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Updated: Aug 11, 2025
System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis
Published on: April 5, 2017
Chemical Validation of Mycobacterium tuberculosis Phosphopantetheine Adenylyltransferase Using Fragment Linking and
Jamal El Bakali1,2, Michal Blaszczyk3,4, Joanna C Evans5,6
1Yusuf Hamied Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.
Abstract:
The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD <20 microM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.
Insights
Researchers identified new drug leads targeting tuberculosis by inhibiting a key enzyme in coenzyme A (CoA) biosynthesis. Fragment linking yielded potent inhibitors with anti-Mycobacterium tuberculosis activity.
Area of Science:
- Biochemistry
- Drug Discovery
- Microbiology
Background:
- The coenzyme A (CoA) biosynthesis pathway is a promising target for novel antimicrobial agents.
- Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), requires new therapeutic strategies.
Purpose of the Study:
- To identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), a crucial enzyme in Mtb CoA biosynthesis.
- To validate MtbPPAT as a druggable target for anti-TB drug development.
Main Methods:
- Fragment screening was employed to identify initial binders to MtbPPAT.
- X-ray crystallography guided the structure-based design and linking of fragments.
- CRISPR interference was used to confirm on-target activity against Mtb.
Main Results:
- Three distinct fragment series binding to different MtbPPAT active site regions were discovered.
- Fragment linking resulted in an active site binder with a KD <20 µM.
- On-target anti-Mtb activity was confirmed for the developed inhibitors.
Conclusions:
- MtbPPAT is a validated drug target for anti-TB therapies.
- Fragment linking is an effective strategy for developing potent MtbPPAT inhibitors.
- This work advances the development of novel anti-TB drugs targeting CoA biosynthesis.
