Designing Inhibitors of Phosphopantetheinyl Transferases Of Pathogenic bacteria – DIPTOP

Many mycobacteria are human pathogens: Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, caused nearly 1.6 million deaths in 2021, and Mycobacterium abscessus (Mab) induces lung infections in patients with cystic fibrosis or weakened defences. Treatments exist but they are long and have many side effects. Moreover, in the case of Mab, the treatments are rendered ineffective by its intrinsic and acquired resistance.
These bacteria each express 2 phosphopantetheinyl transferases (PPTase), essential for their survival, which transfer the phosphopantetheinyl (P-pant) arm of coenzyme A (CoA) to the acyl transporter domain (ACP) of polyketide synthases (PKS), fatty acid synthases and non-ribosomal peptide synthases (NRPS). PptT in Mtb and PptAb in Mab share 69% sequence identity and activate all PKS and NRPS in these bacteria.
A crystallographic screen of 943 fragments (molecular weight about 200 Da) on the PptAb enzyme identified nearly 50 compounds that bind close to CoA. The DIPTOP project aims to optimise the most promising ligands to obtain drug candidates that inhibit PptAb and its ortholog PptT in Mtb, in vitro and in vivo.
These structures highlight three key sites for interaction with the enzyme: a polar region leading to a hydrophobic tunnel in which the P-pant arm of CoA is housed at acidic pH, a lipophilic region in the vicinity of the adenine of CoA, and a polar interaction with glutamate 153. Five compounds with these interactions are selected to be optimised for PptT and/or PptAb inhibition properties and antimicrobial activity on Mtb and/or Mab. They were chosen on the basis of their molecular diversity and synthetic accessibility. Their optimisation will be guided by structural (X-ray crystallography), biophysical (nanoDSF, TRIC, spectral shift, isothermal titration calorimetry), enzymatic (transfer inhibition) and functional (bacterial growth inhibition) data.
The production of recombinant PptT and PptAb in large quantities and with an excellent degree of purity is perfectly mastered, an essential prerequisite for the biophysical and structural characterisations required for the project. New biophysical techniques are currently being deployed at the IPBS (nanoDSF, spectral shift and TRIC) which are particularly well suited to highlighting protein-ligand interactions and determining the dissociation constants of complexes, even with low affinity. The determination of the structure of these proteins, whether in isolated or complex form, is also mastered.
An enzymatic test is available in the laboratory, which allows the transfer reaction of the P-pant arm to an ACP domain to be visualised by electrophoresis. If necessary, a more sensitive test based on fluorescence polarisation, described in the literature, will be used.
The determination of antimicrobial activity on slow growing pathogenic bacteria such as Mtb and Mab for many compounds can be problematic. A growth competition test was therefore developed using M. smegmatis (Msm), a fast-growing non-pathogenic mycobacterium with a cell envelope similar to that of Mab or Mtb. Msm, transformed to express either PptT or PptAb, in fusion with GFP, is cocultured with Msm expressing Sfp, the PPTase from Bacillus subtilis, in fusion with mCherry. These 3 PPTases complement PptMs the PPTase of Msm, deleted in these modified strains. After incubation with the compounds to be evaluated, the nature of the fluorescence observed (none, GFP, mCherry or GFP and mCherry) makes it possible to determine whether the compound effectively targets PptT or PptAb.