PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is essential for maturation of ribosomes, may have a role in lipogenesis, and is implicated in several diseases. A potent, selective, and cell- active PRMT3 inhibitor would be a valuable tool for further investigating PRMT3 biology. Here we report the discovery of the first PRMT3 chemical probe, SGC707, by structure-based optimization of the allosteric PRMT3 inhibitors we reported previously, and thorough characterization of this probe in biochemical, biophysical, and cellular assays. SGC707 is a potent PRMT3 inhibitor (IC50 = 31 ± 2 nm, KD = 53 ± 2 nm) with outstanding selectivity (selective against 31 other methyltransferases and more than 250 non-epigenetic targets). The mechanism of action studies and crystal structure of the PRMT3-SGC707 complex confirm the allosteric inhibition mode. Importantly, SGC707 engages PRMT3 and potently inhibits its methyltransferase activity in cells. It is also bioavailable and suitable for animal studies. This well- characterized chemical probe is an excellent tool to further study the role of PRMT3 in health and disease.

Dipeptidyl peptidase IV (DP-IV) is a cell surface serine dipeptidase that is involved in the regulation of the incretin hormones, glucagon-like peptide (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). There is accumulating evidence that other members of the glucagon family of peptides are also endogenous substrates for this enzyme. To identify candidate substrates for DP-IV, a mass spectrometry-based protease assay was developed that measures cleavage efficiencies (k cat /K m ) of polypeptides in a mixture, using only a few picomoles of each substrate and physiological amounts of enzyme in a single kinetic experiment. Oxyntomodulin and the growth hormone-(1-43) fragment were identified as new candidate in vivo substrates. Pituitary adenylate cyclase-activating polypeptide-(1-38) (PACAP38), a critical mediator of lipid and carbohydrate metabolism, was also determined to be efficiently processed by DP-IV in vitro. The catabolism of exogenously administered PACAP38 in wild type and DP-IVdeficient C57Bl/6 mice was monitored by tandem mass spectrometry. Animals lacking DP-IV exhibited a significantly slower clearance of the circulating peptide with virtually complete suppression of the inactive DP-IV metabolite, PACAP-(3-38). These in vivo results suggest that DP-IV plays a major role in the degradation of circulating PACAP38.

] These authors contributed equally to this work.Supporting information for this article (including detailed synthetic procedures and compound characterization as well as methods for scaffold hopping, crystallization, structure determination, bio-chemical assays, SPR, ITC, PRMT3 InCELL Hunter assay, cellular PRMT3 assay, cell viability assay, and mouse PK studies) is available on the WWW under http://dx

Precision medicine in oncology is the result of an increasing awareness of patient specific clinical features coupled with the development of genomic-based diagnostics and targeted therapeutics. Companion diagnostics designed for specific drug-target pairs were the first to widely utilize clinically applicable tumor biomarkers (e.g. HER2, EGFR), directing treatment for patients whose tumors exhibit a mutation susceptible to a FDA approved targeted therapy (e.g. trastuzumab, erlotinib). Clinically relevant germline mutations in drug metabolizing enzymes and transporters (e.g. TPMT, DPYD) have been shown to impact drug response, providing rationale for individualized dosing to optimize treatment. The use of multigene expression-based assays to analyze an array of prognostic biomarkers have been shown to help direct treatment decisions, especially in breast cancer (e.g. Oncotype DX). More recently, the use of Next-Generation Sequencing to detect many potential “actionable” cancer molecular alterations is further shifting the one gene-one drug paradigm towards a more comprehensive, multi-gene approach. Currently, many clinical trials (e.g. NCI-MATCH, NCI-MPACT) are assessing novel diagnostic tools with a combination of different targeted therapeutics, while also examining tumor biomarkers that were previously unexplored in a variety of cancer histologies. Results from ongoing trials like the NCI-MATCH will help determine the clinical utility and future development of the precision-medicine approach.

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