The synthesis and structure-activity relationship of a series of indole inhibitors of cytosolic phospholipase A2alpha (cPLA2alpha, type IVA phospholipase) are described. Inhibitors of cPLA2alpha are predicted to be efficacious in treating asthma as well as the signs and symptoms of osteoarthritis, rheumatoid arthritis, and pain. The introduction of a benzyl sulfonamide substituent at C2 was found to impart improved potency of these inhibitors, and the SAR of these sulfonamide analogues is disclosed. Compound 123 (Ecopladib) is a sub-micromolar inhibitor of cPLA2alpha in the GLU micelle and rat whole blood assays. Compound 123 displayed oral efficacy in the rat carrageenan air pouch and rat carrageenan-induced paw edema models.

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 EPZ031686 and sulfamide EPZ030456, with cellular potency at a level sufficient to probe the in vitro biology of SMYD3 inhibition. EPZ031686 shows good bioavailability following oral dosing in mice making it a suitable tool for potential in vivo target validation studies. KEYWORDS: SMYD3, oxindole, methyltransferase, KMT, oncology, tool compound S et and Mynd Domain containing 3 (SMYD3) is a lysine methyltransferase (KMT) expressed at high levels in a number of different cancer histologies and is associated with a poor clinical prognosis. 1−10 While no single mechanism has emerged to explain this correlation, a number of studies have implicated SMYD3 in the regulation of gene transcription and signal transduction pathways critical for cell survival in breast, liver, prostate, pancreatic, and lung cancer models. 4,7−9 In addition, considerable evidence has been reported in the literature showing that genetic knockdown of SMYD3 leads to a decrease in proliferation of a variety of cancer cell lines. 4,[7][8][9]11 Two studies, employing RNAi-based technologies, have shown that ablation of SMYD3 in hepatocellular carcinoma cell lines greatly reduces cell viability and that its pro-oncogenic role is dependent on its catalytic activity. 7,9 Moreover, SMYD3 has also been shown to be a critical mediator of transformation induced by a KRAS gain-of-function mutation in both pancreatic and lung adenocarcinoma mouse models; these models were likewise dependent on the catalytic activity of SMYD3. 11 The biological function of SMYD3 is still poorly understood. Early studies of SMYD3 suggested that its primary function is to methylate histones. Indeed, several reports have indicated that SMYD3 modifies histone H3 on lysine 4, 9,12 but have also identified a novel modification of histone H4 on lysine 5. 7 The results of these studies have not yet yielded a clear picture of how SMYD3 might be regulating chromatin, but a recent study has strongly implicated SMYD3 as a direct regulator of MAPK pathways in the cytoplasm and not as a regulator of transcription. MAP3K2 (MEKK2) was shown to be trimethylated at lysine 260 by SMYD3. Modification of this residue leads to enhanced downstream MAPK activation and appears to be critical for mutant KRAS driven oncogenesis. 11 SMYD3's role in cancer cell line proliferation, its effect on known oncogenic signal transduction pathways, and the association of SMYD3 mRNA expression with aggressive transformed phenotypes make SMYD3 an attractive target for therapeutic intervention. We report here the first potent and selective small molecule inhibitors suitable for target validation studies.Compound 1 was identified as a mi...

Compound 1 was previously reported to be a potent inhibitor of cPLA(2)alpha in both artificial monomeric substrate and cell-based assays. However, 1 was inactive in whole blood assays previously used to characterize cyclooxygenase and lipoxygenase inhibitors. The IC(50) of 1 increased dramatically with cell number or lipid/detergent concentration. In an attempt to insert an electrophilic ketone between the indole and benzoic acid moieties, we discovered that increasing the distance between the two moieties gave a compound with activity in the GLU (7-hydroxycoumarinyl-gamma-linolenate) micelle assay, which contains lipid and detergent. Extensive structure-activity relationship work around this lead identified a potent pharmacophore for cPLA(2)alpha inhibition. The IC(50)s between the GLU micelle and rat whole blood assays correlated highly. No correlation was found for other parameters, including lipophilicity or acidity of the required acid functionality. Compounds 25, 39, and 94 emerged as potent, selective inhibitors of cPLA(2)alpha and represent well-validated starting points for further optimization.

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.

The optimization of a class of indole cPLA 2 alpha inhibitors is described herein. The importance of the substituent at C3 and the substitution pattern of the phenylmethane sulfonamide region are highlighted. Optimization of these regions led to the discovery of 111 (efipladib) and 121 (WAY-196025), which are shown to be potent, selective inhibitors of cPLA 2 alpha in a variety of isolated enzyme assays, cell based assays, and rat and human whole blood assays. The binding of these compounds has been further examined using isothermal titration calorimetry. Finally, these compounds have shown efficacy when dosed orally in multiple acute and chronic prostaglandin and leukotriene dependent in vivo models.

The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. AbstractA rapid carboxylic acid-promoted lactone aminolysis is reported. A number of carboxylic acids were found to promote this amide bond-forming transformation, with aliphatic acids being the most efficient.This reaction is an equilibrium process (K eq ~ 1.8), and mechanistic investigations are consistent with mediation of a kinetically important proton-transfer step by the carboxylate, i. e., the conjugate base of the acid employed.

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SET domain-containing protein 2 (SETD2), a histone methyltransferase, has been identified as a target of interest in certain hematological malignancies, including multiple myeloma. This account details the discovery of EPZ-719 , a novel and potent SETD2 inhibitor with a high selectivity over other histone methyltransferases. A screening campaign of the Epizyme proprietary histone methyltransferase-biased library identified potential leads based on a 2-amidoindole core. Structure-based drug design (SBDD) and drug metabolism/pharmacokinetics (DMPK) optimization resulted in EPZ-719 , an attractive tool compound for the interrogation of SETD2 biology that enables in vivo target validation studies.