Directed Evolution of the Nonribosomal Peptide Synthetase BpsA to Enable Recognition by the Human Phosphopantetheinyl Transferase for Counter-Screening Antibiotic Candidates

Abstract

Bacterial type II phosphopantetheinyl transferases (PPTases), required for the activation of many cellular mega-synthases, have been validated as promising drug targets in several pathogens. Activation of the blue-pigment-synthesizing nonribosomal peptide synthetase BpsA by a target PPTase can be used to screen in vitro for new antibiotic candidates from chemical libraries. For a complete screening platform, there is a need to also counter-screen inhibitors for cross-reactivity with the endogenous human Type II PPTase (hPPTase), as this is a likely source of toxicity. As hPPTase is unable to recognize the PCP-domain of native BpsA, we used a combination of directed evolution and rational engineering to generate a triple-substitution variant that is able to be efficiently activated by hPPTase. Our engineered BpsA variant was able to readily detect inhibition of both hPPTase and the equivalent rat PPTase by broad-spectrum PPTase inhibitors, demonstrating its potential for high-throughput counter-screening of novel antibiotic candidates.

Inhibition of Indigoidine Synthesis as a High-Throughput Colourimetric Screen for Antibiotics Targeting the Essential Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT

“…We next sought to benchmark our DMSO-resolubilisation end-point assay against the kinetic assay we had previously described [ 22 ]. For this, we selected two compounds with different inhibition profiles, namely 6-nitroso-1,2-benzopyrone, which has been shown to have activity in vitro against numerous PPTases [ 19 , 22 , 35 ] and in vivo against the fungus Aspergillus fumigatus in a PPTase dependant manner [ 36 ], and the weaker inhibitor sanguinarine chloride, which has been previously reported as an inhibitor of both Sfp from B. subtilis [ 21 ] and M. tuberculosis PptT [ 16 ]. We used identical enzymatic concentrations across both assays to permit a direct comparison.…”

“…Our assay therefore meets a need for a simple high-throughput assay that can be applied to directly screen for inhibitors of PptT or other Type II PPTases, or to guide the development of next-generation drug leads based on promising scaffolds such as ML-267 and 8918. Recently, we have also shown that the PCP-domain of BpsA can be rapidly evolved using error-prone mutagenesis to be recognised by other Type II PPTases that initially cannot activate BpsA [ 35 ]. This provides a platform to enable the rapid integration of multiple PPTases for inhibitory activity, or to counter-screen the endogenous human PPTase to eliminate drug candidates likely to have undesirable off-target effects.…”