title: Identification of a novel inhibitor of isocitrate lyase as a potent antitubercular agent against both active and non-replicating Mycobacterium tuberculosis.
abstract: OBJECTIVE
  Screen and identify novel inhibitors of isocitrate lyase (ICL) as potent antitubercular agents against Mycobacterium tuberculosis and determine their inhibitory characteristics, antitubercular activities and mechanisms of action.
  
  
  METHODS
  Recombinant ICL of M. tuberculosis was expressed and purified, which was used for high-throughput screening (HTS) and the following experiments. A total of 71,765 compounds were screened to identify ICL inhibitors which were then evaluated for their roles as potent antitubercular agents. To determine the inhibitory characteristics of the agents against latent M. tuberculosis in persistent infections, a macrophage model (mouse J774A.1 cell) infected with Mycobacterium marinum BAA-535 strain was built and assessed. The potent antitubercular agents were identified using the macrophage model. Then, the inhibitory intensity and mode of the agents that exhibit on ICL protein of M. tuberculosis were analyzed, and the interaction mechanisms were preliminarily clarified according to the parameters of enzyme kinetics, circular dichroism experiments, fluorescence quenching assay, and molecular docking.
  
  
  RESULTS
  The previously established ICL inhibitor screening model was evaluated to be suitable for HTS assay. Of the 71,765 compounds, 13 of them were identified to inhibit ICL effectively and stably. IMBI-3 demonstrated the most significant inhibitory activity with IC50 of 30.9 μmol/L. Its minimum inhibitory concentration (MIC) for M. tuberculosis, including extensively drug-resistant tuberculosis (XDR-TB) and multidrug-resistant tuberculosis (MDR-TB), were determined in the range of 0.25-1 μg/mL. When IMBI-3 is used in combination with isoniazid, the colony-forming units (CFU) counting of latent M. tuberculosis in J774A.1 macrophage cells decreased significantly as IMBI-3 concentration increased. The inhibition mode of IMBI-3 on ICL was probably competitive inhibition with an inhibition constant (Ki) of approximate 1.85 μmol/L. The interaction between IMBI-3 and ICL of M. tuberculosis was also confirmed by circular dichroism experiments and fluorescence quenching assay. And seven possible active amino acids of ICL of M. tuberculosis were identified in the active site through molecular docking.
  
  
  CONCLUSIONS
  IMBI-3, a novel potent antitubercular agent targeting ICL of M. tuberculosis, was identified and evaluated. It inhibited both log-phase M. tuberculosis in vitro and dormant M. tuberculosis in macrophages. It was the first representative compound of this family with the ICL enzyme inhibition and antimycobacterial activities.
authors.affiliation: The National Laboratory for Screening New Microbial Drugs, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
authors.forename: Yishuang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Zhou
authors.affiliation: The National Laboratory for Screening New Microbial Drugs, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Deng
authors.affiliation: The National Laboratory for Screening New Microbial Drugs, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
authors.forename: Xinghua
authors.initials: X
authors.lastname: Li
authors.affiliation: The National Laboratory for Screening New Microbial Drugs, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
authors.forename: Jianzhou
authors.initials: J
authors.lastname: Meng
authors.affiliation: The National Laboratory for Screening New Microbial Drugs, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Guan
authors.affiliation: The Laboratory of Bacterial Immunology, Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, China.
authors.forename: Chuanyou
authors.initials: C
authors.lastname: Li
authors.affiliation: The National Laboratory for Screening New Microbial Drugs, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China. Electronic address: xiaocl318@163.com.
authors.forename: Chunling
authors.initials: C
authors.lastname: Xiao
mesh_terms: D000995:Antitubercular Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery; D004359:Drug Therapy, Combination; D057166:High-Throughput Screening Assays; D006801:Humans; D007522:Isocitrate Lyase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D055985:Latent Tuberculosis / Q000188:drug therapy* / Q000382:microbiology; D008264:Macrophages / Q000382:microbiology*; D062105:Molecular Docking Simulation; D009165:Mycobacterium Infections, Nontuberculous / Q000188:drug therapy* / Q000382:microbiology; D019910:Mycobacterium marinum / Q000187:drug effects* / Q000201:enzymology / Q000254:growth & development; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology / Q000254:growth & development; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D007522:Isocitrate Lyase
keywords: Antitubercular agent; Inhibitor; Isocitrate lyase; Latent; Mycobacterium tuberculosis