Mechanism of Selective Enzyme Inhibition through Uncompetitive Regulation of an Allosteric Agonist.

Classical uncompetitive inhibitors are potent pharmacological modulators of enzyme function. Since they selectively target enzyme-substrate complexes (E:S), their inhibitory potency is amplified by increasing substrate concentrations. Recently, an unconventional uncompetitive inhibitor, called CE3F4R, was discovered for the exchange protein activated by cAMP isoform 1 (EPAC1). Unlike conventional uncompetitive inhibitors, CE3F4R is uncompetitive with respect to an allosteric effector, cAMP, as opposed to the substrate (i.e., CE3F4R targets the E:cAMP rather than the E:S complex). However, the mechanism of CE3F4R as an uncompetitive inhibitor is currently unknown. Here, we elucidate the mechanism of CE3F4R's action using NMR spectroscopy. Due to limited solubility and line broadening, which pose major challenges for traditional structural determination approaches, we resorted to a combination of protein- and ligand-based NMR experiments to comparatively analyze EPAC mutations, inhibitor analogs, and cyclic nucleotide derivatives that trap EPAC at different stages of activation. We discovered that CE3F4R binds within the EPAC cAMP-binding domain (CBD) at a subdomain interface distinct from the cAMP binding site, acting as a wedge that stabilizes a cAMP-bound mixed-intermediate. The mixed-intermediate includes attributes of both the apo/inactive and cAMP-bound/active states. In particular, the intermediate targeted by CE3F4R traps a CBD's hinge helix in its inactive conformation, locking EPAC into a closed domain topology that restricts substrate access to the catalytic domain. The proposed mechanism of action also explains the isoform selectivity of CE3F4R in terms of a single EPAC1 versus EPAC2 amino acid difference that destabilizes the active conformation of the hinge helix.