Fig 1.
Chemical structures of SMYD2 (A) and SMYD3 (B) inhibitors.
Fig 2.
Characterization of EPZ033294 as an inhibitor of SMYD2.
A) Representative SMYD2 biochemical dose-response curve for EPZ033294. IC50 value and standard deviation of 3.9 ± 0.3 nM was determined from 2 independent experiments. B) EPZ033294 IC50 values as a function of peptide concentration illustrating noncompetitive inhibition. IC50 values with their standard error and the fit line calculated from a single experiment. C) Structure of EPZ033294 (cyan) with SMYD2 (green) and SAM (yellow) (PDB ID 5V3H). Electron density (2Fo−Fc, 1σ) for the compound is shown. Hydrogen bonds are indicated as dashed lines. D) Western blot of BTF3 methylation showing dose dependent effects of EPZ033294 and a cell biochemical IC50 of 2.9 nM. Data shown is representative of three independent experiments.
Table 1.
Biochemical and cellular potencies and physicochemical properties of SMYD2 and SMYD3 inhibitors used in this study.
Fig 3.
Anti-proliferative activity of SMYD2 inhibitors.
(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.
Fig 4.
Characterization of EPZ028862 as an inhibitor of SMYD3.
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).
Fig 5.
Gene ablation techniques show no dependence on SMYD2 or SMYD3 for cancer cell proliferation.
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.