Tyrosine kinases play a central role in the activation of signal transduction pathways and cellular responses that mediate the pathogenesis of rheumatoid arthritis. Imatinib mesylate (imatinib) is a tyrosine kinase inhibitor developed to treat Bcr/Abl-expressing leukemias and subsequently found to treat c-Kit-expressing gastrointestinal stromal tumors. We demonstrate that imatinib potently prevents and treats murine collageninduced arthritis (CIA). We further show that micromolar concentrations of imatinib abrogate multiple signal transduction pathways implicated in RA pathogenesis, including mast cell c-Kit signaling and TNF-α release, macrophage c-Fms activation and cytokine production, and fibroblast PDGFR signaling and proliferation. In our studies, imatinib attenuated PDGFR signaling in fibroblast-like synoviocytes (FLSs) and TNF-α production in synovial fluid mononuclear cells (SFMCs) derived from human RA patients. Imatinib-mediated inhibition of a spectrum of signal transduction pathways and the downstream pathogenic cellular responses may provide a powerful approach to treat RA and other inflammatory diseases.

Compounds that modulate the heat shock protein (HSP) network have potential in a broad range of research applications and diseases. A yeast-based liquid culture assay that measured time-dependent turbidity enabled the high-throughput screening of different Saccharomyces cerevisae strains to identify HSP modulators with unique molecular mechanisms. A focused set of four strains, with differing sensitivities to Hsp90 inhibitors, was used to screen a compound library of 3680 compounds. Computed turbidity curve functions were used to classify strain responses and sensitivity to chemical effects across the compound library. Filtering based on single-strain selectivity identified nine compounds as potential heat shock modulators, including the known Hsp90 inhibitor macbecin. Haploid yeast deletion strains (360), mined from previous Hsp90 inhibitor yeast screens and heat shock protein interaction data, were screened for differential sensitivities to known N-terminal ATP site-directed Hsp90 inhibitors to reveal functional distinctions. Strains demonstrating differential sensitivity (13) to Hsp90 inhibitors were used to prioritize primary screen hit compounds, with NSC145366 emerging as the lead hit. Our follow-up biochemical and functional studies show that NSC145366 directly interacts and inhibits the C-terminus of Hsp90, validating the platform as a powerful approach for early-stage identification of bioactive modulators of heat shock-dependent pathways.

The 90-kDa heat shock protein 90 (Hsp90) is a chaperone protein responsible for the folding and activation of numerous proteins disregulated in various cancers that drive oncogenesis. Consequently, inhibition of Hsp90 is thought to be an attractive anti-cancer therapeutic strategy, though to date, clinical success has been limited. Hsp90 inhibition leads to simultaneous degradation of multiple oncogenic client proteins, giving “polypharmacologic” effects through the targeting of a single, highly networked protein. Hence, the development of small molecule inhibitors of Hsp90 may be effective anti-cancer agents that have reduced likelihood of development of resistance when compared to other less networked single protein targets. This study demonstrates identification and validation of a novel chemotype that perturbs the heat shock protein network. A liquid culture-based phenotypic screen employing Saccharomyces cerevisiae haploid deletions strains with known sensitivities to Hsp90 inhibitors by screening of the NCI diversity II chemical library set. The screen identified 9 hit compounds; 2 previously identified heat shock modulators and 7 novel heat shock modulator hits. To validate these novel compounds as heat shock network selective, we bioinformatically show that the NSC145366 compounds sensitivity profile in NCI tumor cell lines significantly correlates with other Hsp90 inhibitors. Also, biochemical analyses demonstrate NSC145366 has direct interactions with two cytoplasmic isoforms of human Hsp90 both in recombinant protein preparations and in crude tumor cell lysates. In addition, we show that the compound preferentially interacts with the Hsp90 C-terminal dimerization domain as opposed to many of the current N-terminal domain-based inhibitors. Further characterization of the compound interaction with Hsp90 and its effects on the heat shock cellular network are ongoing. This study demonstrates the utility of phenotypic screening to identify heat shock protein network modulators and the possibility inhibiting Hsp90 in a novel and different mode from previous inhibitors. Citation Format: Kourtney M. Goode, Fiona M. Thomas, Pete Pascuzzi, V. Jo Davisson, Tony R. Hazbun. Mechanistic assessment of heat shock network perturbation by novel chemotypes identified using chemogenomic screening. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B89.