Discovery of novel aminoquinazolin-7-yl 6,7-dihydro-indol-4-ones as potent, selective inhibitors of heat shock protein 90.

A novel class of Hsp90 inhibitors, structurally distinct from previously reported scaffolds, was developed from rational design and optimization of a compound library screen hit. These aminoquinazoline derivatives, represented by compound 15 (SNX-6833) or 1-(2-amino-4-methylquinazolin-7-yl)-3,6,6-trimethyl-6,7-dihydro-1H-indol-4(5H)-one, selectively bind to Hsp90 and inhibit its cellular activities at concentrations as low as single digit nanomolar.

Application of chemoproteomics to drug discovery: identification of a clinical candidate targeting hsp90.

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.

Discovery of novel 2-aminobenzamide inhibitors of heat shock protein 90 as potent, selective and orally active antitumor agents.

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.

Novel carbazole and acyl-indole antimitotics.

In the course of our Heat Shock 90 program, certain carbazole compounds were identified which had an off-target antiproliferative activity. To understand the off-target activity, we studied one analog with strong activity. We discovered that it had an effect on tubulin polymerization kinetics and was competitive with colchicine. Additional analogs were made, and a number of potent compounds were identified.

Targeting of multiple signaling pathways by the Hsp90 inhibitor SNX-2112 in EGFR resistance models as a single agent or in combination with erlotinib.

Inhibition of Hsp90 has emerged as a therapeutic strategy to target NSCLC subtypes, which are refractory to epidermal growth factor receptor (EGFR) inhibitor-based treatment. We report on a novel small molecule inhibitor of Hsp90, SNX-2112, and an orally bioavailable prodrug analog, SNX-5422. In cellular models of wild-type or mutant EGFR (L858R and T790M mutations), SNX-2112 alone and in combination with erlotinib inhibited EGF activation of pAKT(473) and pSTAT3(705). pERK1/2 and pS6 were also potently inhibited by similar treatments. SNX-2112 reduced EGF cross-talk and activation of the c-Met receptor by causing c-Met degradation. In NCI-H1975 xenograft models, SNX-5422 showed activity as a single agent and in combination with erlotinib resulted in prolonged animal survival at reduced compound concentrations relative to either compound alone. These results support the advanced evaluation of SNX-5422 as a treatment for non-small cell lung cancer (NSCLC), especially in cases where the cancer is driven by c-Met amplification or mutated EGFR forms that are resistant to EGFR inhibitors.

SNX-2112, a selective Hsp90 inhibitor, potently inhibits tumor cell growth, angiogenesis, and osteoclastogenesis in multiple myeloma and other hematologic tumors by abrogating signaling via Akt and ERK.

Heat-shock protein 90 (Hsp90) acts as a molecular chaperone required for maintaining the conformational stability of client proteins regulating cell proliferation, survival, and apoptosis. Here we investigate the biologic significance of Hsp90 inhibition in multiple myeloma (MM) and other hematologic tumors using an orally available novel small molecule inhibitor SNX-2112, which exhibits unique activities relative to 17-allyamino-17-demethoxy-geldanamycin (17-AAG). SNX-2112 triggers growth inhibition and is more potent than 17-AAG against MM and other malignancies. It induces apoptosis via caspase-8, -9, -3, and poly (ADP-ribose) polymerase cleavage. SNX-2112 inhibits cytokine-induced Akt and extracellular signal-related kinase (ERK) activation and also overcomes the growth advantages conferred by interleukin-6, insulin-like growth factor-1, and bone marrow stromal cells. Importantly, SNX-2112 inhibits tube formation by human umbilical vein endothelial cells via abrogation of eNOS/Akt pathway and markedly inhibits osteoclast formation via down-regulation of ERK/c-fos and PU.1. Finally, SNX-2112, delivered by its prodrug SNX-5422, inhibits MM cell growth and prolongs survival in a xenograft murine model. Our results indicate that blockade of Hsp90 by SNX-2112 not only inhibits MM cell growth but also acts in the bone marrow microenvironment to block angiogenesis and osteoclastogenesis. Taken together, our data provide the framework for clinical studies of SNX-2112 to improve patient outcome in MM and other hematologic malignancies.

Small molecule inhibitors of Hsp90 potently affect inflammatory disease pathways and exhibit activity in models of rheumatoid arthritis.

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-kappaB nuclear translocation was evaluated in cellular systems, using lipopolysaccharide (LPS), tumor necrosis factor alpha, or interleukin-1beta 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-kappaB 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.

Discovery of benzamide tetrahydro-4H-carbazol-4-ones as novel small molecule inhibitors of Hsp90.

