Dissecting the biology of mTORC1 beyond rapamycin.

Rapamycin extends maximal life span and increases resistance to starvation in many organisms. The beneficial effects of rapamycin are thought to be mediated by its inhibitory effects on the mechanistic target of rapamycin complex 1 (mTORC1), although it only partially inhibits the kinase activity of mTORC1. Other mTOR kinase inhibitors have been developed, such as Torin-1, but these readily cross-react with mTORC2. Here, we report the distinct characteristics of a third-generation mTOR inhibitor called RapaLink1. We found that low doses of RapaLink1 inhibited the phosphorylation of all mTORC1 substrates tested, including those whose phosphorylation is sensitive or resistant to inhibition by rapamycin, without affecting mTORC2 activity even after prolonged treatment. Compared with rapamycin, RapaLink1 showed better efficacy for inhibiting mTORC1 and potently blocked cell proliferation and induced autophagy. Moreover, using RapaLink1, we demonstrated that mTORC1 and mTORC2 exerted differential effects on cell glycolysis and glucose uptake. Last, we found that RapaLink1 and rapamycin had opposing effects on starvation resistance in Drosophila. Consistent with the effects of RapaLink1, genetic blockade of mTORC1 activity made flies more sensitive to starvation, reflecting the complexity of the mTORC1 network that extends beyond effects that can be inhibited by rapamycin. These findings extend our understanding of mTOR biology and provide insights into some of the beneficial effects of rapamycin.

Farnesyltransferase-Mediated Delivery of a Covalent Inhibitor Overcomes Alternative Prenylation to Mislocalize K-Ras.

Mutationally activated Ras is one of the most common oncogenic drivers found across all malignancies, and its selective inhibition has long been a goal in both pharma and academia. One of the oldest and most validated methods to inhibit overactive Ras signaling is by interfering with its post-translational processing and subsequent cellular localization. Previous attempts to target Ras processing led to the development of farnesyltransferase inhibitors, which can inhibit H-Ras localization but not K-Ras due to its ability to bypass farnesyltransterase inhibition through alternative prenylation by geranylgeranyltransferase. Here, we present the creation of a neo-substrate for farnesyltransferase that prevents the alternative prenlation by geranylgeranyltransferase and mislocalizes oncogenic K-Ras in cells.

A Kinase Inhibitor Targeted to mTORC1 Drives Regression in Glioblastoma.

Although signaling from phosphatidylinositol 3-kinase (PI3K) and AKT to mechanistic target of rapamycin (mTOR) is prominently dysregulated in high-grade glial brain tumors, blockade of PI3K or AKT minimally affects downstream mTOR activity in glioma. Allosteric mTOR inhibitors, such as rapamycin, incompletely block mTORC1 compared with mTOR kinase inhibitors (TORKi). Here, we compared RapaLink-1, a TORKi linked to rapamycin, with earlier-generation mTOR inhibitors. Compared with rapamycin and Rapalink-1, TORKi showed poor durability. RapaLink-1 associated with FKBP12, an abundant mTOR-interacting protein, enabling accumulation of RapaLink-1. RapaLink-1 showed better efficacy than rapamycin or TORKi, potently blocking cancer-derived, activating mutants of mTOR. Our study re-establishes mTOR as a central target in glioma and traces the failure of existing drugs to incomplete/nondurable inhibition of mTORC1.

Overcoming resistance to HER2 inhibitors through state-specific kinase binding.

The heterodimeric receptor tyrosine kinase complex formed by HER2 and HER3 can act as an oncogenic driver and is also responsible for rescuing a large number of cancers from a diverse set of targeted therapies. Inhibitors of these proteins, particularly HER2, have dramatically improved patient outcomes in the clinic, but recent studies have demonstrated that stimulating the heterodimeric complex, either via growth factors or by increasing the concentrations of HER2 and HER3 at the membrane, significantly diminishes the activity of the inhibitors. To identify an inhibitor of the active HER2-HER3 oncogenic complex, we developed a panel of Ba/F3 cell lines suitable for ultra-high-throughput screening. Medicinal chemistry on the hit scaffold resulted in a previously uncharacterized inhibitor that acts through preferential inhibition of the active state of HER2 and, as a result, is able to overcome cellular mechanisms of resistance such as growth factors or mutations that stabilize the active form of HER2.

Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor.

Precision medicines exert selective pressure on tumour cells that leads to the preferential growth of resistant subpopulations, necessitating the development of next-generation therapies to treat the evolving cancer. The PIK3CA-AKT-mTOR pathway is one of the most commonly activated pathways in human cancers, which has led to the development of small-molecule inhibitors that target various nodes in the pathway. Among these agents, first-generation mTOR inhibitors (rapalogs) have caused responses in 'N-of-1' cases, and second-generation mTOR kinase inhibitors (TORKi) are currently in clinical trials. Here we sought to delineate the likely resistance mechanisms to existing mTOR inhibitors in human cell lines, as a guide for next-generation therapies. The mechanism of resistance to the TORKi was unusual in that intrinsic kinase activity of mTOR was increased, rather than a direct active-site mutation interfering with drug binding. Indeed, identical drug-resistant mutations have been also identified in drug-naive patients, suggesting that tumours with activating MTOR mutations will be intrinsically resistant to second-generation mTOR inhibitors. We report the development of a new class of mTOR inhibitors that overcomes resistance to existing first- and second-generation inhibitors. The third-generation mTOR inhibitor exploits the unique juxtaposition of two drug-binding pockets to create a bivalent interaction that allows inhibition of these resistant mutants.

Structure of the human autophagy initiating kinase ULK1 in complex with potent inhibitors.

