In vitro Evaluierung von Sigma:Core Hemmstoffen der bakteriellen RNA Polymerase

In order to identify and characterize novel inhibitors of bacterial RNA polymerase, an in vitro test system was developed. Therefore, Escherichia coli was used as model organism. The inhibitors should prohibit the protein-protein interaction of the βʹ subunit of the core enzyme of RNA polymerase and the dissociable σ70 factor, and inhibit thereby transcription. This inhibition mechanism shall prevent cross-resistances with clinically used Rifamycins and fidaxomicin. The test system consists of various binding and functional assays, of which most are based on the biophysical surface plasmon resonance technology. The variable assays allow on the one hand the determination of affinity and activity of the inhibitors, and on the other hand the determination of the mode of action. The binding mode was investigated by competition experiments with the σ70 factor and comparative binding studies to wild-type and point-mutated, truncated βʹ proteins. Within the scope of this work, three inhibitor classes of small molecules and one peptide were identified that effectively inhibited the σ70 – core RNA polymerase interaction. By the generation of structure-activity relationships as well as elucidation of the binding mode, the developed test system contributed to the optimization of the compounds.Zur Identifizierung und Charakterisierung neuer Hemmstoffe der bakteriellen RNA Polymerase als potentielle antibiotisch wirksame Arzneistoffe wurde ein in vitro Testsystem aufgebaut. Dabei wurde Escherichia coli als Modellorganismus verwendet. Die Hemmstoffe sollen die Protein-Protein Interaktion zwischen der βʹ Untereinheit des Core-Enzyms der RNA Polymerase und dem dissoziierbaren σ70 Faktor unterbinden und so die Transkription verhindern. Dadurch sollen Kreuzresistenzen mit den in der Klinik eingesetzten Rifamycinen und Fidaxomicin vermieden werden. Das Testsystem besteht aus zahlreichen Bindungs- und funktionalen Assays, die größtenteils auf der biophysikalischen Oberflächenplasmonresonanz-Technologie basieren. Die unterschiedlichen Tests erlauben zum einen die Bestimmung von Affinität und Aktivität, zum anderen die Bestimmung des Bindungsmodus. Der Bindungsmodus wird mit Hilfe von Kompetitionsversuchen mit dem σ70 Faktor und vergleichenden Bindungsstudien an Wildtyp und punktmutierten, verkürzten βʹ Proteinen untersucht. Im Rahmen dieser Arbeit konnten drei Wirkstoffklassen kleiner Moleküle und ein Peptid identifiziert werden, welche die σ70 – Core RNA Polymerase Interaktion effektiv hemmen. Durch die Generierung von Struktur-Wirkungsbeziehungen konnte das entwickelte Testsystem zur Optimierung der Verbindungen und zur Aufklärung von deren Bindungsmodi beitragen

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