Potent mTOR Inhibitor Research Articles

Design and development of Tetrahydro-Quinoline derivatives as dual mTOR-C1/C2 inhibitors for the treatment of lung cancer

Lung cancer is one of the most prevailed cancer worldwide. Many genes get mutated in lung cancer but the involvement of EGFR, KRAS, PTEN and PIK3CA are more common. Unavailability of potent drugs and resistance to the available drugs are major concern in the treatment of lung cancer. In the present research, mTOR was selected as an important alternative target for the treatment of lung cancer which involves the PI3K/AKT/mTOR pathway. We studied binding interactions of AZD-2014 with the mTOR protein to identify important interactions required to design potent mTOR inhibitors which was supported by QSAR studies. Pharmacophore based virtual screening studies provided core scaffold, THQ. Based on molecular docking interactions, 31 THQ derivatives were synthesized and characterized. All compounds were screened for cellular mTOR enzyme assay along with antiproliferative activity against the panel of cancerous cell lines, from which 6 compounds were further screened for colony forming assay. Two most potent compounds, HB-UC-1 and HB-UC-5, were further screened for flow cytometry analysis, gene expression study and western blot analysis. Gene expression study revealed the efficiency of compound HB-UC-1 against both mTORC1 and mTORC2 by affecting downstream regulators of mTORC1 (E4BP4, eIF4EBP1) and mTORC2 (PCK1), respectively. In western blot analysis, both compounds, inhibited phosphorylation of AKT S473 which proved the efficiency these compounds against the mTORC2. These two compounds were further screened for in-vivo biological evaluation. Both compounds increased lifespan of cancer-bearing animals with improvement in mean survival time. Further, in bezopyrene induced lung cancer animal model, both compounds showed effectiveness through the biochemical parameters and histopathological evaluation of the lung tissue. In future, potent hit compound from this series could be modified to develop lead mTOR inhibitors for the treatment of lung cancer.

GDC-0349 inhibits non-small cell lung cancer cell growth

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related human mortality with a clear need for new therapeutic intervention. GDC-0349 is a potent and selective ATP-competitive mTOR inhibitor. In A549 cells and primary human NSCLC cells, GDC-0349 inhibited cell growth, proliferation, cell cycle progression, migration and invasion, while inducing significant apoptosis activation. Although GDC-0349 blocked Akt-mTORC1/2 activation in NSCLC cells, it also exerted cytotoxicity in Akt1-knockout A549 cells. Furthermore, restoring Akt-mTOR activation by a constitutively-active Akt1 only partially attenuated GDC-0349-induced A549 cell apoptosis, indicating the existence of Akt-mTOR-independent mechanisms. In NSCLC cells GDC-0349 induced sphingosine kinase 1 (SphK1) inhibition, ceramide accumulation, JNK activation and oxidative injury. Conversely, N-acetylcysteine, the JNK inhibitor and sphingosine 1-phosphate alleviated GDC-0349-induced NSCLC cell apoptosis. In vivo, daily oral administration of GDC-0349 potently inhibited NSCLC xenograft growth in mice. Akt-mTOR in-activation, SphK1 inhibition, JNK activation and oxidative stress were detected in NSCLC xenograft tissues with GDC-0349 administration. In summary, GDC-0349 inhibits NSCLC cell growth via Akt-mTOR-dependent and Akt-mTOR-independent mechanisms.

Abstract 665: Discovery and preclinical characterization of PQR626: A potent, orally available, and brain-penetrant mTOR inhibitor for the treatment of tuberous sclerosis complex

