Active site directed N-carboxymethyl peptide inhibitors of a soluble metalloendopeptidase from rat brain

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Abstract
A soluble metalloendopeptidase isolated from rat brain preferentially cleaves bonds in peptides having aromatic residues in the P1 and P2 position. An additional aromatic residue in the P3 position greatly increases the binding affinity of the substrate, suggesting the presence of an extended substrate recognition site in the enzyme, capable of binding a minimum of 5 amino acid residues. A series of N-carboxymethyl peptide derivatives structurally related to model substrates and containing a carboxylate group capable of coordinating with the active site Zn atom were synthesized and tested as potential inhibitors. One of these inhibitors, N-[1(RS)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, was a potent competitive inhibitor of the enzyme with a Ki of 1.94 .mu.M. The 2 diastereomers of this inhibitor were separated by high-pressure liquid chromatography. The more potent diastereomer had a Ki of 0.81 .mu.M. The inhibitory potency of the less active diastereomer was lower by 1 order of magnitude. Decreasing the hydrophobicity of the residue binding the S1 subsite of the enzyme by, for example, replacement of the phenylethyl group with a methyl residue decreased the inhibitory potency by almost 2 orders of magnitude. Deletion of the carboxylate group decreased the inhibitory potency by more than 3 orders of magnitude. Shortening the inhibitor chain by a single alanine residue had a similar effect. Binding of the inhibitor to the enzyme increased its thermal stability. The present data together with previous studies with synthetic and natural peptides support the conclusion that the active site of the enzyme contains 2 hydrophobic pockets at the S1 and S3'' subsites. As with other metalloendopeptidases, effective inhibition requires the presence in the inhibitor of a group binding to the substrate recognition site of the enzyme and a group capable of coordinating with the active site metal atom.