Related Experiment Video
Updated: Jun 30, 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 K D <20 μM 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 novel drug targets for tuberculosis by inhibiting the Mycobacterium tuberculosis phosphopantetheine adenylyltransferase (MtbPPAT) enzyme. Linking fragments led to an active site binder with anti-TB activity, validating MtbPPAT as a drug target.
Area of Science:
- Drug discovery and development
- Antimicrobial resistance
- Biochemistry and enzymology
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.
- Mtb phosphopantetheine adenylyltransferase (MtbPPAT) is crucial for CoA biosynthesis in Mtb.
Purpose of the Study:
- To identify inhibitors of MtbPPAT through fragment screening.
- To validate MtbPPAT as a druggable target for anti-TB drug development.
- To explore fragment linking strategies for enhanced MtbPPAT inhibition.
Main Methods:
- Fragment screening of MtbPPAT active site.
- X-ray crystallography for structural guidance.
- Fragment linking to create high-affinity binders.
- CRISPR interference assays to confirm on-target activity.
Main Results:
- Discovery of three distinct fragment series binding to the MtbPPAT active site.
- Successful linking of weakly-binding fragments into a potent inhibitor.
- Achieved an MtbPPAT inhibitor with a dissociation constant (KD) < 20 μM.
- Demonstrated on-target anti-Mtb activity using CRISPR interference.
Conclusions:
- MtbPPAT is a validated drug target for anti-TB therapies.
- Fragment linking is an effective strategy for developing MtbPPAT inhibitors.
- This study advances the development of novel anti-TB drugs targeting CoA biosynthesis.
