Structure–Efficiency Relationship of [1,2,4]Triazol-3-ylamines as Novel Nicotinamide Isosteres that Inhibit Tankyrases

Tankyrases 1 and 2 are members of the poly­(ADP-ribose) polymerase (PARP) family of enzymes that modulate Wnt pathway signaling. While amide- and lactam-based nicotinamide mimetics that inhibit tankyrase activity, such as XAV939, are well-known, herein we report the discovery and evaluation of a novel nicotinamide isostere that demonstrates selectivity over other PARP family members. We demonstrate the utilization of lipophilic efficiency-based structure–efficiency relationships (SER) to rapidly drive the evaluation of this series. These efforts led to a series of selective, cell-active compounds with solubility, physicochemical, and in vitro properties suitable for further optimization

Structure and Property Guided Design in the Identification of PRMT5 Tool Compound EPZ015666

The recent publication of a potent and selective inhibitor of protein methyltransferase 5 (PRMT5) provides the scientific community with <i>in vivo</i>-active tool compound <b>EPZ015666</b> (<b>GSK3235025</b>) to probe the underlying pharmacology of this key enzyme. Herein, we report the design and optimization strategies employed on an initial hit compound with poor <i>in vitro</i> clearance to yield <i>in vivo</i> tool compound <b>EPZ015666</b> and an additional potent <i>in vitro</i> tool molecule <b>EPZ015866</b> (<b>GSK3203591</b>)

EPZ011989, A Potent, Orally-Available EZH2 Inhibitor with Robust in Vivo Activity

Inhibitors of the protein methyltransferase Enhancer of Zeste Homolog 2 (EZH2) may have significant therapeutic potential for the treatment of B cell lymphomas and other cancer indications. The ability of the scientific community to explore fully the spectrum of EZH2-associated pathobiology has been hampered by the lack of in vivo-active tool compounds for this enzyme. Here we report the discovery and characterization of <b>EPZ011989</b>, a potent, selective, orally bioavailable inhibitor of EZH2 with useful pharmacokinetic properties. <b>EPZ011989</b> demonstrates significant tumor growth inhibition in a mouse xenograft model of human B cell lymphoma. Hence, this compound represents a powerful tool for the expanded exploration of EZH2 activity in biology

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

Identification of NVP-TNKS656: The Use of Structure–Efficiency Relationships To Generate a Highly Potent, Selective, and Orally Active Tankyrase Inhibitor

Tankyrase 1 and 2 have been shown to be redundant, druggable nodes in the Wnt pathway. As such, there has been intense interest in developing agents suitable for modulating the Wnt pathway in vivo by targeting this enzyme pair. By utilizing a combination of structure-based design and LipE-based structure efficiency relationships, the core of XAV939 was optimized into a more stable, more efficient, but less potent dihydropyran motif <b>7</b>. This core was combined with elements of screening hits <b>2</b>, <b>19</b>, and <b>33</b> and resulted in highly potent, selective tankyrase inhibitors that are novel three pocket binders. NVP-TNKS656 (<b>43</b>) was identified as an orally active antagonist of Wnt pathway activity in the MMTV-Wnt1 mouse xenograft model. With an enthalpy-driven thermodynamic signature of binding, highly favorable physicochemical properties, and high lipophilic efficiency, NVP-TNKS656 is a novel tankyrase inhibitor that is well suited for further in vivo validation studies

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

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

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