Autophagy is a conserved cellular process that involves the degradation of cellular components for energy maintenance and cytoplasmic quality control that has recently gained interest as a novel target for a variety of human diseases, including cancer. A prime candidate to determine the potential therapeutic benefit of targeting autophagy is the kinase ULK1, whose activation initiates autophagy. Here, we report the first structures of ULK1, in complex with multiple potent inhibitors. These structures show features unique to the enzyme and will provide a path for the rational design of selective compounds as cellular probes and potential therapeutics.

Parallel synthesis and biological evaluation of 837 analogues of procaspase-activating compound 1 (PAC-1).

Procaspase-Activating Compound 1 (PAC-1) is an ortho-hydroxy N-acyl hydrazone that enhances the enzymatic activity of procaspase-3 in vitro and induces apoptosis in cancer cells. An analogue of PAC-1, called S-PAC-1, was evaluated in a veterinary clinical trial in pet dogs with lymphoma and found to have considerable potential as an anticancer agent. With the goal of identifying more potent compounds in this promising class of experimental therapeutics, a combinatorial library based on PAC-1 was created, and the compounds were evaluated for their ability to induce death of cancer cells in culture. For library construction, 31 hydrazides were condensed in parallel with 27 aldehydes to create 837 PAC-1 analogues, with an average purity of 91%. The compounds were evaluated for their ability to induce apoptosis in cancer cells, and through this work, six compounds were discovered to be substantially more potent than PAC-1 and S-PAC-1. These six hits were further evaluated for their ability to relieve zinc-mediated inhibition of procaspase-3 in vitro. In general, the newly identified hit compounds are two- to four-fold more potent than PAC-1 and S-PAC-1 in cell culture, and thus have promise as experimental therapeutics for treatment of the many cancers that have elevated expression levels of procaspase-3.

Pharmacokinetics and derivation of an anticancer dosing regimen for PAC-1, a preferential small molecule activator of procaspase-3, in healthy dogs.

PAC-1 is a preferential small molecule activator of procaspase-3 and has potential to become a novel and effective anticancer agent. The rational development of PAC-1 for translational oncologic applications would be advanced by coupling relevant in vitro cytotoxicity studies with pharmacokinetic investigations conducted in large mammalian models possessing similar metabolism and physiology as people. In the present study, we investigated whether concentrations and exposure durations of PAC-1 that induce cytotoxicity in lymphoma cell lines in vitro can be achievable in healthy dogs through a constant rate infusion (CRI) intravenous delivery strategy. Time- and dose-dependent procaspase-3 activation by PAC-1 with subsequent cytotoxicity was determined in a panel of B-cell lymphoma cells in vitro. The pharmacokinetics of PAC-1 administered orally or intravenously was studied in 6 healthy dogs using a crossover design. The feasibility of maintaining steady state plasma concentration of PAC-1 for 24 or 48 h that paralleled in vitro cytotoxic concentrations was investigated in 4 healthy dogs. In vitro, PAC-1 induced apoptosis in lymphoma cell lines in a time- and dose-dependent manner. The oral bioavailability of PAC-1 was relatively low and highly variable (17.8 ± 9.5%). The achievement and maintenance of predicted PAC-1 cytotoxic concentrations in normal dogs was safely attained via intravenous CRI lasting for 24 or 48 h in duration. Using the dog as a large mammalian model, PAC-1 can be safely administered as an intravenous CRI while achieving predicted in vitro cytotoxic concentrations.

Discovery and canine preclinical assessment of a nontoxic procaspase-3-activating compound.

A critical event in the apoptotic cascade is the proteolytic activation of procaspases to active caspases. The caspase autoactivating compound PAC-1 induces cancer cell apoptosis and exhibits antitumor activity in murine xenograft models when administered orally as a lipid-based formulation or implanted s.c. as a cholesterol pellet. However, high doses of PAC-1 were found to induce neurotoxicity, prompting us to design and assess a novel PAC-1 derivative called S-PAC-1. Similar to PAC-1, S-PAC-1 activated procaspase-3 and induced cancer cell apoptosis. However, S-PAC-1 did not induce neurotoxicity in mice or dogs. Continuous i.v. infusion of S-PAC-1 in dogs led to a steady-state plasma concentration of ∼10 µmol/L for 24 to 72 hours. In a small efficacy trial of S-PAC-1, evaluation of six pet dogs with lymphoma revealed that S-PAC-1 was well tolerated and that the treatments induced partial tumor regression or stable disease in four of six subjects. Our results support this canine setting for further evaluation of small-molecule procaspase-3 activators, including S-PAC-1, a compound that is an excellent candidate for further clinical evaluation as a novel cancer chemotherapeutic.

Procaspase-3 activation as an anti-cancer strategy: structure-activity relationship of procaspase-activating compound 1 (PAC-1) and its cellular co-localization with caspase-3.

A goal of personalized medicine as applied to oncology is to identify compounds that exploit a defined molecular defect in a cancerous cell. A compound called procaspase-activating compound 1 (PAC-1) was reported that enhances the activity of procaspase-3 in vitro and induces apoptotic death in cancer cells in culture and in mouse xenograft models. Experimental evidence indicates that PAC-1 activates procaspase-3 in vitro through chelation of inhibitory zinc ions. Described herein is the synthesis and biological activity of a family of PAC-1 derivatives where key functional groups have been systematically altered. Analysis of these compounds reveals a strong correlation between the in vitro procaspase-3 activating effect and their ability to induce death in cancer cells in culture. Importantly, we also show that a fluorescently labeled version of PAC-1 co-localizes with sites of caspase-3 activity in cancer cells. The data presented herein further bolster the hypothesis that PAC-1 induces apoptosis in cancer cells through the direct activation of procaspase-3, has implications for the design and discovery of next-generation procaspase-3 activating compounds, and sheds light on the anti-apoptotic role of cellular zinc.