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.

Surface plasmon resonance--more than a screening technology: insights in the binding mode of σ70:core RNAP inhibitors.

AIM: Antibiotic resistance has become a major health problem. The σ(70):core interface of bacterial RNA polymerase is a promising drug target. Recently, the coiled-coil and lid-rudder-system of the ß' subunit has been identified as an inhibition hot spot. Materials & methods & Results: By using surface plasmon resonance-based assays, inhibitors of the protein-protein interaction were identified and competition with σ(70) was shown. Effective inhibition was verified in an in vitro transcription and a σ(70):core assembly assay. For one hit series, we found a correlation between activity and affinity. Mutant interaction studies suggest the inhibitors' binding site. CONCLUSION: Surface plasmon resonance is a valuable technology in drug design, that has been used in this study to identify and evaluate σ(70):core RNA polymerase inhibitors.

Discovery of novel bacterial RNA polymerase inhibitors: pharmacophore-based virtual screening and hit optimization.

The bacterial RNA polymerase (RNAP) is a validated target for broad spectrum antibiotics. However, the efficiency of drugs is reduced by resistance. To discover novel RNAP inhibitors, a pharmacophore based on the alignment of described inhibitors was used for virtual screening. In an optimization process of hit compounds, novel derivatives with improved in vitro potency were discovered. Investigations concerning the molecular mechanism of RNAP inhibition reveal that they prevent the protein-protein interaction (PPI) between σ(70) and the RNAP core enzyme. Besides of reducing RNA formation, the inhibitors were shown to interfere with bacterial lipid biosynthesis. The compounds were active against Gram-positive pathogens and revealed significantly lower resistance frequencies compared to clinically used rifampicin.

Peptide-based investigation of the Escherichia coli RNA polymerase σ(70):core interface as target site.

The number of bacterial strains that are resistant against antibiotics increased dramatically during the past decades. This fact stresses the urgent need for the development of new antibacterial agents with novel modes of action targeting essential enzymes such as RNA polymerase (RNAP). Bacterial RNAP is a large multi-subunit complex consisting of a core enzyme (subunits: α(2)ßß'ω) and a dissociable sigma factor (σ(70); holo enzyme: α(2)ßß'ωσ(70)) that is responsible for promoter recognition and transcription initiation. The interface between core RNAP and σ(70) represents a promising binding site. Nevertheless, detailed studies investigating its druggability are rare. Compounds binding to this region could inhibit this protein-protein interaction and thus holo enzyme formation, resulting in inhibition of transcription initiation. Sixteen peptides covering different regions of the Escherichia coli σ(70):core interface were designed; some of them-all derived from σ(70) 2.2 region-led to a strong RNAP inhibition. Indeed, an ELISA-based experiment confirmed the most active peptide P07 to inhibit the σ(70):core interaction. Furthermore, an abortive transcription assay revealed that P07 impedes transcription initiation. In order to study the mechanism of action of P07 in more detail, molecular dynamics simulations and a rational amino acid replacement study were performed, leading to the conclusion that P07 binds to the coiled-coil region in ß' and that its flexible N-terminus inhibits the enzyme by interaction with the ß' lid-rudder-system (LRS). This work revisits the ß' coiled-coil as a hot spot for the protein-protein interaction inhibition and expands it by introduction of the LRS as target site.

Mutation in elongation factor G confers resistance to the antibiotic argyrin in the opportunistic pathogen Pseudomonas aeruginosa.

The natural myxobacterial product argyrin is a cyclic peptide exhibiting immunosuppressive activity as well as antibacterial activity directed against the highly intrinsically resistant opportunistic pathogen Pseudomonas aeruginosa. In this study, we used whole-genome sequencing technology as a powerful tool to determine the mode of action of argyrin. Sequencing of argyrin-resistant P. aeruginosa isolates selected in vitro uncovered six point mutations that distinguished the resistant mutants from their susceptible parental strain. All six mutations were localized within one gene: fusA1, which encodes for the elongation factor EF-G. After the reintroduction of selected mutations into the susceptible wild type, the strain became resistant to argyrin. Surface plasmon resonance experiments confirmed the interaction of argyrin A with FusA1. Interestingly, EF-G has been previously shown to be the target of the anti-Staphylococcus antibiotic fusidic acid. Mapping of the mutations onto a structural model of EF-G revealed that the mutations conveying resistance against argyrin were clustered within domain III on the side opposite to that involved in fusidic acid binding, thus indicating that argyrin exhibits a new mode of protein synthesis inhibition. Although no mutations causing argyrin resistance have been found in other genes of P. aeruginosa, analysis of the sequence identity in EF-G and its correlation with argyrin resistance in different bacteria imply that additional factors such as uptake of argyrin play a role in the argyrin resistance of other organisms.

Influence of DNA template choice on transcription and inhibition of Escherichia coli RNA polymerase.

In recent decades, quantitative transcription assays using bacterial RNA polymerase (RNAP) have been performed under widely diverse experimental conditions. We demonstrate that the template choice can influence the inhibitory potency of RNAP inhibitors. Furthermore, we illustrate that the sigma factor (σ(70)) surprisingly increases the transcription efficiency of templates with nonphysiological nonprokaryotic promoters. Our results might be a useful guideline in the early stages of using RNAP for drug discovery.

A protein arginine N-methyltransferase 1 (PRMT1) and 2 heteromeric interaction increases PRMT1 enzymatic activity.

Protein arginine N-methyltransferases (PRMTs) act in signaling pathways and gene expression by methylating arginine residues within target proteins. PRMT1 is responsible for most cellular arginine methylation activity and can work independently or in collaboration with other PRMTs. In this study, we demonstrate a direct interaction between PRMT1 and PRMT2 using co-immunoprecipitation, bimolecular fluorescence complementation, and enzymatic assays. As a result of this interaction, PRMT2 stimulated PRMT1 activity, affecting its apparent V(max) and K(M) values in vitro and increasing the production of methylarginines in cells. Active site mutations and regional deletions from PRMT1 and -2 were also investigated, which demonstrated that complex formation required full-length, active PRMT1. Although the inhibition of methylation by adenosine dialdehyde prevented the interaction between PRMT1 and -2, it did not prevent the interaction between PRMT1 and a truncation mutant of PRMT2 lacking its Src homology 3 (SH3) domain. This result suggests that the SH3 domain may mediate an interaction between PRMT1 and -2 in a methylation-dependent fashion. On the basis of our findings, we propose that PRMT1 serves as the major methyltransferase in cells by forming higher-order oligomers with itself, PRMT2, and possibly other PRMTs.