A universal strategy for high-yield production of soluble and functional clostridial collagenases in E. coli

Clostridial collagenases are foe and friend: on the one hand, these enzymes enable host infiltration and colonization by pathogenic clostridia, and on the other hand, they are valuable biotechnological tools due to their capacity to degrade various types of collagen and gelatine. However, the demand for high-grade preparations exceeds supply due to their pathogenic origin and the intricate purification of homogeneous isoforms. We present the establishment of an Escherichia coli expression system for a variety of constructs of collagenase G (ColG) and H (ColH) from Clostridium histolyticum and collagenase T (ColT) from Clostridium tetani, mimicking the isoforms in vivo. Based on a setup of five different expression strains and two expression vectors, 12 different constructs were expressed, and a flexible purification platform was established, consisting of various orthogonal chromatography steps adaptable to the individual needs of the respective variant. This fast, cost-effective, and easy-to-establish platform enabled us to obtain at least 10 mg of highly pure mono-isoformic protein per liter of culture, ideally suited for numerous sophisticated downstream applications. This production and purification platform paves the way for systematic screenings of recombinant collagenases to enlighten the biochemical function and to identify key residues and motifs in collagenolysis

Phosphonate as a Stable Zinc-Binding Group for "Pathoblocker" Inhibitors of Clostridial Collagenase H (ColH)

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

Crystallization and preliminary X-ray characterization of the catalytic domain of collagenase G from Clostridium histolyticum

The catalytic domain of collagenase G from C. histolyticum was expressed in E. coli BL21 (DE3) and purified using affinity and size-exclusion column-chromatographic methods. Crystals were obtained at 290 K by the sitting-drop vapour-diffusion method and diffraction data have been collected to 2.75 Å resolution

Phosphonate as Stable Zinc-binding Group for Inhibitors of Clostridial Collagenase H (ColH) as Pathoblocker Agents.

Microbial infections are a significant threat to public health and resistances are 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 catalyzes 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

Discovery of a Potent Inhibitor Class with High Selectivity toward Clostridial Collagenases.

Secreted virulence factors like bacterial collagenases are conceptually attractive targets for fighting microbial infections. However, previous attempts to develop potent compounds against these metalloproteases failed to achieve selectivity against human matrix metalloproteinases (MMPs). Using a surface plasmon resonance-based screening complemented with enzyme inhibition assays, we discovered an N-aryl mercaptoacetamide-based inhibitor scaffold that showed sub-micromolar affinities toward collagenase H (ColH) from the human pathogen Clostridium histolyticum. Moreover, these inhibitors also efficiently blocked the homologous bacterial collagenases, ColG from C. histolyticum, ColT from C. tetani, and ColQ1 from the Bacillus cereus strain Q1, while showing negligible activity toward human MMPs-1, -2, -3, -7, -8, and -14. The most active compound displayed a more than 1000-fold selectivity over human MMPs. This selectivity can be rationalized by the crystal structure of ColH with this compound, revealing a distinct non-primed binding mode to the active site. The non-primed binding mode presented here paves the way for the development of selective broad-spectrum bacterial collagenase inhibitors with potential therapeutic application in humans

Journal of the American Chemical Society / Discovery of a Potent Inhibitor Class with High Selectivity toward Clostridial Collagenases

Secreted virulence factors like bacterial collagenases are conceptually attractive targets for fighting microbial infections. However, previous attempts to develop potent compounds against these metalloproteases failed to achieve selectivity against human matrix metalloproteinases (MMPs). Using a surface plasmon resonance-based screening complemented with enzyme inhibition assays, we discovered an N-aryl mercaptoacetamide-based inhibitor scaffold that showed sub-micromolar affinities toward collagenase H (ColH) from the human pathogen Clostridium histolyticum. Moreover, these inhibitors also efficiently blocked the homologous bacterial collagenases, ColG from C. histolyticum, ColT from C. tetani, and ColQ1 from the Bacillus cereus strain Q1, while showing negligible activity toward human MMPs-1, -2, -3, -7, -8, and -14. The most active compound displayed a more than 1000-fold selectivity over human MMPs. This selectivity can be rationalized by the crystal structure of ColH with this compound, revealing a distinct non-primed binding mode to the active site. The non-primed binding mode presented here paves the way for the development of selective broad-spectrum bacterial collagenase inhibitors with potential therapeutic application in humans.(VLID)354723

N-Aryl-2-iso-butylmercaptoacetamides: the discovery of highly potent and selective inhibitors of Pseudomonas aeruginosa virulence factor LasB and Clostridium histolyticum virulence factor ColH

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