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Thimet oligopeptidase (TOP, EC 3.4.24.15) and neurolysin (NEL, EC 3.4.24.16) are closely related zinc-dependent metalo-oligopeptidases, which take part in the metabolism of oligopeptides (from 5 to 17 amino acid residues) inside and outside cells. Both peptidases are ubiquitously distributed in tissues. TOP is one of the main intracellular peptide-processing enzymes being important for the antigen selection in the MHC Class I presentation route, while NEL function has been more associated with the extracellular degradation of neurotensin. Despite efforts being made to develop specific inhibitors for these peptidases, the most used are: CPP-Ala-Ala-Tyr-PABA, described by Orlowski et al. in 1988, and CPP-Ala-Aib-Tyr-PABA (JA-2) that is an analog more resistant to proteolysis, which development was made by Shrimpton et al. in 2000. In the present work, we describe other analogs of these compounds but, with better discriminatory capacity to inhibit specifically NEL or TOP. The modifications introduced in these new analogs were based on a key difference existent in the extended binding sites of NEL and TOP: the negatively charged Glu469 residue of TOP corresponds to the positively charged Arg470 residue of NEL. These residues are in position to interact with the residue at the P 1 ′ and/or P 2 ′ of their substrates (mimicked by the Ala-Ala/P1′-P2′ residues of the CPP-Ala-Ala-Tyr-PABA). Therefore, exploring this single difference, the following compounds were synthesized: CPP- Asp -Ala-Tyr-PABA, CPP- Arg -Ala-Tyr-PABA, CPP-Ala- Asp -Tyr-PABA, CPP-Ala- Arg -Tyr-PABA. Confirming the predictions, the replacement of each non-charged residue of the internal portion Ala-Ala by a charged residue Asp or Arg resulted in compounds with higher selectivity for NEL or TOP, especially due to the electrostatic attraction or repulsion by the NEL Arg470 or TOP Glu469 residue. The CPP-Asp-Ala-Tyr-PABA and CPP-Ala-Asp-Tyr-PABA presented higher affinities for NEL, and, the CFP-Ala-Arg-Tyr-PABA showed higher affinity for TOP. • Electrostatic differences mapped at the NEL and TOP binding sites were used to design new selective inhibitors. • Inhibitors containing Asp residues at P 1 ′ and P 2 ′ positions presented higher selectivity to NEL. • Inhibitors containing Arg residues at P 1 ′ and P 2 ′ positions presented higher selectivity to TOP. • NEL R470E and TOPE469R mutants confirmed the major contribution of this site for inhibitor binding. • Molecular docking supported the results and provided directions for future developments. [ABSTRACT FROM AUTHOR]

Abstract: Thimet oligopeptidase (EC 3.4.24.15, TOP) is a metallo-oligopeptidase that participates in the intracellular metabolism of peptides. Predictions based on structurally analogous peptidases (Dcp and ACE-2) show that TOP can present a hinge-bend movement during substrate hydrolysis, what brings some residues closer to the substrate. One of these residues that in TOP crystallographic structure are far from the catalytic residues, but, moves toward the substrate considering this possible structural reorganization is His600. In the present work, the role of His600 of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S1 and S1′ specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His600 residue makes important interactions with the substrate, supporting the prediction that His600 moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both K m and k cat, showing the importance of His600 for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His600 in TOP catalysis, transferring a proton to the newly generated NH2-terminus or helping Tyr605 and/or Tyr612 in the intermediate oxyanion stabilization. [Copyright &y& Elsevier]