A Kinetic Alignment of Orthologous Inosine-5′-monophosphate Dehydrogenases

IMP dehydrogenase (IMPDH) catalyzes two very different chemical transformations, a dehydrogenase reaction and a hydrolysis reaction. The enzyme toggles between the open conformation required for the dehydrogenase reaction and the closed conformation of the hydrolase reaction by moving a mobile flap into the NAD site. Despite these multiple functional constraints, the residues of the flap and NAD site are highly diverged, and the equilibrium between open and closed conformations (<i>K</i>_<i>c</i>) varies widely. In order to understand how differences in the dynamic properties of the flap influence the catalytic cycle, we have delineated the kinetic mechanism of IMPDH from the pathogenic protozoan parasite <i>Cryptosporidium parvum</i> (<i>Cp</i>IMPDH), which was obtained from a bacterial source through horizontal gene transfer, and its host counterpart, human IMPDH type 2 (hIMPDH2). Interestingly, the intrinsic binding energy of NAD⁺ differentially distributes across the dinucleotide binding sites of these two enzymes as well as in the previously characterized IMPDH from <i>Tritrichomonas foetus</i> (<i>Tf</i>IMPDH). Both the dehydrogenase and hydrolase reactions display significant differences in the host and parasite enzymes, in keeping with the phylogenetic and structural divergence of their active sites. Despite large differences in <i>K</i>_<i>c</i>, the catalytic power of both the dehydrogenase and hydrolase conformations are similar in <i>Cp</i>IMPDH and <i>Tf</i>IMPDH. This observation suggests that the closure of the flap simply sets the stage for catalysis rather than plays a more active role in the chemical transformation. This work provides the essential mechanistic framework for drug discovery

Characterization of the Enzymatic Activity of SETDB1 and Its 1:1 Complex with ATF7IP

The protein methyltransferase (PMT) SETDB1 is a strong candidate oncogene in melanoma and lung carcinomas. SETDB1 methylates lysine 9 of histone 3 (H3K9), utilizing <i>S</i>-adenosylmethionine (SAM) as the methyl donor and its catalytic activity, has been reported to be regulated by a partner protein ATF7IP. Here, we examine the contribution of ATF7IP to the <i>in vitro</i> activity and substrate specificity of SETDB1. SETDB1 and ATF7IP were co-expressed and 1:1 stoichiometric complexes were purified for comparison against SETDB1 enzyme alone. We employed both radiometric flashplate-based and SAMDI mass spectrometry assays to follow methylation on histone H3 15-mer peptides, where lysine 9 was either unmodified, monomethylated, or dimethylated. Results show that SETDB1 and the SETDB1:ATF7IP complex efficiently catalyze both monomethylation and dimethylation of H3K9 peptide substrates. The activity of the binary complex was 4-fold lower than SETDB1 alone. This difference was due to a decrease in the value of <i>k</i>_cat as the substrate <i>K</i>_M values were comparable between SETDB1 and the SETDB1:ATF7IP complex. H3K9 methylation by SETDB1 occurred in a distributive manner, and this too was unaffected by the presence of ATF7IP. This finding is important as H3K9 can be methylated by HMTs other than SETDB1 and a distributive mechanism would allow for interplay between multiple HMTs on H3K9. Our results indicate that ATF7IP does not directly modulate SETDB1 catalytic activity, suggesting alternate roles, such as affecting cellular localization or mediating interaction with additional binding partners

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

Biochemical and cellular potencies and physicochemical properties of SMYD2 and SMYD3 inhibitors used in this study.

<p>Biochemical and cellular potencies and physicochemical properties of SMYD2 and SMYD3 inhibitors used in this study.</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

Characterization of EPZ028862 as an inhibitor of SMYD3.

<p>A) Representative SMYD3 biochemical dose-response curve for EPZ028862 with a mean IC₅₀ value and standard deviation of 1.80 ± 0.06 nM from 2 experiments. B) Structure of EPZ028862 (cyan) with SMYD3 (green) and SAM (yellow) (PDB ID 5V37); water molecules are represented with red spheres. Electron density (2Fo−Fc, 1σ) for the compound is shown. Hydrogen bonds are indicated as dashed lines. C) Anti-proliferative activity of the SMYD3 inhibitor EPZ028862 across a broad panel of cancer cell lines in 2D culture (left) and in 3D culture (right). The 25 μM value represents the highest dose tested. Each value represents the mean of three replicates. Error bars represent the standard deviation (not readily visible on scale).</p