Abstract Mechanistic target of rapamycin (mTOR), a key player in cell proliferation, growth and survival, is overactivated in tumors and neurological disorders.[1] Rapalogs have recently been explored to alleviate epileptic seizures in Tuberous Sclerosis Complex (TSC).[2] Herein, we combined pharmacophore features of PQR620[3], the first-in-class brain penetrant ATP-competitive mTOR kinase inhibitor showing efficacy in a TSC mouse model, and PQR617[4], a potent, highly selective mTOR inhibitor. An extensive chemical exploration of the morpholine ring led to the discovery of PQR626, a highly potent, selective, brain penetrant inhibitor of mTORC1/2 kinase. In male Sprague Dawley rats and female C57BL/6J mice, PQR626 displayed an excellent brain penetration compared to everolimus, which possessed a limited ability to cross the blood-brain-barrier (brain/plasma levels ~ 1.4:1 vs 1:92). An additional pharmacokinetic comparative study with everolimus and AZD2014 confirmed the superiority of PQR626 [brain/plasma levels: ~ 1:25 (AZD2014), ~ 1:61 (everolimus), ~ 1.8:1 (PQR626)]. PQR626 showed very good tolerability in mice (MTD 100-150 mg/kg). Efficacy studies, using mice with conditional inactivation of the Tsc1 gene primarily in glia (Tsc1GFAPCKO mice), were performed in a dose-range finding study. PQR626 (50 mg/kg, BID - twice a day) showed a significant effect on survival and significantly prevented/decreased mortality as compared to the vehicle. On the basis of its favorable pharmacological parameters, excellent brain penetration, safety profile and efficacy in Tsc1GFAPCKO mice, PQR626 qualifies as a novel mTOR inhibitor with potential application in the treatment of epilepsy and neurological disorders. [1] Wymann M. P. and Schneiter R. Nat. Rev. Mol. Cell Biol. 2008, 9, 162-176. [2] Krueger D. A. et. al. Neurology 2016, 87, 2408-2415. [3] Rageot D. et. al. J Med Chem. 2018, 61 (22), 10084-10105. [4] Borsari C. et. al. J Med Chem. 2019, 62 (18), 8609-8630. Citation Format: Chiara Borsari, Erhan Keles, Denise Rageot, Anna Melone, Thomas Bohnacker, Lucinda Kate Batchelor, Martina De Pascale, Paul Hebeisen, Petra Hillmann, Doriano Fabbro, Matthias Wymann. Discovery and preclinical characterization of PQR626: A potent, orally available, and brain-penetrant mTOR inhibitor for the treatment of tuberous sclerosis complex [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 665.

Upregulation of miR-101 during Influenza A Virus Infection Abrogates Viral Life Cycle by Targeting mTOR Pathway.

Micro RNAs (miRNAs) are a class of small non-coding single-stranded RNA, which play an important role in modulating host-Influenza A virus (IAV) crosstalk. The interplay between influenza and miRNA interaction is defined by a plethora of complex mechanisms, which are not fully understood yet. Here, we demonstrate that in IAV infected A549 cells, a synchronous increase was observed in the expression of mTOR up to 24 hpi and significant downregulation at 48 hpi. Additionally, NP of IAV interacts with mTOR and modulates the levels of mTOR mRNA and protein, thus regulating the translation of host cell. RNA sequencing and qPCR analysis of IAV-infected A549 cells and NP transfected cells revealed that miR-101 downregulates mTOR transcripts at later stages of infection. Ectopic expression of miR-101 mimic led to a decrease in expression of NP, a reduction in IAV titer and replication. Moreover, treatment of the cells with Everolimus, a potent inhibitor of mTOR, resulted in an increase of miR-101 transcript levels, which further suppressed the viral protein synthesis. Collectively, the data suggest a novel mechanism that IAV stimulates mTOR pathway at early stages of infection; however, at a later time-point, positive regulation of miR-101 restrains the mTOR expression, and hence, the viral propagation.

Pharmacophore-guided fragment-based design of novel mammalian target of rapamycin inhibitors: extra precision docking, fingerprint-based 2D and atom-based 3D-QSAR modelling

Rapamycin and their derivatives known as rapalogs were the first-generation mTOR inhibitors which interacted with mTORC1 but not with the mTORC2 protein. Second-generation inhibitors could bind with both and showed excellent anti-proliferative activity. Our aim was to design novel mTOR inhibitors which could bind at both the allosteric and the kinase site. The FRB domain is present in both the mTORC1 and mTORC2 protein complexes. We have employed e-pharmacophore-guided fragment-based design to develop novel mTOR inhibitors. The affinity of designed molecules at both the sites was analysed using molecular docking in extra precision mode. The atom-based 3D-QSAR model was developed to predict the activity while the fingerprint-based 2D-QSAR model was employed to refine an identified hit as potent dual mTOR inhibitor. Ligand ASK23 showed a docking score of −15.452 kcal/mol at the allosteric site (PDB ID 5GPG) while ASK38 showed a docking score of −11.535 kcal/mol at the kinase site (PDB ID 4JT6). Ligand ASK12 showed binding energy of −106.23 kcal/mol at the allosteric site. Refined molecule ASK12a from ASK12 showed the highest predicted activity (pIC50: 6.512). The stability of the best hits and receptor complex was analysed using molecular dynamics simulation studies. Herein we report five potential mTOR dual inhibitors based on the predicted activity, drug-likeness analysis and off-target effects. To the best of our knowledge, this is the first report on pharmacophore-guided fragment-based drug design for mTOR inhibitors. This design strategy can be used for the rational drug design against other proteins for which only apo-structures are available. Communicated by Ramaswamy H. Sarma

