In light of the global antimicrobial-resistance crisis, there is an urgent need for novel bacterial targets and antibiotics with novel modes of action. It has been shown that Pseudomonas aeruginosa elastase (LasB) and Clostridium histolyticum ( Hathewaya histolytica ) collagenase (ColH) play a significant role in the infection process and thereby represent promising antivirulence targets. Here, we report novel N -aryl-3-mercaptosuccinimide inhibitors that target both LasB and ColH, displaying potent activities in vitro and high selectivity for the bacterial over human metalloproteases. Additionally, the inhibitors demonstrate no signs of cytotoxicity against selected human cell lines and in a zebrafish embryo toxicity model. Furthermore, the most active ColH inhibitor shows a significant reduction of collagen degradation in an ex vivo pig-skin model.

Extracellular virulence factors have emerged as attractive targets in the current antimicrobial resistance crisis. The Gram-negative pathogen Pseudomonas aeruginosa secretes the virulence factor elastase B (LasB), which plays an important role in the infection process. Here, we report a submicromolar, non-peptidic, fragment-like inhibitor of LasB discovered by careful visual inspection of structural data. Inspired by the natural LasB substrate, the original fragment was successfully merged and grown. The optimized inhibitor is accessible via simple chemistry and retained selectivity with a substantial improvement in activity, which can be rationalized by the crystal structure of LasB in complex with the inhibitor. We also demonstrate an improved in vivo efficacy of the optimized hit in Galleria mellonella larvae, highlighting the significance of this class of compounds as promising drug candidates.Alternative binding modes are often observed in the realm of fragment-based drug design. [1] Despite the potential to significantly accelerate hit-to-lead optimization, there are few examples of successful fragment linking/merging or systematic exploitation of such invaluable sources of structural information. This is presumably due to a number of conditions that need to be met such as the linker composition and the resulting ADMET properties. [2,3] We [

The development of drug resistance by Mycobacterium tuberculosis and other pathogenic bacteria emphasizes the need for new antibiotics. Unlike animals, most bacteria synthesize isoprenoid precursors through the MEP pathway. 1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the first reaction of the MEP pathway and is an attractive target for the development of new antibiotics. We report here the successful use of a loop truncation to crystallize and solve the first DXPS structures of a pathogen, namely M. tuberculosis (MtDXPS). The main difference found to other DXPS structures is in the active site where a highly coordinated water was found, showing a new mechanism for the enamine-intermediate stabilization. Unlike other DXPS structures, a “fork-like” motif could be identified in the enamine structure, using a different residue for the interaction with the cofactor, potentially leading to a decrease in the stability of the intermediate. In addition, electron density suggesting a phosphate group could be found close to the active site, provides new evidence for the D-GAP binding site. These results provide the opportunity to improve or develop new inhibitors specific for MtDXPS through structure-based drug design.

Target-directed dynamic combinatorial chemistry (tdDCC) enables identification, as well as optimization of ligands for un(der)explored targets such as the anti-infective target 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). We report the use of tdDCC...

Microbial infections are a significant threat to public health, and resistance is on the rise, so new antibiotics with novel modes of action are urgently needed. The extracellular zinc metalloprotease collagenase H (ColH) from Clostridium histolyticum is a virulence factor that catalyses tissue damage, leading to improved host invasion and colonisation. Besides the major role of ColH in pathogenicity, its extracellular localisation makes it a highly attractive target for the development of new antivirulence agents. Previously, we had found that a highly selective and potent thiol prodrug (with a hydrolytically cleavable thiocarbamate unit) provided efficient ColH inhibition. We now report the synthesis and biological evaluation of a range of zinc-binding group (ZBG) variants of this thiol-derived inhibitor, with the mercapto unit being replaced by other zinc ligands. Among these, an analogue with a phosphonate motif as ZBG showed promising activity against ColH, an improved selectivity profile, and significantly higher stability than the thiol reference compound, thus making it an attractive candidate for future drug development.

Kinetic target-guided synthesis represents an efficient hit-identification strategy,i nw hich the protein assembles its own inhibitors from ap oolo fc omplementary building blocks via an irreversible reaction. Herein, we pioneered an in situ Ugi reactionf or the identification of novel inhibitors of am odel enzyme and binders for an important drug target, namely,t he aspartic protease endothiapepsina nd the bacterial b-sliding clamp DnaN, respectively.H ighly sensitive mass-spectrometry methods enabledm onitoring of the protein-templated reaction of four complementaryr eaction partners, which occurredi nabackground-free manner for endothiapepsin or with ac lear amplification of two binders in the presence of DnaN. TheU gi products we identified show low micromolar activity on endothiapepsin or moderate affinity for the b-sliding clamp. We succeeded in expanding the portfolioo fc hemical reactions and biological targets and demonstrated the efficiency and sensitivity of this approach,which can find application on any drug target.

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Antimicrobial resistance is currently one of the serious global public health threats. Unlike the conventional antimicrobial drugs, antivirulence agents disarm rather than kill bacterial pathogens and therefore represent an alternative option to skirt the problem of resistance. Pseudomonas aeruginosa elastase (LasB) and Clostridium histolyticum collagenase (ColH) are extracellular bacterial proteases which play a critical role in the establishment and progression of the respective bacterial infection. In this study, we report the modulation of the α-position of the previously reported N aryl mercaptoacetamide class leading to a new type of highly potent LasB and ColH inhibitors (N aryl 2-iso-butylmercaptoacetamides). In addition to their non-toxicity and high selectivity over several human off-targets, selected derivatives may be considered unprecedented dual inhibitors of both LasB and ColH. Among the prepared derivatives, compound 37 showed the most promising properties: it had a favorable safety profile, maintained the viability and integrity of both skin- and lung-cells treated with P. aeruginosa supernatant, demonstrated in vivo efficacy in Galleria mellonella larvae, and revealed a good volume of distribution and moderate in vivo clearance in mice. Taking together, these results demonstrate that compound 37 is a promising candidate for antivirulence drug development.