, O. Aksoy, R.A. Wong, et al., A kinase inhibitor targeted to mTORC1 drives regression in glioblastoma, Canc. Cell 31 (3) (2017) 424e435.
[287] F. Mu, Y. Jiang, F. Ao, H. Wu, Q. You, Z. Chen, RapaLink-1 plays an antith- rombotic role in antiphospholipid syndrome by improving autophagy both in vivo and vitro, Biochem. Biophys. Res. Commun. 525 (2) (2020) 384e391.
[288] K. Kuroshima, H. Yoshino, S. Okamura, et al., Potential new therapy of Rapalink-1, a new generation mTOR inhibitor, against sunitinib-resistant renal cell carcinoma, Canc. Sci. (2020), https://doi.org/10.1111/cas.14395.
[289] A. Goel, A.B. Kunnumakkara, B.B. Aggarwal, Curcumin as “Curecumin”: from kitchen to clinic, Biochem. Pharmacol. 75 (4) (2008) 787-809.
[290] M. Li, Z. Zhang, D.L. Hill, H. Wang, R. Zhang, Curcumin, a dietary component, has anticancer, chemosensitization, and radiosensitization effects by down- regulating the MDM2 oncogene through the PI3K/mTOR/ETS2 pathway, Canc. Res. 67 (5) (2007) 1988-1996.
[291] S.M. Johnson, P. Gulhati, I. Arrieta, et al., Curcumin inhibits proliferation of colorectal carcinoma by modulating Akt/mTOR signaling, Anticancer Res. 29
(8) (2009) 3185-3190.
[292] G.S. Van Aller, J.D. Carson, W. Tang, et al., Epigallocatechin gallate (EGCG), a major component of green tea, is a dual phosphoinositide-3-kinase/mTOR inhibitor, Biochem. Biophys. Res. Commun. 406 (2) (2011) 194-199.
[293] C.H. Huang, S.J. Tsai, Y.J. Wang, M.H. Pan, J.Y. Kao, T.D. Way, EGCG inhibits protein synthesis, lipogenesis, and cell cycle progression through activation of AMPK in p53 positive and negative human hepatoma cells, Mol. Nutr. Food Res. 53 (9) (2009) 1156-1165.
[294] S.Y. Park, Y. Lee, Y. Kim, et al., Control of AMP-activated protein kinase, Akt, and mTOR in EGCG-treated HT-29 colon cancer cells, Food Sci Biotechnol 22 (2013) 147e151.
[295] S. Liu, Z.L. Xu, L. Sun, et al., (-)-Epigallocatechin-3-gallate induces apoptosis in human pancreatic cancer cells via PTEN, Mol. Med. Rep. 14 (1) (2016) 599- 605.
[296] C.F. Nicoletti, H.B.P. Delfino, M. Pinhel, et al., Impact of green tea epi- gallocatechin-3-gallate on HIF1-a and mTORC2 expression in obese women: anti-cancer and anti-obesity effects? Nutr. Hosp. 36 (2) (2019) 315-320.
[297] J. Meng, Y. Chen, J. Wang, et al., EGCG protects vascular endothelial cells from oxidative stress-induced damage by targeting the autophagy-dependent PI3K-AKT-mTOR pathway, Ann. Transl. Med. 8 (5) (2020) 200.
[298] B. Tian, J. Liu, Resveratrol: a review of plant sources, synthesis, stability, modification and food application, J. Sci. Food Agric. 100 (4) (2020) 1392-
1404.
[299] M. Liu, S.A. Wilk, A. Wang, et al., Resveratrol inhibits mTOR signaling by promoting the interaction between mTOR and DEPTOR, J. Biol. Chem. 285
(47) (2010) 36387e36394.
[300] Y. Zhang, A. Gilmour, Y.H. Ahn, L. de la Vega, A.T. Dinkova-Kostova, The isothiocyanate sulforaphane inhibits mTOR in an NRF2-independent manner, Phytomedicine (2019) 153062.
[301] A. Wiczk, D. Hofman, G. Konopa, A. Herman-Antosiewicz, Sulforaphane, a cruciferous vegetable-derived isothiocyanate, inhibits protein synthesis in human prostate cancer cells, Biochim. Biophys. Acta 1823 (8) (2012) 1295-
1305.
[302] N.M. Shawky, L. Segar, Sulforaphane inhibits platelet-derived growth factor- induced vascular smooth muscle cell proliferation by targeting mTOR/ p70S6kinase signaling independent of Nrf 2 activation, Pharmacol. Res. 119 (2017) 251-264.
[303] E. Juengel, S. Maxeiner, J. Rutz, et al., Sulforaphane inhibits proliferation and invasive activity of everolimus-resistant kidney cancer cells in vitro, Onco- target 7 (51) (2016) 85208e85219.
[304] K. Suppipat, C.S. Park, Y. Shen, X. Zhu, H.D. Lacorazza, Sulforaphane induces cell cycle arrest and apoptosis in acute lymphoblastic leukemia cells, PLoS One 7 (12) (2012), e51251.
[305] A. Pawlik, A. Wiczk, A. Kaczyn´ska, J. Antosiewicz, A. Herman-Antosiewicz,
Sulforaphane inhibits growth of phenotypically different breast cancer cells, Eur. J. Nutr. 52 (8) (2013) 1949-1958.
[306] W. Chen, Y. Luo, L. Liu, et al., Cryptotanshinone inhibits cancer cell prolif- eration by suppressing Mammalian target of rapamycin-mediated cyclin D1 expression and Rb phosphorylation, Canc. Prev. Res. 3 (8) (2010) 1015-1025.
[307] K. Sahin, M. Tuzcu, N. Basak, et al., Sensitization of cervical cancer cells to cisplatin by genistein: the role of NFkB and akt/mTOR signaling pathways, J Oncol 2012 (2012) 461562.
[308] K.Y. Lee, J.R. Kim, H.C. Choi, Genistein-induced LKB1-AMPK activation in- hibits senescence of VSMC through autophagy induction, Vasc. Pharmacol. 81 (2016) 75-82.
[309] K.M. Malloy, J. Wang, L.H. Clark, et al., Novasoy and genistein inhibit endo- metrial cancer cell proliferation through disruption of the AKT/mTOR and MAPK signaling pathways, Am J Transl Res 10 (3) (2018) 784-795.
[310] S. Miwa, N. Sugimoto, N. Yamamoto, et al., Caffeine induces apoptosis of osteosarcoma cells by inhibiting AKT/mTOR/S6K, NF-kB and MAPK path- ways, Anticancer Res. 32 (9) (2012) 3643-3649.
[311] H. Liu, J. Song, Y. Zhou, et al., Methylxanthine derivatives promote autophagy in gastric cancer cells targeting PTEN, Anti Canc. Drugs 30 (4) (2019) 347-
355.
[312] D. Tewari, P. Patni, A. Bishayee, A.N. Sah, A. Bishayee, Natural products tar- geting the PI3K-Akt-mTOR signaling pathway in cancer: a novel therapeutic strategy, Semin. Canc. Biol. S1044e579X (19) (2019), 30405-5.