The mammalian target of rapamycin (mTOR) is centrally involved in cell growth, metabolism, and angiogenesis. While showing clinical efficacy in a subset of tumors, rapamycin and rapalogs are specific and allosteric inhibitors of mTOR complex 1 (mTORC1), but they do not directly inhibit mTOR complex 2 (mTORC2), an emerging player in cancer. Here, we report chemical structure and biological characterization of three pyrazolopyrimidine ATP-competitive mTOR inhibitors, WAY-600, WYE-687, and WYE-354 (IC 50 , 5-9 nmol/L), with significant selectivity over phosphatidylinositol 3-kinase (PI3K) isofoms (>100-fold). Unlike the rapalogs, these inhibitors acutely blocked substrate phosphorylation by mTORC1 and mTORC2 in vitro and in cells in response to growth factor, amino acids, and hyperactive PI3K/AKT. Unlike the inhibitors of PI3K or dual-pan PI3K/mTOR, cellular inhibition of P-S6K1(T389) and P-AKT(S473) by the pyrazolopyrimidines occurred at significantly lower inhibitor concentrations than those of P-AKT(T308) (PI3K-PDK1 readout), showing mTOR selectivity in cellular setting. mTOR kinase inhibitors reduced AKT downstream function and inhibited proliferation of diverse cancer cell lines. These effects correlated with a strong G 1 cell cycle arrest in both the rapamycin-sensitive and rapamycin-resistant cells, selective induction of apoptosis, repression of global protein synthesis, and down-regulation of angiogenic factors. When injected into tumor-bearing mice, WYE-354 inhibited mTORC1 and mTORC2 and displayed robust antitumor activity in PTENnull tumors. Together, our results highlight mechanistic differentiation between rapalogs and mTOR kinase inhibitors in targeting cancer cell growth and survival and provide support for clinical development of mTOR kinase inhibitors as new cancer therapy. [Cancer Res 2009;69(15):6232-40]

BACE1 is an aspartyl protease responsible for cleaving amyloid precursor protein to liberate Abeta, which aggregates leading to plaque deposits implicated in Alzheimer's disease. We have identified small-molecule acylguanidine inhibitors of BACE1. Crystallographic studies show that these compounds form unique hydrogen-bonding interactions with the catalytic site aspartic acids and stabilize the protein in a flap-open conformation. Structure-based optimization led to the identification of potent analogs, such as 10d (BACE1 IC(50) = 110 nM).

SAR on HTS hits 1 and 2 led to the potent, Notch-1-sparing GSI 9, which lowered brain Abeta in Tg2576 mice at 100 mg/kg po. Converting the metabolically labile methyl groups in 9 to trifluoromethyl groups afforded the more stable analogue 10, which had improved in vivo potency. Further side chain modification afforded the potent Notch-1-sparing GSI begacestat (5), which was selected for development for the treatment of Alzheimer's disease.

A novel series of HCV NS5B RNA-dependent RNA polymerase inhibitors containing a pyrano[3,4-b]indole scaffold is described leading to the discovery of compound 16, a highly potent and selective inhibitor that is active in the replicon system.

Heat shock protein 90 (Hsp90) is a molecular chaperone that is responsible for activating many signaling proteins and is a promising target in tumor biology. We have identified small-molecule benzisoxazole derivatives as Hsp90 inhibitors. Crystallographic studies show that these compounds bind in the ATP binding pocket interacting with the Asp93. Structure based optimization led to the identification of potent analogues, such as 13, with good biochemical profiles.

Hepatitis C virus (HCV) infection is the cause of significant long-term morbidity and mortality. While often asymptomatic, the majority of HCV infections result in chronic hepatitis that can progress to cirrhosis, end-stage liver disease, and hepatocellular carcinoma. It is estimated that about 170 million people worldwide, approximately 3% of the world's population, are infected with HCV (45). Presently, there is no specific antiviral agent directed against HCV and no vaccine for prevention of hepatitis C infection. The current approved treatments, interferon monotherapy or interferon in combination with ribavirin, have limited benefits. Interferon alone achieves a sustained viral response in only 10 to 20% of patients (32), while the recommended therapy of pegylated interferon in combination with ribavirin results in a sustained viral response in 54% of patients (23). The response rate is lower in patients who are infected with HCV genotype 1b (ϳ34%) (23,47). Adverse side effects, such as severe flu-like symptoms, depression, psychoses, and anemia, are associated with these treatments, causing approximately 20% of patients to discontinue therapy (13,17,33). Consequently, there is an urgent need for the development of an HCV-specific antiviral that is more effective, less toxic, and easier to administer than the present therapy.HCV, a member of the Flaviviridae family, is a positivesense, single-stranded RNA virus with a genome size of ϳ9.4 kb (26, 39). The genome RNA encodes a polyprotein of 3,010 to 3,011 amino acid residues in the order NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. This polyprotein is processed by host and viral proteases (18,34). The nonstructural protein 5B (NS5B) is a virus-encoded RNA-dependent RNA polymerase (RdRp) that is responsible for replication of the viral RNA genome. A functional counterpart of NS5B does not exist in mammalian cells. For this reason, an inhibitor of NS5B could serve as an effective and selective agent for treating HCV infection.The enzymatic activity of the NS5B enzyme in vitro has been extensively characterized (9,19,21,40). Both the full-length and carboxyl-terminally truncated forms of NS5B have been shown to be functionally active in the presence of HCV or exogenous RNA templates. Although HCV replicates inefficiently in cell culture, viral replication, including the activity of the NS5B polymerase, can be studied in a cell culture system

The mammalian target of rapamycin (mTOR) is a central regulator of cell growth, metabolism, and angiogenesis and an emerging target in cancer research. High throughput screening (HTS) of our compound collection led to the identification of 3-(4-morpholin-4-yl-1-piperidin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenol (5a), a modestly potent and nonselective inhibitor of mTOR and phosphoinositide 3-kinase (PI3K). Optimization of compound 5a, employing an mTOR homology model based on an X-ray crystal structure of closely related PI3Kgamma led to the discovery of 6-(1H-indol-5-yl)-4-morpholin-4-yl-1-[1-(pyridin-3-ylmethyl)piperidin-4-yl]-1H-pyrazolo[3,4-d]pyrimidine (5u), a potent and selective mTOR inhibitor (mTOR IC(50) = 9 nM; PI3Kalpha IC(50) = 1962 nM). Compound 5u selectively inhibited cellular biomarker of mTORC1 (P-S6K, P-4EBP1) and mTORC2 (P-AKT S473) over the biomarker of PI3K/PDK1 (P-AKT T308) and did not inhibit PI3K-related kinases (PIKKs) in cellular assays. These pyrazolopyrimidines represent an exciting new series of mTOR-selective inhibitors with potential for development for cancer therapy.

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