title: Phenyl Esters Are Potent Inhibitors of Caseinolytic Protease P and Reveal a Stereogenic Switch for Deoligomerization.
abstract: Caseinolytic protease P (ClpP) represents a central bacterial degradation machinery that is involved in cell homeostasis and pathogenicity. The functional role of ClpP has been studied by genetic knockouts and through the use of beta-lactones, which remain the only specific inhibitors of ClpP discovered to date. Beta-lactones have served as chemical tools to manipulate ClpP in several organisms; however, their potency, selectivity and stability is limited. Despite detailed structural insights into the composition and conformational flexibility of the ClpP active site, no rational efforts to design specific non-beta-lactone inhibitors have been reported to date. In this work, an unbiased screen of more than 137 000 compounds was used to identify five phenyl ester compounds as highly potent ClpP inhibitors that were selective for bacterial, but not human ClpP. The potency of phenyl esters largely exceeded that of beta-lactones in ClpP peptidase and protease inhibition assays and displayed unique target selectivity in living S. aureus cells. Analytical studies revealed that while phenyl esters are cleaved like native peptide substrates, they remain covalently trapped as acyl-enzyme intermediates in the active site. The synthesis of 36 derivatives and subsequent structure-activity relationship (SAR) studies provided insights into conserved structural elements that are important for inhibition potency and acylation reactivity. Moreover, the stereochemistry of a methyl-substituent at the alpha position to the ester, resembling amino acid side chains in peptide substrates, impacted ClpP complex stability, causing either dissociation into heptamers or retention of the tetradecameric state. Mechanistic insights into this intriguing stereo switch and the phenyl ester binding mode were obtained by molecular docking experiments.
authors.affiliation: †Center for Integrated Protein Science at the Department of Chemistry, Technische Universität München, Lichtenbergstrasse 4, Garching, D-85747, Germany.
authors.forename: Mathias W
authors.initials: MW
authors.lastname: Hackl
authors.affiliation: †Center for Integrated Protein Science at the Department of Chemistry, Technische Universität München, Lichtenbergstrasse 4, Garching, D-85747, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Lakemeyer
authors.affiliation: †Center for Integrated Protein Science at the Department of Chemistry, Technische Universität München, Lichtenbergstrasse 4, Garching, D-85747, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Dahmen
authors.affiliation: ‡Center for Integrated Protein Science at the Department of Life Sciences, Technische Universität München, Emil-Erlenmeyer-Forum 8, D-85354 Freising, Germany.
authors.forename: Manuel
authors.initials: M
authors.lastname: Glaser
authors.affiliation: †Center for Integrated Protein Science at the Department of Chemistry, Technische Universität München, Lichtenbergstrasse 4, Garching, D-85747, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Pahl
authors.affiliation: †Center for Integrated Protein Science at the Department of Chemistry, Technische Universität München, Lichtenbergstrasse 4, Garching, D-85747, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Lorenz-Baath
authors.affiliation: †Center for Integrated Protein Science at the Department of Chemistry, Technische Universität München, Lichtenbergstrasse 4, Garching, D-85747, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Menzel
authors.affiliation: §Max-Planck-Institut für Molekulare Physiologie, Abteilung Chemische Biologie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Sievers
authors.affiliation: ⊥Department of Chemistry, Universität Konstanz, Universitätsstrasse 10, D-78457 Konstanz, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Böttcher
authors.affiliation: ‡Center for Integrated Protein Science at the Department of Life Sciences, Technische Universität München, Emil-Erlenmeyer-Forum 8, D-85354 Freising, Germany.
authors.forename: Iris
authors.initials: I
authors.lastname: Antes
authors.affiliation: §Max-Planck-Institut für Molekulare Physiologie, Abteilung Chemische Biologie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany.|∥Technische Universität Dortmund, Fakultät für Chemie und Chemische Biologie, Otto-Hahn-Strasse 6, D-44221 Dortmund, Germany.
authors.forename: Herbert
authors.initials: H
authors.lastname: Waldmann
authors.affiliation: †Center for Integrated Protein Science at the Department of Chemistry, Technische Universität München, Lichtenbergstrasse 4, Garching, D-85747, Germany.
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Sieber
mesh_terms: D020134:Catalytic Domain; D002626:Chemistry, Pharmaceutical / Q000379:methods; D015195:Drug Design; D049071:Endopeptidase Clp / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D004952:Esters / Q000737:chemistry*; D006706:Homeostasis; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry*; D007610:Kallikreins / Q000737:chemistry*; D007700:Kinetics; D008089:Listeria monocytogenes / Q000201:enzymology; D062105:Molecular Docking Simulation; D010447:Peptide Hydrolases / Q000737:chemistry; D011487:Protein Conformation; D013211:Staphylococcus aureus / Q000201:enzymology*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
chemical_list: D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D004952:Esters; D007527:Isoenzymes; D010447:Peptide Hydrolases; D007610:Kallikreins; C083038:proteinase P; C483572:ClpP protease, E coli; C568646:ClpP protein, human; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp