CCG-1423: A small-molecule inhibitor of RhoA transcriptional signaling

ArticleinMolecular Cancer Therapeutics 6(8):2249-60 · August 2007with63 Reads
DOI: 10.1158/1535-7163.MCT-06-0782 · Source: PubMed
Abstract
Lysophosphatidic acid receptors stimulate a Galpha(12/13)/RhoA-dependent gene transcription program involving the serum response factor (SRF) and its coactivator and oncogene, megakaryoblastic leukemia 1 (MKL1). Inhibitors of this pathway could serve as useful biological probes and potential cancer therapeutic agents. Through a transcription-based high-throughput serum response element-luciferase screening assay, we identified two small-molecule inhibitors of this pathway. Mechanistic studies on the more potent CCG-1423 show that it acts downstream of Rho because it blocks SRE.L-driven transcription stimulated by Galpha(12)Q231L, Galpha(13)Q226L, RhoA-G14V, and RhoC-G14V. The ability of CCG-1423 to block transcription activated by MKL1, but not that induced by SRF-VP16 or GAL4-VP16, suggests a mechanism targeting MKL/SRF-dependent transcriptional activation that does not involve alterations in DNA binding. Consistent with its role as a Rho/SRF pathway inhibitor, CCG-1423 displays activity in several in vitro cancer cell functional assays. CCG-1423 potently (<1 mumol/L) inhibits lysophosphatidic acid-induced DNA synthesis in PC-3 prostate cancer cells, and whereas it inhibits the growth of RhoC-overexpressing melanoma lines (A375M2 and SK-Mel-147) at nanomolar concentrations, it is less active on related lines (A375 and SK-Mel-28) that express lower levels of Rho. Similarly, CCG-1423 selectively stimulates apoptosis of the metastasis-prone, RhoC-overexpressing melanoma cell line (A375M2) compared with the parental cell line (A375). CCG-1423 inhibited Rho-dependent invasion by PC-3 prostate cancer cells, whereas it did not affect the Galpha(i)-dependent invasion by the SKOV-3 ovarian cancer cell line. Thus, based on its profile, CCG-1423 is a promising lead compound for the development of novel pharmacologic tools to disrupt transcriptional responses of the Rho pathway in cancer.
    • "For the high-throughput screen and counter-screen, HEK293 cells were co-transfected with an SRE-Luciferase reporter plasmid and GPR56 7TM or GNA13 Q226L pcDNA3.1 plasmids, respectively. Vehicle-treated cells were the positive controls (top limit of the assays), and cells treated with latrunculin B, which disrupts the signaling axis downstream of both GPR56 or Gα13 were the negative controls (bottom limit of the assays) (Evelyn et al., 2007). The Z' score for the screening assay was 0.67 and 0.8 for the counter-screening assay (Supplemental Figure 1) (Zhang et al., 1999). "
    [Show abstract] [Hide abstract] ABSTRACT: Adhesion GPCRs (aGPCRs) have emerging roles in development and tissue maintenance and are the most prevalent GPCR sub-class mutated in human cancers, but to date, no drugs have been developed to target them in any disease. aGPCRs have large extracellular domains (ECDs) containing a conserved sub-domain that mediates constitutive self-cleavage at a site near the start of the 7-transmembrane domain (7TM). The two resultant receptor protomers, ECD or N-terminal fragment (NTF), and the 7TM or C-terminal fragment remain non-covalently bound at the plasma membrane in an inactive receptor state. We recently demonstrated that NTF dissociation liberates the 7TM N-terminal stalk, which acts as a tethered-peptide agonist permitting receptor-dependent heterotrimeric G protein activation. In many cases, natural aGPCR ligands are extracellular matrix (ECM) proteins that bind the NTF and may act to dissociate it to reveal the tethered agonist. Given the perceived difficulty in working with ECM proteins to create pharmacological aGPCR probes, we developed a SRE-luciferase-based high-throughput screening approach to identify GPR56/ADGRG1 small-molecule inhibitors. A 2000 compound library comprising known drugs and natural products was screened for inhibitors of GPR56-dependent SRE activation that did not inhibit constitutively active Gα13-dependent SRE activation. Three compounds were identified and Dihydromunduletone (DHM), a rotenoid derivative, was selected for further validation using cell-free aGPCR/heterotrimeric G protein GTPγS binding reconstitution assays. DHM inhibited GPR56 and GPR114/ADGRG5 which have similar tethered agonists, but did not inhibit the aGPCR GPR110/ADGRF1, Class A M3 muscarinic acetylcholine, or β2 adrenergic GPCRs. DHM inhibited tethered- or synthetic-peptide agonist-stimulated GPR56, but did not inhibit basal GPR56 activity, demonstrating that it antagonizes the peptide agonist. These data demonstrate identification of DHM as a novel aGPCR antagonist and potentially useful chemical probe that may be developed as a future aGPCR therapeutic.
