Katayoun Jessen

SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo

The MET receptor tyrosine kinase has emerged as an important target for the development of novel cancer therapeutics. Activation of MET by mutation or gene amplification has been linked to kidney, gastric, and lung cancers. In other cancers, such as glioblastoma, autocrine activation of MET has been demonstrated. Several classes of ATP-competitive inhibitor have been described, which inhibit MET but also other kinases. Here, we describe SGX523, a novel, ATP-competitive kinase inhibitor remarkable for its exquisite selectivity for MET. SGX523 potently inhibited MET with an IC50 of 4 nmol/L and is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical assays. Crystallographic study revealed that SGX523 stabilizes MET in a unique inactive conformation that is inaccessible to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation, and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. Our results show that SGX523 is the most selective inhibitor of MET catalytic activity described to date and is thus a useful tool to investigate the role of MET kinase in cancer without the confounding effects of promiscuous protein kinase inhibition. [Mol Cancer Ther 2009;8(12):3181–90]

SGX393 inhibits the CML mutant Bcr-AblT315I and preempts in vitro resistance when combined with nilotinib or dasatinib

Imatinib inhibits Bcr-Abl, the oncogenic tyrosine kinase that causes chronic myeloid leukemia. The second-line inhibitors nilotinib and dasatinib are effective in patients with imatinib resistance resulting from Bcr-Abl kinase domain mutations. Bcr-AblT315I, however, is resistant to all Abl kinase inhibitors in clinical use and is emerging as the most frequent cause of salvage therapy failure. SGX393 is a potent inhibitor of native and T315I-mutant Bcr-Abl kinase that blocks the growth of leukemia cell lines and primary hematopoietic cells expressing Bcr-AblT315I, with minimal toxicity against Bcr-Abl-negative cell lines or normal bone marrow. A screen for Bcr-Abl mutants emerging in the presence of SGX393 revealed concentration-dependent reduction in the number and range of mutations. Combining SGX393 with nilotinib or dasatinib preempted emergence of resistant subclones, including Bcr-AblT315I. These findings suggest that combination of a T315I inhibitor with the current clinically used inhibitors may be useful for reduction of Bcr-Abl mutants in Philadelphia chromosome-positive leukemia.

The discovery and mechanism of action of novel tumor-selective and apoptosis-inducing 3,5-diaryl-1,2,4-oxadiazole series using a chemical genetics approach.

A novel series of 3,5-diaryl-oxadiazoles was identified as apoptosis-inducing agents through our cell and chemical genetics–based screening assay for compounds that induce apoptosis using a chemical genetics approach. Several analogues from this series including MX-74420 and MX-126374 were further characterized. MX-126374, a lead compound from this series, was shown to induce apoptosis and inhibit cell growth selectively in tumor cells. To elucidate the mechanism(s) by which this class of compounds alters the signal transduction pathway that ultimately leads to apoptosis, expression profiling using the Affymetrix Gene Chip array technology was done along with other molecular and biochemical analyses. Interestingly, we have identified several key genes (cyclin D1, transforming growth factor-β1, p21, and insulin-like growth factor-BP3) that are altered in the presence of this compound, leading to characterization of the pathway for activation of apoptosis. MX-126374 also showed significant inhibition of tumor growth as a single agent and in combination with paclitaxel in murine tumor models. Using photoaffinity labeling, tail-interacting protein 47, an insulin-like growth factor-II receptor binding protein, was identified as the molecular target. Further studies indicated that down-regulation of tail-interacting protein 47 in cancer cells by small interfering RNA shows a similar pathway profile as compound treatment. These data suggest that 3,5-diaryl-oxadiazoles may be a new class of anticancer drugs that are tumor-selective and further support the discovery of novel drugs and drug targets using chemical genetic approaches.

Abstract B148: INK128 is a potent and selective TORC1/2 inhibitor with broad oral antitumor activity

mTOR Ser/Thr protein kinase operates in two distinct multi‐protein complexes, TORC1 and TORC2, which together regulate growth, metabolism, angiogenesis and survival by integrating nutrient and hormonal environmental signals. The activity of mTOR is frequently up‐regulated in human cancer by constitutive mitogen stimuli or oncogenic mutations upstream of TORC1 and TORC2. Rapamycin provides mechanistic rationale and clinical proof of concept for the therapeutic value of targeting mTOR in human cancer, but it also provided insights into how ATP‐competitive TORC1/2 inhibitors have the potential to demonstrate superior efficacy. Through rational drug design we have discovered INK128, a potent, selective TORC1/2 inhibitor with excellent drug‐like properties. INK128 inhibits mTOR kinase (sub‐nanomolar) in an ATP‐dependent fashion and demonstrates a high degree of selectivity against closely related kinases as well as against a panel of more than 400 kinases. INK128 inhibits both the phosphorylation of S6 and 4EBP1, the downstream substrates of TORC1, and selectively inhibits AKT phosphorylation at Ser473, the downstream substrate of TORC2, in vitro and in vivo. Interestingly, potent inhibition was also observed in cell lines resistant to rapamycin and pan‐PI3K inhibitors. Daily, oral administration of INK128 (alone or in combination) inhibited angiogenesis and tumor growth in multiple xenograft models with predicted dose:exposure PK/PD relationship. We attribute the superior activity of INK128 to the fact that it is a more effective inhibitor of TORC1 relative to rapamycin. We further demonstrated that TORC1/2 inhibitors cause death of murine and human leukemia cells in models of pre‐B acute lymphoblastic leukemia. In vivo, oral daily treatment with TORC1/2 inhibitors delayed leukemia onset and augmented the effects of ABL kinase inhibitors. Unexpectedly, these novel TORC1/2 inhibitors had much weaker effects than rapamycin on proliferation and function of normal lymphocytes. These findings establish that transformed lymphocytes are selectively sensitive to active‐site TORC1/2 inhibitors and further support the development of such compounds for leukemia therapy in addition to solid tumors. In summary, INK128 is a potent, selective, and orally active TORC1/2 dual inhibitor positioned to enter clinical development. TORC1/2 inhibitors are mechanistically distinct from rapamycin and offer a compelling approach to the treatment of cancer by targeting translational control, cell metabolism, growth and angiogenesis. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B148.

