title: Investigation of the mycobacterial enzyme HsaD as a potential novel target for anti-tubercular agents using a fragment-based drug design approach.
abstract: OBJECTIVE
  With the emergence of extensively drug-resistant tuberculosis, there is a need for new anti-tubercular drugs that work through novel mechanisms of action. The meta cleavage product hydrolase, HsaD, has been demonstrated to be critical for the survival of Mycobacterium tuberculosis in macrophages and is encoded in an operon involved in cholesterol catabolism, which is identical in M. tuberculosis and M. bovis BCG.
  
  
  METHODS
  We generated a mutant strain of M. bovis BCG with a deletion of hsaD and tested its growth on cholesterol. Using a fragment based approach, over 1000 compounds were screened by a combination of differential scanning fluorimetry, NMR spectroscopy and enzymatic assay with pure recombinant HsaD to identify potential inhibitors. We used enzymological and structural studies to investigate derivatives of the inhibitors identified and to test their effects on growth of M. bovis BCG and M. tuberculosis.
  
  
  RESULTS
  The hsaD deleted strain was unable to grow on cholesterol as sole carbon source but did grow on glucose. Of seven chemically distinct 'hits' from the library, two chemical classes of fragments were found to bind in the vicinity of the active site of HsaD by X-ray crystallography. The compounds also inhibited growth of M. tuberculosis on cholesterol. The most potent inhibitor of HsaD was also found to be the best inhibitor of mycobacterial growth on cholesterol-supplemented minimal medium.
  
  
  CONCLUSIONS
  We propose that HsaD is a novel therapeutic target, which should be fully exploited in order to design and discover new anti-tubercular drugs.
  
  
  BACKGROUND
  This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc.
authors.affiliation: Faculty of Science, Engineering and Computing, Kingston University London, Kingston upon Thames, UK.
authors.forename: Ali
authors.identifier: http://orcid.org/0000-0002-4627-8185
authors.initials: A
authors.lastname: Ryan
authors.affiliation: Faculty of Science, Engineering and Computing, Kingston University London, Kingston upon Thames, UK.
authors.forename: Elena
authors.initials: E
authors.lastname: Polycarpou
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford, UK.|School of Medicine, Koç University, Istanbul, Turkey.
authors.forename: Nathan A
authors.initials: NA
authors.lastname: Lack
authors.affiliation: Mycobacteria Research Laboratory, Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London, London, UK.|Centre for Clinical Microbiology, University College London, Royal Free Campus, London, UK.|Mycobacterial Metabolism and Antibiotic Research Laboratory, The Francis Crick Institute, Mill Hill Laboratory, London, UK.
authors.forename: Dimitrios
authors.identifier: http://orcid.org/0000-0003-0472-7576
authors.initials: D
authors.lastname: Evangelopoulos
authors.affiliation: Faculty of Science, Engineering and Computing, Kingston University London, Kingston upon Thames, UK.
authors.forename: Christian
authors.initials: C
authors.lastname: Sieg
authors.affiliation: Faculty of Science, Engineering and Computing, Kingston University London, Kingston upon Thames, UK.
authors.forename: Alice
authors.initials: A
authors.lastname: Halman
authors.affiliation: Mycobacteria Research Laboratory, Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London, London, UK.
authors.forename: Sanjib
authors.initials: S
authors.lastname: Bhakta
authors.affiliation: Faculty of Science, Engineering and Computing, Kingston University London, Kingston upon Thames, UK.
authors.forename: Olga
authors.initials: O
authors.lastname: Eleftheriadou
authors.affiliation: Centre for Clinical Microbiology, University College London, Royal Free Campus, London, UK.
authors.forename: Timothy D
authors.identifier: http://orcid.org/0000-0003-4658-8594
authors.initials: TD
authors.lastname: McHugh
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford, UK.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Keany
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford, UK.
authors.forename: Edward D
authors.initials: ED
authors.lastname: Lowe
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford, UK.
authors.forename: Romain
authors.initials: R
authors.lastname: Ballet
authors.affiliation: School of Pharmacy, University of Jordan, Amman, Jordan.
authors.forename: Areej
authors.initials: A
authors.lastname: Abuhammad
authors.affiliation: Department of Microbiology and Immunology, Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: William R
authors.initials: WR
authors.lastname: Jacobs
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, UK.|Division of Biological Chemistry & Drug Discovery, School of Life Sciences, University of Dundee, James Black Centre, Dundee, UK.
authors.forename: Alessio
authors.identifier: http://orcid.org/0000-0002-8654-1670
authors.initials: A
authors.lastname: Ciulli
authors.affiliation: Faculty of Science, Engineering and Computing, Kingston University London, Kingston upon Thames, UK.|Department of Pharmacology, University of Oxford, Oxford, UK.
authors.forename: Edith
authors.identifier: http://orcid.org/0000-0002-1441-4322
authors.initials: E
authors.lastname: Sim
mesh_terms: D000995:Antitubercular Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D006867:Hydrolases / Q000037:antagonists & inhibitors* / Q000172:deficiency / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D015394:Molecular Structure; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D000995:Antitubercular Agents; D004791:Enzyme Inhibitors; D006867:Hydrolases; C527647:HsaD protein, Mycobacterium tuberculosis