    Article · Jun 2016
    • "These results were confirmed in human foreskin fibroblasts and in cutaneous wound healing experiments where isoxazole promoted more rapid wound healing compared to control-treated animals [132]. A similar screen for modulators of RhoA-mediated signaling led to the identification of CCG-1432 [158]. Compounds related to CCG-1432 bind the nuclear localization signal within the RPEL domain of MRTFs, inhibiting importin-dependent nuclear translocation [159] . "
    [Show abstract] [Hide abstract] ABSTRACT: Cardiac fibroblasts help maintain the normal architecture of the healthy heart and are responsible for scar formation and the healing response to pathological insults. Various genetic, biomechanical, or humoral factors stimulate fibroblasts to become contractile smooth muscle-like cells called myofibroblasts that secrete large amounts of extracellular matrix. Unfortunately, unchecked myofibroblast activation in heart disease leads to pathological fibrosis, which is a major risk factor for the development of cardiac arrhythmias and heart failure. A better understanding of the molecular mechanisms that control fibroblast plasticity and myofibroblast activation is essential to develop novel strategies to specifically target pathological cardiac fibrosis without disrupting the adaptive healing response. This review highlights the major transcriptional mediators of fibroblast origin and function in development and disease. The contribution of the fetal epicardial gene program will be discussed in the context of fibroblast origin in development and following injury, primarily focusing on Tcf21 and C/EBP. We will also highlight the major transcriptional regulatory axes that control fibroblast plasticity in the adult heart, including transforming growth factor β (TGFβ)/Smad signaling, the Rho/myocardin-related transcription factor (MRTF)/serum response factor (SRF) axis, and Calcineurin/transient receptor potential channel (TRP)/nuclear factor of activated T-Cell (NFAT) signaling. Finally, we will discuss recent strategies to divert the fibroblast transcriptional program in an effort to promote cardiomyocyte regeneration. This article is a part of a Special Issue entitled "Fibrosis and Myocardial Remodeling".
    Article · Dec 2015
    • "In this study, we stereoselectively synthesized optically pure isomers of CCG-1423 and related compounds (Fig 1), which were developed by Neubig and co-workers. They focused on the inhibitory potential in Rho/ MRTF-A/SRF-mediated pathway [11,171819. We then validated their biological activities and analyzed their binding to MRTF-A by molecular docking simulations. "
    [Show abstract] [Hide abstract] ABSTRACT: CCG-1423 suppresses several pathological processes including cancer cell migration, tissue fibrosis, and the development of atherosclerotic lesions. These suppressions are caused by inhibition of myocardin-related transcription factor A (MRTF-A), which is a critical factor for epithelial-mesenchymal transition (EMT). CCG-1423 can therefore be a potent inhibitor for EMT. CCG-1423 and related compounds, CCG-100602 and CCG-203971 possess similar biological activities. Although these compounds are comprised of two stereoisomers, the differences in their biological activities remain to be assessed. To address this issue, we stereoselectively synthesized optically pure isomers of these compounds and validated their biological activities. The S-isomer of CCG-1423 rather than the R-isomer exhibited modestly but significantly higher inhibitory effects on the cellular events triggered by MRTF-A activation including serum response factor-mediated gene expression and cell migration of fibroblasts and B16F10 melanoma cells. Accordingly, the S-isomer of CCG-1423 more potently blocked the serum-induced nuclear import of MRTF-A than the R-isomer. No such difference was observed in cells treated with each of two stereoisomers of CCG-100602 or CCG-203971. We previously reported that the N-terminal basic domain (NB), which functions as a nuclear localization signal of MRTF-A, is a binding site for CCG-1423. Consistent with the biological activities of two stereoisomers of CCG-1423, docking simulation demonstrated that the S-isomer of CCG-1423 was more likely to bind to NB than the R-isomer. This is a first report demonstrating the stereospecific biological activities of CCG-1423.
    Full-text · Article · Aug 2015
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