Tricks for receptors applied to an enzyme: insights to MGAT2 inhibitors using binding assays with a novel probe

Acyl coenzyme A: monoacylglycerol acyltransferase 2 (MGAT2) catalyzes the committed step of the monoacylglycerol pathway for the synthesis of triacylglycerol in the small intestine.  MGAT2 is an emerging target for the treatment of metabolic diseases such as obesity, type 2 diabetes and dyslipidemia.  Multiple pharmaceutical companies have launched medicinal chemistry programs and several MGAT2 inhibitors have been described in the patent literature.  However, due to the membrane bound nature of the enzyme and the hydrophobicity of its substrates, in vitro assay techniques used for traditional kinetic studies on soluble enzymes cannot be applied. To circumvent this obstacle, we synthesized the novel, soluble radioligand [ 3 H]-Compound A and established steady state and kinetic binding assay protocols.   In the steady state radioligand displacement assay, diverse novel chemotypes that were identified in a high-throughput screen were found to compete binding of [ 3 H]-Compound A, indicating the broad utility of [ 3 H]-Compound A for testing various classes of MGAT2 inhibitors.  In the dynamic binding assays, the kinetic values of MGAT2 inhibitors such as K D , K on , K off and the residence times of inhibitors on MGAT2 enzyme were derived. Inasmuch as pharmacodynamic effects for enzyme inhibitors are driven by the physical interaction of the enzyme and inhibitors, the current binding assays provide a unique platform for identifying MGAT2 inhibitors of longer residence time as therapeutic candidates.