A potent radiolabeled human renin inhibitor [3H]SR42128: enzymatic, kinetic, and binding studies to renin and other aspartic proteases

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Abstract
The in vitro binding of [3H]SR42128 (Iva-Phe-Nle-Sta-Ala-Sta-Arg), a potent inhibitor of human renin activity, to purified human renin and a number of other aspartic proteases was examined. SR42128 was found to be a competitive inhibitor of human renin, with a Ki of 0.35 nM at pH 5.7 and 2.0 nM at pH 7.4; it was thus more effective at pH 5.7 than at pH 7.4. Scatchard analysis of the interaction binding of [3H]SR42128 to human renin indicated that binding was reversible and saturable at both pH 5.7 and pH 7.4. There was a single class of binding sites, and the KD was 0.9 nM at pH 5.7 and 1 nM at pH 7.4. The association rate was 10 times more rapid at pH 5.7 than at pH 7.4, but there was no difference between the rates of dissociation of the enzyme-inhibitor complex at the two pHs. The effect of pH on the binding of [3H]SR42128 to human renin, cathepsin D, pepsin, and gastricsin was also examined over the pH range 3-8. All the aspartic proteases had a high affinity for the inhibitor at low pH. However, at pH 7.4, [3H]SR42128 was bound only to human renin and to none of the other aspartic proteases. Competitive binding studies with [3H]SR42128 and a number of other inhibitors on human renin or cathepsin D were used to examine the relationships between structure and activity in these systems. The study as a whole indicates that pH plays a major role in the binding of [3H]SR42128 to aspartic proteases and that the nature of the inhibitor residue reacting with the renin S2 subsites is of critical importance for the specificity of the renin-inhibitor interaction.

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