Abstract 4501: INK1117: A potent and orally efficacious PI3Kα-selective inhibitor for the treatment of cancer

Objective: Discovery, characterization and preclinical evaluation of PI3Kalpha (PI3Kα) selective small molecule kinase inhibitors for the treatment of solid tumors. Background: The PI3K pathway is one of the most frequently dysregulated pathways in human cancer. Activating mutations in the PIK3CA gene, encoding the p110 catalytic subunit of PI3Kα, have been identified as a major mechanism of inducing oncogenic PI3K signaling. The high frequency of PIK3CA mutations suggests that PI3Kα inhibitors may have therapeutic utility in genetically defined tumor populations. The discovery of isoform-selective PI3Kα inhibitors has proven to be difficult due to the highly homologous nature of all PI3K isoforms and structurally related kinases. Multiple non-selective class I PI3K (pan-PI3K) inhibitors have entered clinical development. PI3Kα-selective inhibitors may permit more potent inhibition of PI3Kα while minimizing side effects and permitting more alternatives for combination therapy relative to pan-PI3K pathway inhibitors. Results: Through structure-guided drug design we have discovered INK1117, a novel, potent and selective PI3Kα inhibitor with good oral bioavailability. INK1117 potently inhibits PI3Kα and demonstrates a greater than 100-fold selectivity relative to other class I PI3K family members and mTOR as well as a high degree of selectivity against a large panel of protein kinases. INK1117 blocks proliferation of tumor cell lines bearing PIK3CA mutations, and inhibits cellular phosphorylation and activity of AKT. However, INK1117 shows much less activity in PTEN-deficient tumor cells, which typically display constitutive PI3K pathway activation independent of PI3Kα. INK1117 blocks VEGF signaling and angiogenesis in vitro and in vivo. Daily, oral administration of INK1117 potently inhibits tumor growth in xenograft models bearing PIK3CA oncogenic mutations and, comparable to in vitro studies, INK1117 was not efficacious in tumor models with PTEN and/or KRAS mutations. In contrast to pan-PI3K inhibitors, INK1117 does not appear to impair glucose homeostasis or insulin response in glucose/insulin tolerance tests in rodents. Additionally, INK1117 does not significantly impair B and T cell function in vitro and in vivo. These two factors support the hypothesis that INK1117 may be better tolerated than pan-PI3K inhibitors, particularly when used in combination with other agents. Conclusion: INK1117 is a potent and orally efficacious PI3Kα isoform-selective kinase inhibitor with excellent drug-like properties. Selectively targeting PI3Kα offers a new therapeutic approach for the treatment of cancers with PIK3CA activation and preclinical data suggest that isoform-selective inhibitors may provide equivalent efficacy to pan-PI3K inhibitors in selected tumors with a more favorable safety profile. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4501. doi:10.1158/1538-7445.AM2011-4501

Abstract 1668: Pharmacodynamic biomarker development for INK128, a potent and selective inhibitor of TORC1/2 for the treatment of cancer

Pharmacokinetic / pharmacodynamic (PK/PD) modeling is a valuable strategy to achieve target inhibition as well as predictable and meaningful therapeutic efficacy. The mammalian target of rapamycin (mTOR) comprises two protein complexes, TORC1 and TORC2, which together regulate cell growth, metabolism, angiogenesis, and cell survival. Because TORC1 and TORC2 play a crucial role in several pathways that are frequently dysregulated in human cancer, the mTOR kinase is a compelling target for oncology drug development. Through rational drug design we have identified INK128, a potent and selective small molecule, ATP-competitive, active-site TORC1/2 kinase inhibitor with excellent drug-like properties. Inhibition of phosphorylation of S6 and 4EBP1, downstream markers of TORC1 signaling, was selected for PD analysis in peripheral blood cells (PBCs), skin tissue, and tumor tissue biopsy in mice xenograft tumor models. Time- and dose-dependent inhibition of S6 and 4EBP1 was demonstrated in PBCs by phospho-flow (FACS) analysis. Immunohistochemistry and immunoblot analysis demonstrated a correlation between S6 and 4EBP1 inhibition in tumors or skin tissue and antitumor effect. Additionally, site-selective inhibition of AKT phosphorylation at Ser473, the downstream substrate of TORC2, was also demonstrated in tumors and skin biopsies in mouse xenograft models. Our results demonstrate that daily, oral administration of INK128 selectively inhibits PI3K/AKT/mTOR signaling at the level of TORC1/2, and show that INK128 inhibits growth, and in some cases induces regression, of various tumor xenograft models. Results from these studies display a clear pharmacokinetic and pharmacodynamic relationship. Moreover, the activity of several of these downstream markers can be reproducibly measured in human peripheral blood cells and may permit development of a PK/PD model that might assist to predict PBC and skin tissue PD marker inhibition time-profiles in patients. In summary, INK128 presents a compelling, biomarker-guided approach for the treatment of a variety of cancer by targeting TORC1/2 signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1668.