David J. Matthews

Efficiency of signalling through cytokine receptors depends critically on receptor orientation

Human erythropoietin is a haematopoietic cytokine required for the differentiation and proliferation of precursor cells into red blood cells. It activates cells by binding and orientating two cell-surface erythropoietin receptors (EPORs) which trigger an intracellular phosphorylation cascade. The half-maximal response in a cellular proliferation assay is evoked at an erythropoietin concentration of 10 pM (ref. 3), 10−2 of its K d value for erythropoietin–EPOR binding site 1 (Kd ≈ 1 nM), and 10−5 of the K d for erythropoietin–EPOR binding site 2 (Kd ≈ 1 μM). Overall half-maximal binding (IC50) of cell-surface receptors is produced with ∼0.18 nM erythropoietin, indicating that only ∼6% of the receptors would be bound in the presence of 10 pM erythropoietin. Other effective erythropoietin-mimetic ligands that dimerize receptors can evoke the same cellular responses, but much less efficiently, requiring concentrations close to their K d values (∼0.1 μM). The crystal structure of erythropoietin complexed to the extracellular ligand-binding domains of the erythropoietin receptor, determined at 1.9 Å from two crystal forms, shows that erythropoietin imposes a unique 120° angular relationship and orientation that is responsible for optimal signalling through intracellular kinase pathways.

Abstract 3737: The PI3K/mTOR inhibitor PWT33597 regresses 786-0 renal xenografts

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL PWT33597 is a balanced dual inhibitor of phosphatidylinositol 3-kinase alpha (PI3K alpha) and mTOR, currently in clinical development. Inhibition of signaling downstream of PI3K and mTOR by [PWT33597][1] has been demonstrated, and [PWT33597][1] is strongly efficacious in multiple xenograft models tested to date with different PIK3CA and PTEN genotypes. Inhibitors of mTORC1 (rapalogs) are approved for treatment of advanced renal cell carcinoma (RCC), but their effect is restricted to a subset of patients, and is not sustained. Inhibitors of VEGFR signaling such as sorafenib and sunitinib have also demonstrated clinical activity in RCC and are approved in this indication. Based on the proven role of rapalogs in RCC, we tested [PWT33597][1] in a renal xenograft model with the hypothesis that inhibition of both mTORC1 and mTORC2, as well as PI3K, may offer increased efficacy by direct targeting of multiple signaling nodes including VEGFR signaling. Dual inhibition would also be expected to decrease release of negative feedback loops triggered by either mTOR or PI3K inhibition alone. [PWT33597][1] was tested in 786-0 (VHL -/-, PTEN -/-) xenografts in comparison to rapamycin (mTORC1 inhibitor), and sorafenib (VEGFR/RAF inhibitor). While sorafenib and rapamycin showed tumor growth inhibition (TGI), the maximal effect with sorafenib was 64% TGI, and rapamycin had a largely cytostatic effect. The overall efficacy observed with [PWT33597][1] was superior, with 93% TGI observed. [PWT33597][1] was also tested in comparison to a pan-PI3K inhibitor (GDC-0941, 49% TGI) and demonstrated superior efficacy. Immunohistochemical analysis of tumors for cleaved caspase 3 (CC3) as an indicator of apoptosis showed that a single dose of [PWT33597][1] induced a several fold increase in CC3 staining, and this effect was also apparent after 18 days of dosing. Based on these results we tested the ability of [PWT33597][1] to regress large 786-0 tumors (approx. 500 mm3), and a rapid decrease in tumor size was observed in 100% of animals. Additional cellular and xenograft studies in RCC models are ongoing to further define the mechanism of action of [PWT33597][1], and will be presented. Taken together, these data provide rationale for testing [PWT33597][1] in patients with RCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3737. doi:1538-7445.AM2012-3737 [1]: /lookup/external-ref?link_type=GENPEPT&access_num=PWT33597&atom=%2Fcanres%2F72%2F8_Supplement%2F3737.atom

Abstract 1644: Design and discovery of PWT33597 (VDC-597), a dual inhibitor of PI3-kinase alpha and mTOR

Phosphoinositide-3-kinase (PI3K) is an important mediator of tumor cell growth, survival and proliferation. In particular, PI3K alpha is important for signaling downstream of receptor tyrosine kinases and is also frequently amplified or mutationally activated in tumors, suggesting that selective inhibitors of this isoform may have therapeutic utility in the treatment of cancer. Downstream of PI3K, the mTOR kinase also plays a critical role in cellular growth and metabolism, and inhibitors of mTOR have demonstrated clinical benefit in several tumor types. We report here the design, discovery and characterization of PWT33597 (VDC-597), a dual inhibitor of PI3K alpha and mTOR, which entered human clinical trials in 2011. Starting with the known pan-Class I PI3-kinase inhibitor ZSTK474, we identified the methanesulfonylpiperazine analogue, 2-(difluoromethyl)-1-[4-[4-(methylsulfonyl)-1-piperazinyl]-6-(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole as a promising lead compound with activity against both PI3K alpha (IC 50 = 21 nM) and PI3K delta (IC 50 = 18 nM). The addition of a methoxy group at the 4-position of the benzimidazole group led to a more selective inhibitor of PI3K alpha (IC 50 = 6 nM versus 41 nM for PI3K delta), although with reduced solubility. A search for more soluble analogues identified SN 32976 as a selective inhibitor of PI3K alpha (IC 50 = 28 nM) over both PI3K delta (IC 50 = 287 nM) and mTOR (IC 50 = 227 nM), with good aqueous solubility. SN 32976 displayed good oral bioavailability and was significantly more active than ZSTK474 against a U87 MG human tumor xenograft model in mice. A search for more metabolically stable analogues subsequently identified PWT33597, which maintained the selectivity for PI3K alpha (IC 50 = 26 nM) over PI3K delta (IC 50 = 291 nM) but now also displayed activity against mTOR in biochemical assays (IC 50 = 21 nM). PWT33597 had good pharmacokinetic properties in multiple preclinical species, was not extensively metabolized in vivo and showed little potential for interaction with cytochrome P450 enzymes. Human clinical trials of PWT33597 were completed in 2012, and it is now undergoing further studies in veterinary cancers (as VDC-597). Citation Format: Gordon W. Rewcastle, Jack U. Flanagan, Anna C. Giddens, Swarna A. Gamage, Sophia KY Tsang, Jackie D. Kendall, Bruce C. Baguley, Christina M. Buchanan, David J. Matthews, Marie O9Farrell, Stephen MF Jamieson, William A. Denny, Peter R. Shepherd. Design and discovery of PWT33597 (VDC-597), a dual inhibitor of PI3-kinase alpha and mTOR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1644. doi:10.1158/1538-7445.AM2014-1644