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 the first 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. Abstract Figure