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Tuberculosis is a global health problem caused by infection with the Mycobacterium tuberculosis (Mtb) bacteria. Although antibiotic treatment has dramatically reduced the impact of tuberculosis on the population, the existence and spreading of drug resistant strains urgently demands the development of new drugs that target Mtb in a different manner than currently used antibiotics. The prokaryotic ubiquitin-like protein (Pup) proteasome system is an attractive target for new drug development as it is unique to Mtb and related bacterial genera. Using a Pup-based fluorogenic substrate, we screened for inhibitors of Dop, the Mtb depupylating protease, and identified I-OMe-Tyrphostin AG538 (1) and Tyrphostin AG538 (2). The hits were validated and determined to be fast-reversible, non-ATP competitive inhibitors. We synthesized >25 analogs of 1 and 2 and show that several of the synthesized compounds also inhibit the depupylation actions of Dop on native substrate, FabD-Pup. Importantly, the pupylation activity of PafA, the sole Pup ligase in Mtb, was also inhibited by some of these compounds., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The Nedd8 activating enzyme (NAE) launches the transfer of the ubiquitin-like protein Nedd8 through an enzymatic cascade to covalently modify a diverse array of proteins, thus regulating their biological functions in the cell. The C-terminal peptide of Nedd8 extends deeply into the active site of NAE and plays an important role in the specific recognition of Nedd8 by NAE. We used phage display to profile C-terminal mutant sequences of Nedd8 that could be recognized by NAE for the activation reaction. We found that NAE can accommodate diverse changes in the Nedd8 C-terminal sequence (⁷¹ LALRGG⁷⁶), including Arg and Ile replacing Leu71, Leu, Ser, and Gln replacing Ala72, and substitutions by bulky aromatic residues at positions 73 and 74. We also observed that short peptides corresponding to the C-terminal sequences of the Nedd8 variants can be activated by NAE to form peptide~NAE thioester conjugates. Once NAE is covalently loaded with these Nedd8-mimicking peptides, it can no longer activate full-length Nedd8 for transfer to the neddylation targets, such as the cullin subunits of cullin-RING E3 ubiquitin ligases (CRLs). We have thus developed a new method to inhibit protein neddylation by Nedd8-mimicking peptides., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A litter of pups: The synthesis and in vitro evaluation of new Pup-based fluorogenic substrates for Dop, the mycobacterial depupylase, are described. A full-length Pup-amidomethylcoumarin conjugate as well as an amino-terminus-truncated analogue exhibited high sensitivity and specificity towards hydrolysis by Dop. The substrates developed here might find application as high-throughput screening assay reagents for the identification of Dop inhibitors., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)