Discovery of AMP mimetics that exhibit high inhibitory potency and specificity for AMP deaminase

The first potent, specific, and cell-penetrable AMP deaminase (AMPDA) inhib itors were discovered through an investigation of 3-substituted 3,6,7,8-tet rahydroinlidazo[4,5-d][ 1,3]diazepin-8-ol analogues. Inhibition constants f or the most potent inhibitors were 10(5)-fold lower than the KM for the sub strate AMP. High affinity required the presence of both the 8-hydroxyl and the 3-substituent and is postulated to arise from a cooperative interaction that reduces binding entropy costs and enables the diazepine base to adopt a binding conformation that mimics the transition-state (TS) structure. Th e high specificity of the inhibitor series for AMPDA relative to other AMP- binding enzymes (> 10(5)) is attributed in part to the diazepine base which favors interactions with residues used to stabilize the TS structure and p recludes interactions typically used by AMP-binding enzymes to bind AMP. In contrast, discrimination between AMPDA and adenosine deaminase (ADA), two enzymes postulated to stabilize a similar TS structure, is highly dependent on the 3-substituent. Replacement of the ribose group in the potent ADA in hibitor coformycin (K-i (ADA) = 10(-11) M vs K-i (AMPDA) = 3 x 10(-6) M) wi th 3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthylethyl led to a > 10(10)-fold change in specificity (K-i (ADA) > 10(-3) M vs K-i, (AMPDA) = 2 x 10(-9) M ). Inhibitors from the series readily penetrate cells and inhibit intracell ular AMPDA activity. Incubation of isolated rat hepatocytes with AMPDA inhi bitors had no effect on secondary metabolite levels during normoxic conditi ons but led to increased adenosine production and adenylate sparing under c onditions that induce net ATP breakdown. These results suggest that inhibit ors of AMPDA may represent site- and event-specific drugs that could preven t or attenuate ischemic tissue damage resulting from a stroke or a heart at tack.