Identification of a Novel Oxadiazole Inhibitor of Mammalian Target of Rapamycin

We performed a biochemical screen against mTOR using in‐house small molecule library. Two novel, structurally distinct hits were identified. Among them, a novel oxadiazole scaffold compound (2) suppressed the phosphorylation of both S6K1 and Akt1 in HeLa cells. Docking study suggested that 2 is ATP‐competitive and shows a pi‐pi interaction with Trp2239 and hydrogen bonds with Trp2239 and Thr2245. Through derivatization, a slightly more potent analogue (2a) was identified with IC50 of 9.6 μM. Our study provides a starting point for discovery of novel potent mTOR inhibitors.

Abstract 3919: GSK458, a novel oral dual PI3K/mTOR Inhibitor, is effective in preclinical model of T-cell lymphoma alone and in combination therapies

Abstract Purpose: Despite T cell lymphoma (TCL) responses well at the initial chemotherapy, the outcome of TCL remains poor when it becomes systematic disease with refractory or relapse (r/r) after several cycle of treatment. Constitutively activated PI3K-mTOR pathway was found variety of cancers including TCL, and dual inhibition of PI3k-mTOR has been shown preclinically and clinically successful in B-cell lymphoma. However, the activity of dual inhibition of PI3k-mTOR in T-cell malignancies remain utterly unknown. GSK458 is a potent oral dual inhibitor of pan PI3K (α, β, γ and δ) and mTOR (mTOR1 and mTOR2). Preclinical study showed GSK458 had broad antitumor activity in vitro and in vivo. In 2016, phase I clinical trial of GSK458 was competed and the dosage was durable in multiple tumor types. Here we extensively characterized the activity and the mechanism of action of GSK458 in preclinical model of T-cell lymphoma alone and in combination therapies. Methods: This study included a panel of T cell lymphoma lines as well as primary samples derived from lymphoma patient, including J45 and SupT-1 are PTCL as T-cell lymphoblastic lymphoma; whereas MJ, HH and H9 are CTCL represented Mycosis fungoides (MF), aggressive leukemic MF and the most aggressive form of CTCL Sezary syndrome (SS) respectively. In vitro viability assay was performed as single agent of GSK458 and in combination with other drugs. Differences in cellular metabolic difference were examined by ATP levels, low mitochondria potential and glucose update utilizing Cell-Titer Glo assays, DiOC6 and 2-NDG flow analysis, respectively. Cellular apoptosis and cell cycle distribution were evaluated by Annexin V and propidium iodide staining followed by flow cytometry. PI3K and mTOR downstream pathway phosphorylation status were detected by flow cytometry. Apoptosis proteins were detected by western blot. The regulative T cells were evaluated by flow cytometry. Results: GSK458 had potent anti-tumor activity as single agent and potentiated the activity of chemotherapy or small inhibitors, such as doxorubicin, carboplatin, venetoclax, brentuximab, romdespsin and proteasome inhibitors in TCL pre-clinical models. GSK458 sufficiently blocked the downstream targets of PI3K-mTOR, such as phosphorylation status of Akt serine473 and threonine308; phosphorylation of mTOR and GSK3β. At metabolic level, GSK458 reduced ATP production, decrease glucose uptake, lowered mitochondrial membrane potential and triggered apoptosis in TCL. Moreover, GSK458 arrested the cell cycle at G1. In our study, we found after exposure to GSK458, the percentage of regulatory T cell among the total CD4+ T cells were decreased. Conclusion: Based on these preclinical results, GSK458 appeared as a novel and promising drug that is possible to develop clinical trial in T-cell lymphoma. (Supported by Roswell Park Cancer Institute Alliance Foundation Grant) Citation Format: Juan J. Gu, Lianjuan yang, Jing He, Weina Shen, Cory Mavis, Francisco Hernandez-Ilizaliturri. GSK458, a novel oral dual PI3K/mTOR Inhibitor, is effective in preclinical model of T-cell lymphoma alone and in combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3919.

