Mechanistic Characterization of Long Residence Time Inhibitors of Diacylglycerol Acyltransferase 2 (DGAT2).

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in triacylglycerol (TAG) synthesis. Genetic knockdown or pharmacological inhibition of DGAT2 leads to a decrease in very-low-density lipoprotein TAG secretion and hepatic lipid levels in rodents, indicating DGAT2 may represent an attractive therapeutic target for treatment of hyperlipidemia and hepatic steatosis. We have previously described potent and selective imidazopyridine DGAT2 inhibitors with high oral bioavailability. However, the detailed mechanism of DGAT2 inhibition has not been reported. Herein, we describe imidazopyridines represented by PF-06424439 (1) and 2 as long residence time inhibitors of DGAT2. We demonstrate that 1 and 2 are slowly reversible, time-dependent inhibitors, which inhibit DGAT2 in a noncompetitive mode with respect to the acyl-CoA substrate. Detailed kinetic analysis demonstrated that 1 and 2 inhibit DGAT2 in a two-step binding mechanism, in which the initial enzyme-inhibitor complex (EI) undergoes an isomerization step resulting in a much higher affinity complex (EI*) with overall apparent inhibition constants ( K_i*^app values) of 16.7 and 16.0 nM for 1 and 2, respectively. The EI* complex dissociates with dissociation half-lives of 1.2 and 1.0 h for 1 and 2, respectively. A binding assay utilizing ¹²⁵I-labeled imidazopyridine demonstrated that the level of imidazopyridine binding to DGAT2 mutant enzymes, H161A and H163A, dramatically decreased to 11-17% of that of the wild-type enzyme, indicating that these residues are critical for imidazopyridines to bind to DGAT2. Taken together, imidazopyridines may thus represent a promising lead series for the development of DGAT2 inhibitors that display an unprecedented combination of potency, selectivity, and in vivo efficacy.

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