In Silico/In Vitro Hit-to-Lead Methodology Yields SMYD3 Inhibitor That Eliminates Unrestrained Proliferation of Breast Carcinoma Cells

Abstract

SMYD3 is a lysine methyltransferase that regulates the expression of over 80 genes and is required for the uncontrolled proliferation of most breast, colorectal, and hepatocellular carcinomas. The elimination of SMYD3 restores normal expression patterns of these genes and halts aberrant cell proliferation, making it a promising target for small molecule inhibition. In this study, we sought to establish a proof of concept for our in silico/in vitro hit-to-lead enzyme inhibitor development platform and to identify a lead small molecule candidate for SMYD3 inhibition. We used Schrodinger® software to screen libraries of small molecules in silico and the five compounds with the greatest predicted binding affinity within the SMYD3 binding pocket were purchased and assessed in vitro in direct binding assays and in breast cancer cell lines. We have confirmed the ability of one of these inhibitors, Inhibitor-4, to restore normal rates of cell proliferation, arrest the cell cycle, and induce apoptosis in breast cancer cells without affecting wildtype cell behavior. Our results provide a proof of concept for this fast and affordable small molecule hit-to-lead methodology as well as a promising candidate small molecule SMYD3 inhibitor for the treatment of human cancer.

Anticancer Impact of Nitric Oxide (NO) and NO Combination with SMYD-3 Inhibitor on Breast Carcinomas

“…The overall goal of this study was to determine the relative efficacy of NO, delivered by GSNO only (Group 1), and the combination therapeutic, SMYD-3 inhibitor (inhibitor-4) and NO from GSNO (Group 2) on human breast carcinomas MCF7 and MDA-MB-231. The impact of inhibitor-4 alone was assessed in a separate study conducted by our collaborators, Alshiraihi et al [29]. Various methods were used to determine efficacy, including cell viability assays (MTT and CTB), colony formation assays, LIVE/DEAD cytotoxicity assays, and cell apoptosis assays (Caspase-Glo 3/7 and Annexin V/PI).…”

“…In previous studies, the RSNO, GSNO, was shown to exhibit tumoricidal characteristics [30][31][32]. Likewise, a separate study indicated that a SYMD3 inhibitor, inhibitor-4, demonstrated potent impacts on two breast cancer lines, MCF7 and MDA-MB-231 (both lines linked to overexpression of SMYD3) [29]. By combining the two for a combination therapeutic, GSNO and inhibitor-4, we sought to surpass the efficacy of each individual therapeutic on MCF7 and MDA-MB-231 breast cancer lines.…”

“…Consequently, SMYD3 overexpression has been linked to increased proliferation, transformation, and metastasis of cancer cells [26,27]. Multiple studies highlight that when SMYD3 is inhibited, a substantial decrease in proliferative capacity of breast cell lines is observed [23,28,29].…”

“…This manuscript contains the results pertaining to NO, delivered by 1 mM GSNO, hereafter described as Group 1, and the combination therapeutic, SMYD3 inhibitor (200 µM inhibitor-4) and NO (from 1 mM GSNO), hereafter described as Group 2. As previously mentioned, the findings pertaining to inhibitor-4 were investigated in a prior study [29]. Glutathione (GSH), the precursor to GSNO, was applied to cells as a functional control, highlighting NO as the active therapeutic (not GSNO).…”

Playing on the Dark Side: SMYD3 Acts as a Cancer Genome Keeper in Gastrointestinal Malignancies

“…Moreover, the existing drug Diperodon has been reported as a new allosteric ligand interacting with SMYD3 and represents a good starting point for the design of compounds acting as modulators of noncatalytic SMYD3 functions [30]. In addition to these, recent studies identified new selective SMYD3is (e.g., BAY-6530, covalent inhibitors 1-4) [26,31], thereby contributing to the ongoing efforts to develop effective SMYD3is that might be used as anticancer drugs.…”