No abstract

A novel class of heat shock protein 90 (Hsp90) inhibitors was developed from an unbiased screen to identify protein targets for a diverse compound library. These indol-4-one and indazol-4-one derived 2-aminobenzamides showed strong binding affinity to Hsp90, and optimized analogues exhibited nanomolar antiproliferative activity across multiple cancer cell lines. Heat shock protein 70 (Hsp70) induction and specific client protein degradation in cells on treatment with the inhibitors supported Hsp90 inhibition as the mechanism of action. Computational chemistry and X-ray crystallographic analysis of selected member compounds clearly defined the protein-inhibitor interaction and assisted the design of analogues. 4-[6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl]-2-[(trans-4-hydroxycyclohexyl)amino]benzamide (SNX-2112, 9) was identified as highly selective and potent (IC(50) Her2 = 11 nM, HT-29 = 3 nM); its prodrug amino-acetic acid 4-[2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-trifluoromethyl-4,5,6,7-tetrahydro-indazol-1-yl)-phenylamino]-cyclohexyl ester methanesulfonate (SNX-5422, 10) was orally bioavailable and efficacious in a broad range of xenograft tumor models (e.g. 67% growth delay in a HT-29 model) and is now in multiple phase I clinical trials.

Many tumors are dependent on de novo fatty acid synthesis to maintain cell growth. Fatty acid synthase (FASN) catalyzes the final synthetic step of this pathway, and its upregulation is correlated with tumor aggressiveness. The consequences and adaptive responses of acute or chronic inhibition of essential enzymes such as FASN are not fully understood. Herein we identify Fasnall, a thiophenopyrimidine selectively targeting FASN through its co-factor binding sites. Global lipidomics studies with Fasnall showed profound changes in cellular lipid profiles, sharply increasing ceramides, diacylglycerols, and unsaturated fatty acids as well as increasing exogenous palmitate uptake that is deviated more into neutral lipid formation rather than phospholipids. We also showed that the increase in ceramide levels contributes to some extent in the mediation of apoptosis. Consistent with this mechanism of action, Fasnall showed potent anti-tumor activity in the MMTV-Neu model of HER2(+) breast cancer, particularly when combined with carboplatin.

Summary Tumor necrosis factor α (TNFα) has both positive and negative roles in human disease. In certain cancers, TNFα is infused locally to promote tumor regression, but dose-limiting inflammatory effects limit broader utility. In autoimmune disease, anti-TNFα antibodies control inflammation in most patients, but these benefits are offset during chronic treatment. TAK1 acts as a key mediator between survival and cell death in TNFα-mediated signaling. Here, we describe Takinib, a potent and selective TAK1 inhibitor that induces apoptosis following TNFα stimulation in cell models of rheumatoid arthritis and metastatic breast cancer. We demonstrate that Takinib is an inhibitor of autophosphorylated TAK1 that binds within the ATP binding pocket, yet is non-competitive, and inhibits by slowing down the rate-limiting step of TAK1 activation. Overall, Takinib is an attractive starting point for the development of inhibitors that sensitize cells to TNFα-induced cell death, with general implications for cancer and autoimmune disease treatment.

Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors.

DAPK1 and ZIPK (also called DAPK3) are closely related serine/threonine protein kinases that regulate programmed cell death and phosphorylation of non-muscle and smooth muscle myosin. We have developed a fluorescence linked enzyme chemoproteomic strategy (FLECS) for the rapid identification of inhibitors for any element of the purinome and identified a selective pyrazolo[3,4-d]pyrimidinone (HS38) that inhibits DAPK1 and ZIPK in an ATP-competitive manner at nanomolar concentrations. In cellular studies, HS38 decreased RLC20 phosphorylation. In ex vivo studies, HS38 decreased contractile force generated in mouse aorta and rabbit ileum, and calyculin A stimulated arterial muscle by decreasing RLC20 and MYPT1 phosphorylation. The inhibitor also promoted relaxation in Ca2+-sensitized vessels. A close structural analogue (HS43) with 5-fold lower affinity for ZIPK produced no effect on cells or tissues. These findings are consistent with a mechanism of action wherein HS38 specifically targets ZIPK in smooth muscle. The discovery of HS38 provides a lead scaffold for the development of therapeutic agents for smooth muscle related disorders and a chemical means to probe the function of DAPK1 and ZIPK across species.

A chemoproteomics-based drug discovery strategy is presented that utilizes a highly parallel screening platform, encompassing more than 1000 targets, with a focused chemical library prior to target selection. This chemoproteomics-based process enables a data-driven selection of both the biological target and chemical hit after the screen is complete. The methodology has been exemplified for the purine binding proteome (proteins utilizing ATP, NAD, FAD). Screening of an 8000 member library yielded over 1500 unique protein-ligand interactions, which included novel hits for the oncology target Hsp90. The approach, which also provides broad target selectivity information, was used to drive the identification of a potent and orally active Hsp90 inhibitor, SNX-5422, which is currently in phase 1 clinical studies.

No abstract