title: Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity.
abstract: The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X3X2X1-aminomethylcoumarin) and by determining specificity constants (kcat/Km), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X1 position, basic residues or Trp in the X2 position, and Pro ≫ Ala > Trp in the X3 position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents.
authors.affiliation: From the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Tatos
authors.initials: T
authors.lastname: Akopian
authors.affiliation: From the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Olga
authors.initials: O
authors.lastname: Kandror
authors.affiliation: Department of Biochemistry, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139.
authors.forename: Christopher
authors.initials: C
authors.lastname: Tsu
authors.affiliation: Department of Developmental, Molecular, and Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, and.
authors.forename: Jack H
authors.initials: JH
authors.lastname: Lai
authors.affiliation: Department of Developmental, Molecular, and Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, and.
authors.forename: Wengen
authors.initials: W
authors.lastname: Wu
authors.affiliation: Department of Developmental, Molecular, and Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, and.
authors.forename: Yuxin
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Developmental, Molecular, and Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, and.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhao
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts 02115.
authors.forename: Annie
authors.initials: A
authors.lastname: Park
authors.affiliation: From the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Lisa
authors.initials: L
authors.lastname: Wolf
authors.affiliation: Department of Biochemistry, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139.
authors.forename: Lawrence R
authors.initials: LR
authors.lastname: Dick
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts 02115.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Rubin
authors.affiliation: Department of Developmental, Molecular, and Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, and.
authors.forename: William
authors.initials: W
authors.lastname: Bachovchin
authors.affiliation: From the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, Alfred_Goldberg@hms.harvard.edu.
authors.forename: Alfred L
authors.initials: AL
authors.lastname: Goldberg
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D001897:Boronic Acids / Q000737:chemistry* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000254:growth & development; D009842:Oligopeptides / Q000737:chemistry* / Q000494:pharmacology; D019151:Peptide Library; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001897:Boronic Acids; D009097:Multienzyme Complexes; D009842:Oligopeptides; D019151:Peptide Library; D015842:Serine Proteinase Inhibitors; C119658:ClpP2 protein, bacteria; D012697:Serine Endopeptidases
keywords: Drug Development; Enzyme Inhibitor; Enzyme Mechanism; Infectious Disease; Peptide Chemical Synthesis; Protein Degradation