Temsirolimus as a dual inhibitor of retinoblastoma and angiogenesis via targeting mTOR signalling

Targeting the mammalian target of rapamycin (mTOR) is a promising strategy for cancer therapy. Temsirolimus, a FDA-approved anticancer drug with efficacy in certain solid tumors and hematologic malignancies, is a potent mTOR inhibitor. In this work, we are the first to provide preclinical evidence that temsirolimus is an attractive candidate for retinoblastoma treatment as a dual inhibitor of retinoblastoma and angiogenesis. We show that temsirolimus selectively inhibits growth, survival and migration of retinoblastoma cells while sparing normal retinal and fibroblast cells, with IC50 value that is within the clinically achievable range. Temsirolimus potently inhibits retinal angiogenesis via targeting biological functions of retinal endothelial cells. Our mechanism analysis demonstrates that temsirolimus inhibits retinoblastoma and angiogenesis via suppressing mTOR signalling and secretion of proangiogenic cytokines. In line with in vitro data, we further demonstrate the inhibitory effects of temsirolimus on retinoblastoma and angiogenesis in in vivo xenograft mouse model. Our findings provide a preclinical rationale to explore temsirolimus as a strategy to treat retinoblastoma and highlight the therapeutic value of targeting mTOR in retinoblastoma.

MTOR is a Mechanistic Target of Muscle and Cancer Cross‐Talk with Exercise

INTRODUCTIONThe mTOR signaling pathway regulates growth and cellular proliferation, which is vital for normal cell function. However, when this pathway is dysregulated, such as in cancer, it can lead to hyperactivation of signals which promote cell proliferation and protect against cell death. DEPTOR, a potent endogenous mTOR inhibitor, generally has low expression in many cancers. We have unique preliminary findings that myokines released during electrically‐stimulated muscle contraction of hemicorpus‐hindlimb perfused rats affect mTOR signaling and DEPTOR protein content in breast cancer cells. When MCF‐7 cancer cells were treated with perfusate collected during muscle contraction, a significant inhibition of proliferation was noted alongside diminished mTOR activity and elevated DEPTOR expression.PURPOSETo investigate changes in anabolic signaling and resultant protein synthesis rates in MCF7 human breast cancer cells exposed to pharmacologically administered mTOR inhibitors.METHODSMCF7 cells were cultured in 10cm plates until 60% confluence in standard Growth Media (GM). Cells were treated with either Rapamycin or Torin1 inhibitors (100nM or 250nM, respectively) in 10 ml of GM. Cells for protein synthesis analysis were additionally treated with heavy water (2H2O) to yield a 4% final volume. All groups were incubated in respective media for 24hrs and prepped for western blot or rates of protein synthesis analysis.RESULTSDEPTOR protein expression increased in both rapamycin and Torin1 treated cells (2‐fold & 7‐fold, respectively). Both Rapamycin and Torin 1 treated cells show decreased daily rates of protein synthesis compared to cell control (14.41, 9.09 vs 20.46 %/d, respectively).CONCLUSIONOur results suggest that diminished DEPTOR protein content and concomitant mTOR hyperactivity are important contributors to MCF7 cellular proliferation, in culture. Pharmacological administration of mTOR inhibitors suppresses protein synthesis and consequent anabolic signaling, with a rescue of DEPTOR protein content, which, in part, is consistent with our previous findings using exercise perfusate.Support or Funding InformationThis work was funded in part by the College of Health and Education and the Huffines Institute for Sports Medicine at Texas A&M University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

GSK458 Is a Novel Dual PI3K/mTOR Inhibitor with Preclinical Antitumor Activity in T Cell Lymphomas As a Single Agent and in Combination Therapy

