Arthur Van Aerschot

Tuberculosis (TB) remains a major health threat, while the increasing occurrence of drug‐resistant strains underscores the need for new antitubercular drugs. A promising strategy to combat TB is based on disrupting the mycobacterial redox homeostasis by inhibiting an NADPH‐dependent oxidoreductase, mycothione reductase (Mtr). Using high‐throughput screening, we recently identified potent and selective Mtr inhibitors. Here we report high‐resolution X‐ray structures of Mtr from Mycobacterium tuberculosis and Mycobacterium xenopi, including the M. tuberculosis enzyme complexed with a novel inhibitor, Respiri‐1093. Our findings demonstrate that Respiri‐1093 competes with the NADP(H) binding rather than mycothione binding. Analysis of the binding site explains the observed selectivity of the inhibitor towards the M. tuberculosis enzyme. These results provide a structural basis for rational drug design.

Mycothiol (MSH), the major cellular thiol in Mycobacterium tuberculosis (Mtb), plays an essential role in the resistance of Mtb to various antibiotics and oxidative stresses. MshC catalyzes the ATP-dependent ligation of 1-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol (GlcN-Ins) with l-cysteine (l-Cys) to form l-Cys-GlcN-Ins, the penultimate step in MSH biosynthesis. The inhibition of MshC is lethal to Mtb. In the present study, five new cysteinyl-sulfonamides were synthesized, and their binding affinity with MshC was evaluated using a thermal shift assay. Two of them bind the target with EC₅₀ values of 219 and 231 µM. Crystal structures of full-length MshC in complex with these two compounds showed that they were bound in the catalytic site of MshC, inducing dramatic conformational changes of the catalytic site compared to the apo form. In particular, the observed closure of the KMSKS loop was not detected in the published cysteinyl-sulfamoyl adenosine-bound structure, the latter likely due to trypsin treatment. Despite the confirmed binding to MshC, the compounds did not suppress Mtb culture growth, which might be explained by the lack of adequate cellular uptake. Taken together, these novel cysteinyl-sulfonamide MshC inhibitors and newly reported full-length apo and ligand-bound MshC structures provide a promising starting point for the further development of novel anti-tubercular drugs targeting MshC.

To correctly aminoacylate tRNA^Leu, leucyl-tRNA synthetase (LeuRS) catalyzes three reactions: activation of leucine by ATP to form leucyl-adenylate (Leu-AMP), transfer of this amino acid to tRNA^Leu and post-transfer editing of any mischarged product. Although LeuRS has been well characterized biochemically, detailed structural information is currently only available for the latter two stages of catalysis. We have solved crystal structures for all enzymatic states of Neisseria gonorrhoeae LeuRS during Leu-AMP formation. These show a cycle of dramatic conformational changes, involving multiple domains, and correlate with an energetically unfavorable peptide-plane flip observed in the active site of the pre-transition state structure. Biochemical analyses, combined with mutant structural studies, reveal that this backbone distortion acts as a trigger, temporally compartmentalizing the first two catalytic steps. These results unveil the remarkable effect of this small structural alteration on the global dynamics and activity of the enzyme.

Crystallographic screening with drug-like fragments was performed on main capsid protein VP1 of BK polyomavirus. 144 binding fragments clustering to six pockets were established, opening up a perspective of designing capsid assembly inhibitors.

Human cytosolic prolyl-tRNA synthetase (HcProRS) catalyses the formation of the prolyl-tRNA^Pro, playing an important role in protein synthesis. Inhibition of HcProRS activity has been shown to have potential benefits in the treatment of fibrosis, autoimmune diseases and cancer. Recently, potent pyrazinamide-based inhibitors were identified by a high-throughput screening (HTS) method, but no further elaboration was reported. The pyrazinamide core is a bioactive fragment found in numerous clinically validated drugs and has been subjected to various modifications. Therefore, we applied a virtual screening protocol to our in-house library of pyrazinamide-containing small molecules, searching for potential novel HcProRS inhibitors. We identified a series of 3-benzylaminopyrazine-2-carboxamide derivatives as positive hits. Five of them were confirmed by a thermal shift assay (TSA) with the best compounds 3b and 3c showing EC₅₀ values of 3.77 and 7.34 µM, respectively, in the presence of 1 mM of proline (Pro) and 3.45 µM enzyme concentration. Co-crystal structures of HcProRS in complex with these compounds and Pro confirmed the initial docking studies and show how the Pro facilitates binding of the ligands that compete with ATP substrate. Modelling 3b into other human class II aminoacyl-tRNA synthetases (aaRSs) indicated that the subtle differences in the ATP binding site of these enzymes likely contribute to its potential selective binding of HcProRS. Taken together, this study successfully identified novel HcProRS binders from our anti-tuberculosis in-house compound library, displaying opportunities for repurposing old drug candidates for new applications such as therapeutics in HcProRS-related diseases.

Aminoacyl-tRNA synthetases (aaRSs) catalyze the esterification of tRNA with a cognate amino acid and are essential enzymes in all three kingdoms of life. Due to their important role in the translation of the genetic code, aaRSs have been recognized as suitable targets for the development of small molecule anti-infectives. In this review, following a concise discussion of aaRS catalytic and proof-reading activities, the various inhibitory mechanisms of reported natural and synthetic aaRS inhibitors are discussed. Using the expanding repository of ligand-bound X-ray crystal structures, we classified these compounds based on their binding sites, focusing on their ability to compete with the association of one, or more of the canonical aaRS substrates. In parallel, we examined the determinants of species-selectivity and discuss potential resistance mechanisms of some of the inhibitor classes. Combined, this structural perspective highlights the opportunities for further exploration of the aaRS enzyme family as antimicrobial targets.

Aminoacyl-tRNA synthetases (aaRSs) have become viable targets for the development of antimicrobial agents due to their crucial role in protein translation. A series of six amino acids were coupled to the purine-like 7-amino-5-hydroxymethylbenzimidazole nucleoside analogue following an optimized synthetic pathway. These compounds were designed as aaRS inhibitors and can be considered as 1,3-dideazaadenine analogues carrying a 2-hydroxymethyl substituent. Despite our intentions to obtain N¹-glycosylated 4-aminobenzimidazole congeners, resembling the natural purine nucleosides glycosylated at the N⁹-position, we obtained the N³-glycosylated benzimidazole derivatives as the major products, resembling the respective purine N⁷-glycosylated nucleosides. A series of X-ray crystal structures of class I and II aaRSs in complex with newly synthesized compounds revealed interesting interactions of these “base-flipped” analogues with their targets. While the exocyclic amine of the flipped base mimics the reciprocal interaction of the N³-purine atom of aminoacyl-sulfamoyl adenosine (aaSA) congeners, the hydroxymethyl substituent of the flipped base apparently loses part of the standard interactions of the adenine N¹ and the N⁶-amine as seen with aaSA analogues. Upon the evaluation of the inhibitory potency of the newly obtained analogues, nanomolar inhibitory activities were noted for the leucine and isoleucine analogues targeting class I aaRS enzymes, while rather weak inhibitory activity against the corresponding class II aaRSs was observed. This class bias could be further explained by detailed structural analysis.