Direct capture, inhibition and crystal structure of HsaD (Rv3569c) from M. tuberculosis by Cycliphostin & Cyclipostins analogs

ABSTRACT: Tuberculosis (TB) which is one of the deadliest infectious disease worldwide, is caused by the inhalation of the pathogenic bacteria Mycobacterium tuberculosis. Since many years now, the emergence of multiple (MDR), extensively (XDR) and totally (TDR) drug resistant strains amplifies the incidences of TB resulting in failure of treatment and death. However, no new TB drug classes have been developed or approved for drug susceptible TB since the current 6-month four-drug combination was introduced in the 1970s. Most of the promising new molecules in development are either repurposed drug compounds or new derivatives of known anti-mycobacterial drugs that mainly target the bacteria cell wall biosynthesis. It is now acknowledged that microbial lipolytic enzymes are involved in bacterial growth, cell wall biosynthesis, carbon sources management and virulence during infections by M. tuberculosis. Although no drug is reported to target specifically enzymes involved in mycobacterial intracellular lipid metabolism; our results have clearly shown that mycobacterial enzymes can be potential therapeutic targets. Consequently, specific inhibitors of such enzymes might turn out to be valuable anti-tuberculous agents. We previously reported that the Cyclophostin & Cyclipostins (CyC) analogs are a new family of potent antimycobacterial molecules which react specifically and covalently with (Ser/Cys)-based enzymes. Competitive ABPP approach with CyC17-pretreated M. tb lysate and the ActivX Desthiobiotin-FP activity-based probe (ABP) allowed identifying 23 distinct proteins as potential targets of this inhibitor, that were all (Ser/Cys)-based enzymes, most of them participating in M. tb lipid metabolism and cell wall biosynthesis. However, this approach for target identification in cell lysates is mostly indirect with the risk of false positive hits, and cannot be applied in vivo for various technical reasons, the main one being the need for a large number of compounds and cells. To overcome these issues, we report here the synthesis of new CyC alkyne-containing inhibitors (i.e., CyCyne) analogous to CyC17, and their use for the direct fishing of target proteins in living M. tb via bio-orthogonal click-chemistry activity-based protein profiling (CC-ABPP) strategy. In particular, we further explore and validate, through a combination of biochemical and structural approaches, the specificity of inhibition of the HsaD activity by the CyC analogs.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Bacteria Mycobacterium Tuberculosis H37rv

DISEASE(S): Tuberculosis

SUBMITTER: AUDEBERT Stephane  

LAB HEAD: Dr Jean-François CAVALIER

PROVIDER: PXD034921 | Pride | 2022-09-20

REPOSITORIES: Pride