Introduction: T-cell lymphoma (TCL) accounts approximately 15% of all Non-Hodgkin's lymphoma cases. TCL are often divided into either systemic or cutaneous TCL (CTCL). The management of TCL systemic or cutaneous is challenging because the emergence of chemotherapy resistance that lead to early death (systemic T-cell lymphomas) or chronic debilitating clinical course (CTCL). To improve the clinical outcomes and reduce treatment-related toxicity, research need to be done focusing on understanding and targeting the molecular mechanism driving TCL subtypes. Recently, studies showed that the PI3K AKT/mTOR pathway was activated in TCL. GSK458 is a potent oral dual inhibitor of pan PI3K (α, β, γ and δ) and mTOR (mTOR1 and mTOR2). Preclinical studies in B-cell lymphomas showed GSK458 had broad antitumor activity in vitro and in vivo. In 2016, Phase I clinical trial of GSK458 was competed at the maximal dosage of 2.5mg twice daily. However, the effects of GSK458 on T-cell malignancies remain utterly unknown. Here, we evaluated the activity of GSK458 in preclinical T cell lymphoma models. Methods: We used a panel of T-cell lymphoma cell lines representing PTCL (J45), T-cell lymphoblastic lymphoma (SupT-1), and Mycosis Fungoides (MF)(MJ, HH and H9). TCL cell lines were exposed to escalating doses of GSK458 (1nM-100µM) without or with chemotherapeutic agents (doxorubicin, cisplatin, carboplatin, and dexamethasone); Bcl-2 inhibitor (Venetoclax); proteasome inhibitors ( bortezomib, carfilzomib, Ixazomib); or HDAC inhibitors (SAHA) for 48 and 72 hrs. Differences in cell viability, ATP levels, low mitochondria potential, glucose update, apoptosis and cell cycle distribution were evaluated utilizing PrestoBlue, Cell-Titer Glo assays, DiOC6, 2-NDG, Annexin V and propidium iodide staining followed by flow cytometric analysis, respectively. IC50 was calculated by GraphPad. PI3K and mTOR downstream pathway phosphorylation status, such as p-AKT Ser473, p-AKT Thr308, p-mTOR and p-GSK3β were detected by internally staining of FITC conjugated-antibodies followed by flow cytometry. Apoptosis proteins (MCL-1, PARP, p53, XIAP etc.) were detected by western blot. The additive/synergistic activity of GSK458 was detected by presto blue assay and Coefficient of synergy was calculated using CalcuSyn. Results:In vitro exposure of TCL cell lines to GSK458 demonstrated a dose- and time-dependent cell death. The IC50 of the cells were ranged from 3nM to 1.05uM at 72 hours. At 72h, GSK458 10nM lowered cellular mitochondrial potential, ATP levels and glucose uptake. GSK458 induced apoptosis and arrested the cell cycle at G1. At molecular level, GSK458 reduced phosphorylation status of AKT ser473 and Thr308, mTOR and GSK3β. Interestingly, GSK458 inhibited Mcl-1 expression level. GSK458 exhibited synergistic activity when combined with doxorubicin and dexamethasone. To a lesser degree, GSK458 enhanced the anti-tumor activity of Venetoclax, proteasome and HDAc inhibitors. Conclusion: GSK458 is active as a single agent or in combination with chemotherapy agents or small molecule inhibitors in a variety of T-cell pre-clinical models representing forms of systemic or cutaneous T-cell lymphoma. GSK458 was able to inhibit phosphorylation of AKT, mTOR and GSK3β, which may be the mechanism to reduce ATP production and glucose uptake in the cancer cells. Moreover, GSK458 arrested cell cycle at G1 arrest. Our data supports the clinical evaluation of GSK458 in relapsed/refractory T-cell lymphoma patients. (Supported by Roswell Park Cancer Institute Alliance Foundation Grant) DisclosuresNo relevant conflicts of interest to declare.

Lack of muscle mTOR kinase activity causes early onset myopathy and compromises whole-body homeostasis.

