Target Identification for and Optimization of Two Novel Series of Rho-mediated Gene Transcription Inhibitors with Anti-Fibrotic Properties
Kahl, Dylan
2019
Abstract
Wound healing is characterized by the excessive deposition of extracellular matrix (ECM) components. Fibrosis is a devastating result of the improper regulation of the wound healing process, and it contributes to ~45% of deaths in the developed world. A major hallmark of fibrosis is the fibroblast-to-myofibroblast transition. Myofibroblasts have an increased production of ECM components and impart contractile forces that cause architectural distortion to the surrounding tissue. These forces recruit signal transduction by various extracellular signaling pathways, including transforming growth factor β (TGF-β), lysophosphatidic acid (LPA), endothelin, and connective tissue growth factor (CTGF), ultimately activating the Rho family of GTPases and their downstream kinase, Rho-associated coiled-coil containing protein kinase (ROCK). This signaling leads to myocardin-related transcription factor (MRTF)/serum response factor (SRF)-mediate gene transcription of SRF-associated cytoskeletal genes, ultimately causing fibroblast differentiation into myofibroblast cells. Since Rho/MRTF/SRF-mediated signaling is known to regulate pro-fibrotic gene expression arising from multiple extracellular signaling pathways, inhibition of this common pathway is hypothesized to be a particularly effective way to treat/prevent fibrosis. In 2007, two distinct series—a 5-aryl-1,3,4-oxadiazol-2-ylthiopropionic acid (58150-) and a bis-amide (222740-) series—of novel inhibitors of Rho/MRTF/SRF-mediated gene expression were identified by the Neubig lab through a phenotypic high-throughput screening campaign. Both series have produced inhibitors that show promising results in multiple in vitro and in vivo models of fibrosis. Due to the phenotypic nature of the assay, the target(s) for both series are unknown. Recently, potential biological targets for both series have been identified through proteomics studies using immobilized agarose resin high affinity pull-down probes. Target validation efforts for both series were conducted in a collaborative effort between the Neubig, Larsen, and Martin labs; and, the successful identification of pirin as a biological target for the 222740-series is reported. Additionally, through structure-based drug design (SBDD) efforts using pirin co-crystal structures, the cellular potency for the series was improved about ten-fold, and additional novel bis-amide scaffolds were discovered. Also, despite the inability to validate a target for the 58150-series, the cellular activity from hit-to-lead was rapidly improved by over 5 orders of magnitude (180 nM to 1 pM). The levels of cellular potency and depth of SAR, coupled with the relatively low molecular weight (< 400 g/mol) of the series, suggests that binding to the unknown molecular target may occur through a novel covalent mechanism. In support of this hypothesis, modifications made to the scaffold that would presumably reduce covalent reactivity produced inhibitors with significantly diminished activities. Nevertheless, the series has no observable cytotoxicity up to 100 µM. As a result, two highly potent and orally bioavailable anti-fibrotic agents for the 58150-series were developed that dose-dependently reduce CTGF gene expression in vitro and significantly and dose-dependently diminish the development of bleomycin-induced dermal fibrosis in mice in vivo.Subjects
Advancement of two novel series of Rho-mediated gene transcription inhibitors with anti-fibrotic properties
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