Latency, thermal stability, and identification of an inhibitory compound of mirolysin, a secretory protease of the human periodontopathogen Tannerella forsythia

• Main Authors: Krzysztof M. Zak, Mark J. Bostock, Irena Waligorska, Ida B. Thøgersen, Jan J. Enghild, Grzegorz M. Popowicz, Przemyslaw Grudnik, Jan Potempa, Miroslaw Ksiazek
• Format: Article
• Language: English
• Published: Taylor & Francis Group 2021-01-01
• Series: Journal of Enzyme Inhibition and Medicinal Chemistry
• Subjects: periodontitis; proteolysis; tannerella forsythia; nmr-based fragment screening; protease inhibitors
• Online Access: http://dx.doi.org/10.1080/14756366.2021.1937619
• ISSN: 1475-6366; 1475-6374

Mirolysin is a secretory protease of Tannerella forsythia, a member of the dysbiotic oral microbiota responsible for periodontitis. In this study, we show that mirolysin latency is achieved by a “cysteine-switch” mechanism exerted by Cys23 in the N-terminal profragment. Mutation of Cys23 shortened the time needed for activation of the zymogen from several days to 5 min. The mutation also decreased the thermal stability and autoproteolysis resistance of promirolysin. Mature mirolysin is a thermophilic enzyme and shows optimal activity at 65 °C. Through NMR-based fragment screening, we identified a small molecule (compound (cpd) 9) that blocks promirolysin maturation and functions as a competitive inhibitor (Ki = 3.2 µM), binding to the S1′ subsite of the substrate-binding pocket. Cpd 9 shows superior specificity and does not interact with other T. forsythia proteases or Lys/Arg-specific proteases.