BackgroundThe protein kinase mechanistic target of rapamycin (mTOR) controls cellular growth and metabolism. Although balanced mTOR signalling is required for proper muscle homeostasis, partial mTOR inhibition by rapamycin has beneficial effects on various muscle disorders and age‐related pathologies. Besides, more potent mTOR inhibitors targeting mTOR catalytic activity have been developed and are in clinical trials. However, the physiological impact of loss of mTOR catalytic activity in skeletal muscle is currently unknown.MethodsWe have generated the mTORmKOKI mouse model in which conditional loss of mTOR is concomitant with expression of kinase inactive mTOR in skeletal muscle. We performed a comparative phenotypic and biochemical analysis of mTORmKOKI mutant animals with muscle‐specific mTOR knockout (mTORmKO) littermates.ResultsIn striking contrast with mTORmKO littermates, mTORmKOKI mice developed an early onset rapidly progressive myopathy causing juvenile lethality. More than 50% mTORmKOKI mice died before 8 weeks of age, and none survived more than 12 weeks, while mTORmKO mice died around 7 months of age. The growth rate of mTORmKOKI mice declined beyond 1 week of age, and the animals showed profound alterations in body composition at 4 weeks of age. At this age, their body weight was 64% that of mTORmKO mice (P < 0.001) due to significant reduction in lean and fat mass. The mass of isolated muscles from mTORmKOKI mice was remarkably decreased by 38–56% (P < 0.001) as compared with that from mTORmKO mice. Histopathological analysis further revealed exacerbated dystrophic features and metabolic alterations in both slow/oxidative and fast/glycolytic muscles from mTORmKOKI mice. We show that the severity of the mTORmKOKI as compared with the mild mTORmKO phenotype is due to more robust suppression of muscle mTORC1 signalling leading to stronger alterations in protein synthesis, oxidative metabolism, and autophagy. This was accompanied with stronger feedback activation of PKB/Akt and dramatic down‐regulation of glycogen phosphorylase expression (0.16‐fold in tibialis anterior muscle, P < 0.01), thus causing features of glycogen storage disease type V.ConclusionsOur study demonstrates a critical role for muscle mTOR catalytic activity in the regulation of whole‐body growth and homeostasis. We suggest that skeletal muscle targeting with mTOR catalytic inhibitors may have detrimental effects. The mTORmKOKI mutant mouse provides an animal model for the pathophysiological understanding of muscle mTOR activity inhibition as well as for mechanistic investigation of the influence of skeletal muscle perturbations on whole‐body homeostasis.

Continuous low-dose everolimus shrinkage tuberous sclerosis complex-associated renal angiomyolipoma: a 48-month follow-up study

Tuberous sclerosis complex (TSC) is a rare disease that causes multisystem benign neoplasm, induced by dysregulation of the mammalian target of the rapamycin pathway (mTOR). This study aimed to examine...

TCT-471 BIOVITESSE – Multicenter prospective First In Man study of a new polymer free drug-coated stent with ultrathin struts: One month optical coherence tomography results of Cohort 1

A potent anti-proliferative mTOR inhibitor has been developed by Biotronik CRC Inc. which is 1’000-fold more lipophilic than sirolimus. In pre-clinical testing, stents coated with a polymer-free, abluminal-biased coating including this drug achieved effective reduction in neoinitimal hyperplasia

Abstract 5460: Discovery and evaluation of WX047, a potent and oral active phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dual inhibitor

Abstract Background: The PI3K/AKT/mTOR signaling pathway is important in regulating cell cycle, which is directly related to cellular quiescence, proliferation and longevity. Dysfunctional signaling of the PI3K/AKT/mTOR pathway in cancer, and its crucial role in cell growth and survival have made it a much desired target for cancer therapeutics. Methods: By using structure-based design, the 4H-pyrido[1,2-a]pyrimidin-4-one derivative as an early lead was discovered. Further SAR studies optimized the in vitro and PK properties and led to the identification of WX047 as a potent dual inhibitor of PI3K and mTOR. Antitumor efficacy of WX047 was evaluated in the PC-3M xenograft tumor model. Results: A series of 7-(pyridin-3-yl)-4H-pyrido[1,2-a]pyrimidin-4-one derivatives were synthesized and evaluated for in vitro and in vivo activities. Among them, WX047 was identified as a potent dual PI3K and mTOR inhibitor with IC50 for mTOR of 19.17 nM and IC50 for PI3Kα of 3.13 nM. The cellular activity of WX047 was determined in PC-3M cells by western blot analysis of p-Akt and p-70S6K with p-Akt and p-70S6K inhibition IC50 of 16 nM and 65 nM respectively. WX047 had low clearance and high oral bioavailability relative to those of the reference compound GSK2126458. In addition, WX047 also demonstrated significant antitumor efficacy in the PC-3M xenograft tumor models (79% TGI@10mpk, PO, QD). Conclusions: WX047 has been identified as an orally active PI3K and mTOR dual inhibitor. It displays significant antitumor efficacy in the PC-3M xenograft tumor model. These support moving the compound forward to clinical investigation. Citation Format: Ning Li, Amy Guan, Lei Huang, Dongling Hao, Bo Gao, Jikui Sun, Nengyang Shih, Lingwei Kong, Peipei Jiang, Yi Li, Dan Yao, Yuxin Qin, Tao Yu, Chengde Wu, Shuhui Chen, Hongyu Yuan, Xiulian Lu. Discovery and evaluation of WX047, a potent and oral active phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dual inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5460.

