Chemical Validation of <i>Mycobacterium tuberculosis</i> Phosphopantetheine Adenylyltransferase Using Fragment Linking and CRISPR Interference**

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 ( Mtb PPAT), 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 Mtb PPAT 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 &lt;20 μM and on‐target anti‐ Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating Mtb PPAT as a potential drug target and designing a Mtb PPAT‐targeting anti‐TB drug.