Distinct Specificities of Mycobacterium tuberculosis and Mammalian Proteasomes for N-Acetyl Tripeptide Substrates

Gang Lin,Christopher Tsu,Lawrence Dick,Xi K Zhou,Carl Nathan

doi:10.1074/jbc.m805324200

Abstract

The proteasome of Mycobacterium tuberculosis (Mtb) is a validated and drug-treatable target for therapeutics. To lay ground-work for developing peptide-based inhibitors with a useful degree of selectivity for the Mtb proteasome over those of the host, we used a library of 5,920 N-acetyl tripeptide-aminomethylcoumarins to contrast the substrate preferences of the recombinant Mtb proteasome wild type and open gate mutant, the Rhodococcus erythropolis proteasome, and the bovine proteasome with activator PA28. The Mtb proteasome was distinctive in strictly preferring P1 = tryptophan, particularly in combination with P3 = glycine, proline, lysine or arginine. Screening results were validated with Michalis-Menten kinetic analyses of 21 oligopeptide aminomethyl-coumarin substrates. Bortezomib, a proteasome inhibitor in clinical use, and 17 analogs varying only at P1 were used to examine the differential impact of inhibitors on human and Mtb proteasomes. The results with the inhibitor panel confirmed those with the substrate panel in demonstrating differential preferences of Mtb and mammalian proteasomes at the P1 amino acid. Changing P1 in bortezomib from Leu to m-CF(3)-Phe led to a 220-fold increase in IC(50) against the human proteasome, whereas changing a P1 Ala to m-F-Phe decreased the IC(50) 400-fold against the Mtb proteasome. The change of a P1 Ala to m-Cl-Phe led to an 8000-fold shift in inhibitory potency in favor of the Mtb proteasome, resulting in 8-fold selectivity. Combinations of preferred amino acids at different sites may thus improve the species selectivity of peptide-based inhibitors that target the Mtb proteasome.

Highlights

Mycobacterium tuberculosis (Mtb),4 the single leading cause of death from bacterial infection, is growing even more danger
The Mtb proteasome is a nonredundant pathway by which Mtb protects itself against nitrosative stress and metabolic stringency in vitro [12], and it is essential for Mtb to survive in mice, even if they are immunodeficient [13]
This information, combined with the ability of small, drug-like compounds to enter Mtb, inhibit its proteasome and kill the bacterium [12], focuses interest on developing selective proteasome inhibitors as potential leads for chemotherapeutics

Summary

Introduction

Mycobacterium tuberculosis (Mtb), the single leading cause of death from bacterial infection, is growing even more danger-. The Mtb proteasome is emerging as a potential anti-tuberculosis drug target. It defends the bacterium against host nitrosative stress [12] and is essential for Mtb to persist in mice [13]. Prokaryotic proteasomes usually include only one or two types of ␣ and ␤ subunit, and the active sites are usually chymotrypsin-like with oligopeptide substrates. In the present work we used a mutant form of the recombinant Mtb proteasome (Mtb20SOG) in which deletion of the N-terminal octapeptide from the ␣ subunits mimics the presumed “open gate” configuration and increases specific activity toward oligopeptide substrates by approximately an order of magnitude [14]

Methods

Results

Conclusion