Hsp90 maintains the conformational stability of multiple proteins implicated in oncogenesis and has emerged as a target for chemotherapy. We report here the discovery of a novel small molecule scaffold that inhibits Hsp90. X-ray data show that the scaffold binds competitively at the ATP site on Hsp90. Cellular proliferation and client assays demonstrate that members of the series are able to inhibit Hsp90 at nanomolar concentrations.

Dynamics of T- and B-lymphocyte turnover in a natural host of simian immunodeficiency virus.

Increased lymphocyte turnover is a hallmark of pathogenic lentiviral infection. To investigate perturbations in lymphocyte dynamics in natural hosts with nonpathogenic simian immunodeficiency virus (SIV) infection, the nucleoside analog bromodeoxyuridine (BrdU) was administered to six naturally SIV-infected and five SIV-negative sooty mangabeys. As a measure of lymphocyte turnover, we estimated the mean death rate by fitting a mathematical model to the fraction of BrdU-labeled cells during a 2-week labeling and a median 10-week delabeling period. Despite significantly lower total T- and B-lymphocyte counts in SIV-infected sooty mangabeys than in SIV-negative mangabeys, the turnover rate of B lymphocytes and CD4(+) and CD8(+) T lymphocytes was not increased in the SIV-infected animals. A small, rapidly proliferating CD45RA(+) memory subset and a large, slower-proliferating CD45RA(-) central memory subset of CD4(+) T lymphocytes identified in the peripheral blood of sooty mangabeys also did not show evidence of increased turnover in the context of SIV infection. Independently of SIV infection, the turnover of CD4(+) T lymphocytes in sooty mangabeys was significantly higher (P < 0.01) than that of CD8(+) T lymphocytes, a finding hitherto not reported in rhesus macaques or humans. The absence of aberrant T-lymphocyte turnover along with an inherently high rate of CD4(+) T-lymphocyte turnover may help to preserve the pool of central memory CD4(+) T lymphocytes in viremic SIV-infected sooty mangabeys and protect against progression to AIDS.

Induction of a virus-specific effector-memory CD4+ T cell response by attenuated SIV infection.

We investigated simian immunodeficiency virus (SIV)-specific CD4+ T cell responses in rhesus macaques chronically infected with attenuated or pathogenic SIV strains. Analysis of SIVDeltanef-infected animals revealed a relatively high frequency of SIV-specific CD4+ T cells representing 4-10% of all CD4+ T lymphocytes directed against multiple SIV proteins. Gag-specific CD4+ T cells in wild-type SIV-infected animals were 5-10-fold lower in frequency and inversely correlated with the level of plasma viremia. SIV-specific CD4+ cells from SIVDeltanef animals were predominantly CD27-CD28-CD45RAlowCCR7-CCR5-, consistent with an effector-memory subset, and included a fully differentiated CD45RA+CCR7- subpopulation. In contrast, SIV-specific CD4+ T cells from SIV-infected animals were mostly CD27+CD28+CD45RA-CCR7+CCR5+, consistent with an early central memory phenotype. The CD45RA+CCR7-CD4+ subset from SIVDeltanef animals was highly enriched for effector CD4+ T cells, as indicated by the perforin expression and up-regulation of the lysosomal membrane protein CD107a after SIV Gag stimulation. SIV-specific CD4+ T cells in attenuated SIV-infected animals were increased in frequency in bronchioalveolar lavage and decreased in lymph nodes, consistent with an effector-memory T cell population. The ability of SIVDeltanef to induce a high frequency virus-specific CD4+ T cell response with direct effector function may play a key role in protective immunity produced by vaccination with attenuated SIV strains.

The use of high-content screening for the discovery and characterization of compounds that modulate mitotic index and cell cycle progression by differing mechanisms of action.

The advent of high-content screening has expanded the ability of researchers to identify and quantify compound effects on a number of cellular events in a manner that allows for the rapid screening of chemical libraries. We have validated an approach for characterizing inhibitors of Aurora kinase family members using high-content screening by determining compound effects on the levels of the mitotic marker phospho-histone H3 (Ser10). Analysis of the data from these experiments led us to the discovery of a series of related compounds that increased the level of cells staining positive for the mitotic marker, indicating a block of cell cycle progression at M-phase. We then reconfigured the Aurora kinase inhibition assay and validated a high-content approach to identify compounds that block progression through M-phase. We were able to take advantage of the flexibility within the high-content screening platform to measure DNA content, activation of apoptosis, and effects on beta-tubulin staining patterns, all of which directly led to the identification of the cellular target of this new class of compounds.