SCREEN FOR INHIBITORS OF PPTT, A MULTI-STEP TARGET FOR CELL WALL SYNTHESIS IN M. TUBERCULOSIS

Modification of acyl carrier proteins (ACP) or domains by the covalent binding of 4’ phosphopantetheine (4’PP) moiety is a fundamental condition for activation of fatty acid synthases (FASes) and polyketide synthases (PKSes). Binding of 4’PP is mediated by 4` phosphopantetheinyl transfersases (PPTases). Mycobacterium tuberculosis (Mtb) possesses two essential PPTases: acyl carrier protein synthase (Mtb AcpS) activating the multi-domain fatty acid synthase I (FAS I) and Mtb PptT, an Sfp-type broad spectrum PPTase activating PKSes. To date, it has not been determined which of the two Mtb PPTases, activates the meromycolate extension ACP, Mtb AcpM, en-route to the production of mycolic acids of the mycobacterial cell wall.

Methods and results. By using SDS-PAGE band shift assay and mass spectrometry analysis, we found that Mtb PptT is the PPTase that activates Mtb AcpM. PptT activates a blue pigment synthase BPSA which is a non ribosomal peptide synthase(NRPS)from Streptomyces lavandulae. Activated BpsA produces indigoidine, a blue pigment enabling a flexible assessment of PptT activity and screen for new inhibitors of PptT. We established a simple highthrougput screen colorimetric assay for PptT inhibitors using activation of Bpsa by PptT and tested 57,000 compounds from various chemical libraries in search for PptT inhibitors. Thirty six “hits” were defined as specific inhibitors of PptT with a reprodicible IC₅₀ in vitro.

The anti-mycobacterial activity of these compound was tested using broth macrodilution assay against replicating M. bovis BCG bacilli. Eight lead compounds with an in vitro activity againts Mtb PptT have an in vivo activity for Mtb complex bacilli.

Conclusion

PptT- BpsA coupled assay can be used for highthroughput screen for novel Mtb PptT inhibitors which have the potential for anti-tuberculous therapy.