Investigation of the mycobacterial enzyme HsaD as a potential novel target for anti‐tubercular agents using a fragment‐based drug design approach

Abstract

BACKGROUND AND PURPOSEWith 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. EXPERIMENTAL APPROACHWe 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. KEY RESULTSThe 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 AND IMPLICATIONSWe propose that HsaD is a novel therapeutic target, which should be fully exploited in order to design and discover new antitubercular drugs.

Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis

“…The hydrolase HsaD was first described as participating in cholesterol catabolism 53 and then found to be essential for intramacrophage survival of M. tb 51 . HsaD has recently been proposed as a novel therapeutic target and awaits further developments 54 .…”

“…The increased MIC 50 value of the strain overexpressing HsaD prompted us to explore the potential interactions occurring at the enzyme’s active site following CyC 17 binding. In silico molecular docking experiments were conducted, as described previously 17 using the recently reported crystal structures of HsaD bound to three different inhibitors 54 : 3,5-dichloro-4-hydroxybenzoic acid (PDB id: 5JZS), 3,5-dichloro-4-hydroxybenzenesulphonic acid (PDB id: 5JZ9) and 3,5-dichloro-benzenesulfonamide (PDB id: 5JZB).…”

In silico Strategies to Support Fragment-to-Lead Optimization in Drug Discovery

“…The three confirmed fragment initial hits were structurally characterized by X-ray crystallography and fragment soaking. A small series of compounds based on these hits were further tested for activity both in vitro and ex vivo with promising results (Ryan et al, 2017). Another target of Mtb where FBDD has been applied is the pantothenate synthetase (Pts) where a similar sized fragment library of 1,250 rule-of-three compliant fragments was investigated.…”

Drug metabolism and antibiotic resistance in micro‐organisms

“…The authors wish to acknowledge that they are co-authors of the article by Ryan et al (2017). Ryan is also an author of a review (Ryan, 2017) and a co-author of the paper by Da Silva et al 2017.…”