Exploration of Neutral Endopeptidase Active Site by a Series of New Thiol-Containing Inhibitors

Article abstract of DOI:10.1021/jm00053a011

With the aim of characterizing the active site of the neutral endopeptidase and especially its putative S₁ subsite, two series of new thiol inhibitors designed to interact with the S₁, S'₁, and S'₂ subsites of the enzyme have been synthesized.These molecules correspond to the general formula HSCH(R₁)CH(R₂)CONHCH(R₃)COOH (series I) and HSCH(R₁)CH(R₂)CONHCH(R₃)CONHCH(R₄)COOH (series II).Due to the synthetic pathway used, these inhibitors were obtained as mixtures of four stereoisomers.HPLC separation of the stereoisomers of 17 HSCHCH(CH2Ph)CONHCH(CH3)COOH allowed the stereochemical dependence of the inhibitory potency to be determined.The most active isomer 17b (IC50 = 3.6 nM) is assumed to have the S,S,S stereochemistry as deduced from both NMR and HPLC data.Although none of the inhibitors obtained were significantly more active than thiorphan, HSCH2CH(CH2Ph)CONHCH2COOH (IC50 = 4 nM), which interacts only with the S'₁ and S'₂ subsites of NEP, their enhanced hydrophobicity is expected to improve their pharmacokinetic properties.All these compounds displayed low affinities for ACE (IC₅₀S > 1 μM).The determination of the IC₅₀S of two inhibitors of series II for NEP and for a mutated enzyme in which Arg¹⁰² was replaced by Glu¹⁰² allowed their mode of binding to the active site of NEP to be characterized.The R₂ and R₃ chains fit the S'₁-S'₂ subsites, while the R₄ group is probably located outside the active site.Taken together these results indicate that the R₁ chain of these inhibitors creates no additional stabilizing interactions with the active site of NEP.Two hypotheses may account for this: there is no hydrophophobic S₁ subsite in NEP or the inhibitors have structures which are too constrained for optimized interactions with the active site.