Small molecule inhibitors and CRISPR/Cas9 mutagenesis demonstrate that SMYD2 and SMYD3 activity are dispensable for autonomous cancer cell proliferation.

A key challenge in the development of precision medicine is defining the phenotypic consequences of pharmacological modulation of specific target macromolecules. To address this issue, a variety of genetic, molecular and chemical tools can be used. All of these approaches can produce misleading results if the specificity of the tools is not well understood and the proper controls are not performed. In this paper we illustrate these general themes by providing detailed studies of small molecule inhibitors of the enzymatic activity of two members of the SMYD branch of the protein lysine methyltransferases, SMYD2 and SMYD3. We show that tool compounds as well as CRISPR/Cas9 fail to reproduce many of the cell proliferation findings associated with SMYD2 and SMYD3 inhibition previously obtained with RNAi based approaches and with early stage chemical probes

Novel Oxindole Sulfonamides and Sulfamides: EPZ031686, the First Orally Bioavailable Small Molecule SMYD3 Inhibitor

SMYD3 has been implicated in a range of cancers; however, until now no potent selective small molecule inhibitors have been available for target validation studies. A novel oxindole series of SMYD3 inhibitors was identified through screening of the Epizyme proprietary histone methyltransferase-biased library. Potency optimization afforded two tool compounds, sulfonamide <b>EPZ031686</b> and sulfamide <b>EPZ030456</b>, with cellular potency at a level sufficient to probe the <i>in vitro</i> biology of SMYD3 inhibition. <b>EPZ031686</b> shows good bioavailability following oral dosing in mice making it a suitable tool for potential <i>in vivo</i> target validation studies

Small molecule inhibitors and CRISPR/Cas9 mutagenesis demonstrate that SMYD2 and SMYD3 activity are dispensable for autonomous cancer cell proliferation - Fig 1

<p><b>Chemical structures of SMYD2 (A) and SMYD3 (B) inhibitors</b>.</p

Anti-proliferative activity of SMYD2 inhibitors.

<p>(A) Correlation plots of (left) cellular methylation IC₅₀ as a function of biochemical IC₅₀ and (right) cell proliferation IC₅₀ as a function of cellular methylation IC₅₀ for SMYD2 inhibitors. (B) Western blot of BTF3 methylation showing dose dependent effects of EPZ032597. Data is representative of two independent experiments. (C) The effect of EPZ032597 on proliferation in a broad panel of cancer cell lines. (D) The effect LLY507 on proliferation of a broad panel of cancer cell lines. Values for C) and D) are the average of three biological replicates; error bars represent standard deviations (not readily visible on scale for all points). The 10 μM value represents the highest dose tested.</p

Gene ablation techniques show no dependence on SMYD2 or SMYD3 for cancer cell proliferation.

<p>Waterfall plot representing LogP RSA scores for sgRNAs targeting A) SMYD2 and B) SMYD3. 313 cell lines were infected with a library of 6500 sgRNAs targeting 600 different genes. LogP RSA scores represent depletion of guides from an infected cell population. Each bar represents a different cell line. Bars are colored by cancer subtype. C) Percent confluency of Hep3B cells infected with CRISPR viruses containing CAS9 and sgRNAs targeting HBE-1, EZH2 (negative controls) or SMYD3. Cell density was evaluated using an Incucyte Zoom. Growth curves were initiated 24 days following virus infection and puromycin selection. Plotted data is the average of three biological replicates. Error bars represent standard deviation (not readily visible on scale). D) SMYD3 western blot of lysates derived from Hep3B cells infected with CAS9 and SMYD3 sgRNA. Parental Hep3Bs and Hep3Bs stably infected with HBE-1, EZH2 (negative controls) or SMYD3 were lysed and probed for SMYD3 levels by western. GAPDH levels were evaluated as a loading control.</p