The most potent low molecular weight inhibitors of pancreatic RNase superfamily enzymes reported to date are synthetic derivatives of adenosine 5'-pyrophosphate. Here we have investigated the effects of six natural nucleotides that also incorporate this moiety (NADP(-), NADPH, ATP, Ap(3)A. Ap(4)A. and Ap(5)A) on the activities of RNase A and two of its homologues, eosinophil-derived neurotoxin and angiogenin. With eosinophil-derived neurotoxin and angiogenin, Ap5A is comparable to the tightest binding inhibitors identified preciously (K-i values at pH 5.9 are 370 nM and 100 muM, respectively): it ranks among the strongest small antagonists of RNase A as well (K-i = 230 nM). The K-i for NADPH with angiogenin is similar to that of Ap(5)A. These findings suggest that Ap(5)A and NADPH may serve as useful new leads for inhibitor design, Examination of inhibition under physiological conditions indicates that NADPH. ATP. and Ap(5)A may suppress intracellular RNase activity significantly in vivo. (C) 2002 Elsevier Science (USA). All rights reserved.