Objective. To evaluate the ability of SNX-7081, a novel small molecule inhibitor of Hsp90, to block components of inflammation, including cytokine production, protein kinase activity, and angiogenic signaling. A close analog was evaluated in preclinical in vivo models of rheumatoid arthritis (RA).Methods. SNX-7081 binding to Hsp90 was characterized in Jurkat cells and RA synovial fibroblasts (RASFs). Inhibition of NF-B nuclear translocation was evaluated in cellular systems, using lipopolysaccharide (LPS), tumor necrosis factor ␣, or interleukin-1␤ stimulation. Suppression of cytokine production in THP-1 cells, human umbilical vein endothelial cells, and RASFs was studied. Disruption of MAPK signaling cascades by SNX-7081 following growth factor stimulation was assessed. SNX-7081 was tested in 2 relevant angiogenesis assays: platelet-derived growth factor activation of fibroblasts and LPS-induced nitric oxide (NO) release in J774 macrophages. A close analog, SNX-4414, was evaluated in rat collagen-induced arthritis and adjuvant-induced arthritis, following oral treatment.Results. SNX-7081 showed strong binding affinity to Hsp90 and expected induction of Hsp70. NF-B nuclear translocation was blocked by SNX-7081 at nanomolar concentrations, and cytokine production was potently inhibited. Growth factor activation of ERK and JNK signaling was significantly reduced by SNX-7081. NO production was also sharply inhibited. In animal models, SNX-4414 fully inhibited paw swelling and improved body weight. Scores for inflammation, pannus formation, cartilage damage, and bone resorption returned to normal.Conclusion. The present results demonstrate that a small molecule Hsp90 inhibitor can impact inflammatory disease processes. The strong in vivo efficacy observed with SNX-4414 provides preclinical validation for consideration of Hsp90 inhibitors in the treatment of RA.

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.

alpha-Piperidine-beta-sulfone hydroxamate derivatives were explored that are potent for matrix metalloproteinases (MMP)-2, -9, and -13 and are sparing of MMP-1. The investigation of the beta-sulfones subsequently led to the discovery of hitherto unknown alpha-sulfone hydroxamates that are superior to the corresponding beta-sulfones in potency for target MMPs, selectivity vs MMP-1, and exposure when dosed orally. alpha-Piperidine-alpha-sulfone hydroxamate 35f (SC-276) was advanced through antitumor and antiangiogenesis assays and was selected for development. Compound 35f demonstrates excellent antitumor activity vs MX-1 breast tumor in mice when dosed orally as monotherapy or in combination with paclitaxel.

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.

α-Sulfone-α-piperidine and α-tetrahydropyranyl hydroxamates were explored that are potent inhibitors of MMP's-2, -9, and -13 that spare MMP-1, with oral efficacy in inhibiting tumor growth in mice and left-ventricular hypertrophy in rats and in the bovine cartilage degradation ex vivo explant system. α-Piperidine 19v (SC-78080/SD-2590) was selected for development toward the initial indication of cancer, while α-piperidine and α-tetrahydropyranyl hydroxamates 19w (SC-77964) and 9i (SC-77774), respectively, were identified as backup compounds.

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.

Water transport across lipid membranes is fundamental to all forms of life and plays a major role in health and disease. However, not only typical water facilitators like aquaporins facilitate water flux, but also transporters, ion channels or receptors represent potent water pathways. The efforts directed towards a mechanistic understanding of water conductivity determinants in transmembrane proteins, the development of water flow inhibitors, and the creation of biomimetic membranes with incorporated membrane proteins or artificial water channels depend on reliable and accurate ways of quantifying water permeabilities Pf. A conventional method is to subject vesicles to an osmotic gradient in a stopped-flow device: Fast recordings of scattered light intensity are converted into the time course of vesicle volume change. Even though an analytical solution accurately acquiring Pf from scattered light intensities exists, approximations potentially misjudging Pf by orders of magnitude are used. By means of computational and experimental data we point out that erroneous results such as that the single channel water permeability pf depends on the osmotic gradient are direct results of such approximations. Finally, we propose an empirical solution of which calculated permeability values closely match those calculated with the analytical solution in the relevant range of parameters.

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