Highly Selective, Potent, and Oral mTOR Inhibitor for Treatment of Cancer as Autophagy Inducer.

On the basis of novel pyrazino[2,3-c]quinolin-2(1H)-one scaffold, we designed and identified a highly selective, potent and oral mTOR inhibitor, 9m. Compound 9m showed low nanomolar activity against mTOR (IC50 = 7 nM) and greater selectivity over the related PIKK family kinases, which demonstrated only modest activity against 3 out of the 409 protein kinases. In vitro assays, compound 9m exhibited high potency against human breast and cervical cancer cells and induced tumor cell cycle arrest and autophagy. 9m inhibited cellular phosphorylation of mTORC1 (pS6 and p4E-BP1) and mTORC2 (pAKT (S473)) substrates. In T-47D xenograft mouse model, oral administration of compound 9m led to significant tumor regression without obvious toxicity. In addition, this compound showed good pharmacokinetics. Collectively, due to its high potency and selectivity, compound 9m could be used as a mTOR drug candidate.

Dual EGFR and PI3k/mTOR Targeting of Head and Neck Cancer in Combination With Radiation Therapy

Previous research has identified the PI3K/Akt/mTOR and the EGFR/Ras/Raf/MAPK as key pathways that are dysregulated in head and neck cancer (HNSCC). In this study we describe the use of two novel small molecular agents, Dacomitinib and Gedatolisib, in combination with radiation treatment (RT) to target EGFR and PI3K as novel clinical treatment strategy in a murine xenograft model of human HNSCC. Dacomitinib, an oral once-daily, irreversible pan-HER inhibitor and Gedatolisib, a highly potent dual inhibitor of PI3Kα, PI3Kγ and mTOR administered by intravenous infusion were provided by Pfizer. The drugs were studied alone and in combination with radiation in a panel of low passage HNSCC cell lines in vitro and in vivo using MTT assays, clonogenic assays, western blotting, RT-PCR and tumor growth delay using a 3 week fractionated schedule of daily doses of 2 Gy. There is a close relationship between EGFR overexpression and sensitivity to Dacomitinib with UT-SCC-14 and UT-SCC-16A being more sensitive than UT-SCC-15 and UT-SCC-24A, which do not overexpress EGFR. Gedatolisib showed a classical dose response in both the UT-SCC-14 and UT-SCC-15 cell lines with an IC50 of 0.075μM and 0.2μM, respectively. Gedatolisib was effective at reducing phosphorylation of PI3K, mTOR and AKT in both cell lines. In tumor growth delay experiments with the UT-SCC-14 cell line, Gedatolisib caused a 15 day growth delay to double the tumor volume while Dacomitinib delayed tumor doubling time by 32 days; the combination of the two drugs was similar to Dacomitinib alone. Gedatolisib and radiation delays were similar to radiation alone while combinations involving Dacomitinib and radiation caused profound growth delay and complete cure of 60% of tumors in ongoing studies. Dacomitinib is an extremely potent EGFR targeting agent in the UT-SCC-14 tumor model while Gedatolisib was less effective. Further research is ongoing to establish whether the relationship between EGFR expression levels and Dacomitinib activity is observed in vivo. If this relationship can be established, then a Phase I study using EGFR expression to stratify patients to Dacomitinib treatment would be warranted.

