Structural and Kinetic Characterization of a Novel N-acetylated Aliphatic Amine Metabolite of the PRMT Inhibitor, EPZ011652

Abstract

Pharmacokinetic and metabolite identification studies were conducted to understand the clearance pathways of EPZ011652 [(2-aminoethyl)(methyl)({3-[4-(propan-2-yloxy)phenyl]-1H-pyrazol-4-yl} methyl)amine], a potent protein arginine N-methyltransferase inhibitor. Metabolic clearance was the major pathway of EPZ011652 elimination in rats with structural elucidation of metabolites via liquid chromatography -mass spectrometry (LC-MS n ) accurate mass measurement revealing the formation of a novel aliphatic N-acetylated metabolite (M1) located on the terminal nitrogen of the ethylenediamine side chain. EPZ015564, a synthetic standard of the N-acetyl product, was prepared and was also generated by human and rat, but not dog hepatocytes. In rat hepatocytes, on incubation with EPZ011652, the concentration of EPZ015564 initially increased before decreasing with incubation time, suggesting that the metabolite is itself a substrate for other metabolizing enzymes, in agreement with the identification of metabolites M2, M3, and M4 in rat bile, all N-acetylated metabolites, undergoing sequential phase I (demethylation, oxidation) or phase II (sulfation) reactions. Reaction phenotyping with recombinant human N-acetyltransferase (NAT) isoforms revealed that both NAT1 and NAT2 are capable of acetylating EPZ011652, although with different catalytic efficiencies. Kinetic profiles of EPZ015564 formation followed classic Michaelis-Menten behavior with apparent K m values of >1000 mM for NAT1 and 165 6 14.1 mM for NAT2. The in vitro intrinsic clearance for EPZ011652 by NAT2 (110 mL/min/mg) was 500-fold greater than by NAT1. In summary, we report the unusual N-acetylation of an aliphatic amine and discuss the implications for drug discovery and clinical development.

MetScore: Site of Metabolism Prediction Beyond Cytochrome P450 Enzymes

“…Molecules 18 and 23 show isoxazole ring cleavages. Compound 12 undergoes glucuronidation and sulfation after Ar−O−Ar ether cleavage (no examples in the training data) and molecule 14 undergoes the “unusual N‐acetylation of an aliphatic amine”. For epacadostat 8 , metabolite M11 results from gut microbiota and M12 is reported not to be observable in microsome or hepatocyte assays.…”

Beyond PAINs: Chemotype Sensitivity of Protein Methyltransferases in Screens

“…While analogues of the cofactor, S -adenosylmethionine (SAM), have been reported, the high polarity and resultant poor permeability of this adenosine chemotype may limit its utility in target validation using cell-based assays, although the DOT1L inhibitor, E-5676, clearly demonstrates that this can be overcome. For substrate competitive inhibition, the quinazolines associated with G9a and SETD8 and the methyllysine channel moiety found on PRMT inhibitors , are notable exceptions. As a consequence, likely “true” actives are not easily recognized based solely on biochemical activity, but require additional confirmation in orthogonal binding assays and/or biochemical mechanism of action (MoA) studies.…”