MTOR Controls Mitochondrial Dynamics and Cell Survival via MTFP1

The mechanisms that link environmental and intracellular stimuli to mitochondrial functions, including fission/fusion, ATP production, metabolite biogenesis, and apoptosis, are not well understood. Here, we demonstrate that the nutrient-sensing mechanistic/mammalian target of rapamycin complex 1 (mTORC1) stimulates translation of mitochondrial fission process 1 (MTFP1) to control mitochondrial fission and apoptosis. Expression of MTFP1 is coupled to pro-fission phosphorylation and mitochondrial recruitment of the fission GTPase dynamin-related protein 1 (DRP1). Potent active-site mTOR inhibitors engender mitochondrial hyperfusion due to the diminished translation of MTFP1, which is mediated by translation initiation factor 4E (eIF4E)-binding proteins (4E-BPs). Uncoupling MTFP1 levels from the mTORC1/4E-BP pathway upon mTOR inhibition blocks the hyperfusion response and leads to apoptosis by converting mTOR inhibitor action from cytostatic to cytotoxic. These data provide direct evidence for cell survival upon mTOR inhibition through mitochondrial hyperfusion employing MTFP1 as a critical effector of mTORC1 to govern cell fate decisions.

Abstract 137: FT-1518, a new generation selective and potent mTORC1 and mTORC2 inhibitor: an in vitro and in vivo profile

Abstract Mammalian target of rapamycin (mTOR) is a clinically validated target in the treatment of cancer. mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2 which regulate cell growth, metabolism, proliferation, and survival. Rapamycin analogues target only the mTORC1 complex but do not affect the mTORC2 complex, which is an important driver for cancer cell growth and survival. The new generation of “Selective” mTOR inhibitors, blocking both mTORC1 and mTORC2 signaling might increase the efficacy and safety while expanding the therapeutic potential of these anticancer agents. Herein we describe FT-1518, a low nanomolar potent, kinase and PI3K sub family selective mTOR inhibitor. FT-1518 not only exhibited high oral bioavailability in preclinical species but has demonstrated excellent microsomal stability with no inhibitory activity towards undesired CYPs. FT-1518 showed high sustained tumor exposure and target Inhibition in a single oral dose xenograft model. FT-1518 depicted very good growth inhibitory activity across a large panel of hematologic and solid tumor cell lines with most activities falling into low nanomolar range. mTOR kinase inhibition in cells, by FT-1518, resulted in more potent inhibition of the mTOR pathway biomarkers (mTORC 1 &amp; 2 biomarkers [pAkt(S473) and pS6(S240/244) or p70 S6K), no inhibition of PI3K biomarker [pAkt(T308)], and improved anti-proliferative activity as compared with rapamycin. FT-1518 exhibited dose-dependent and higher tumor growth inhibition (TGI) in multiple solid tumor xenografts compared with rapalogs and is poised to enter the clinic with a favorable toxicology profile. Citation Format: Alain C. Mita, Monica M. Mita, Anthony D. William, Khalid Pasha, Chandra Siddamadappa, Kevin Zikaras, Felix T. Garzon. FT-1518, a new generation selective and potent mTORC1 and mTORC2 inhibitor: an in vitro and in vivo profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 137. doi:10.1158/1538-7445.AM2017-137

Potent Anticancer and mTOR Inhibitor Activities of Some Synthesized Linear and Macrocyclic Pyridine Derivatives

A series of linear and macrocyclic 2,6-disubstituted pyridine derivatives 1–7 were previously prepared and evaluated as analgesic and anticonvulsant agents. Herein these derivatives were screened as anti pancreatic cancer, anti-GIT cancer and mTOR inhibitor activities. The tested compounds were found to have in vivo and in vitro potent anti-pancreatic cancer and anti-GIT cancer activities. The mechanism of action of these compounds was evaluated as mTOR inhibitors.

Structure-activity relationships study of mTOR kinase inhibition using QSAR and structure-based drug design approaches.

The discovery of clinically relevant inhibitors of mammalian target of rapamycin (mTOR) for anticancer therapy has proved to be a challenging task. The quantitative structure–activity relationship (QSAR) approach is a very useful and widespread technique for ligand-based drug design, which can be used to identify novel and potent mTOR inhibitors. In this study, we performed two-dimensional QSAR tests, and molecular docking validation tests of a series of mTOR ATP-competitive inhibitors to elucidate their structural properties associated with their activity. The QSAR tests were performed using partial least square method with a correlation coefficient of r2=0.799 and a cross-validation of q2=0.714. The chemical library screening was done by associating ligand-based to structure-based approach using the three-dimensional structure of mTOR developed by homology modeling. We were able to select 22 compounds from two databases as inhibitors of the mTOR kinase active site. We believe that the method and applications highlighted in this study will help future efforts toward the design of selective ATP-competitive inhibitors.