4-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)-N-(4-methoxypyridin-2-yl)piperazine-1-carbothioamide (ML267), a Potent Inhibitor of Bacterial Phosphopantetheinyl Transferase That Attenuates Secondary Metabolism and Thwarts Bacterial Growth

Abstract

4′-Phosphopantetheinyl transferases (PPTases) catalyze a post-translational modification essential to bacterial cell viability and virulence. We present the discovery and medicinal chemistry optimization of 2-pyridinyl-N-(4-aryl)piperazine-1-carbothioamides, which exhibit submicromolar inhibition of bacterial Sfp-PPTase with no activity toward the human orthologue. Moreover, compounds within this class possess antibacterial activity in the absence of a rapid cytotoxic response in human cells. An advanced analogue of this series, ML267 (55), was found to attenuate production of an Sfp-PPTase-dependent metabolite when applied to Bacillus subtilis at sublethal doses. Additional testing revealed antibacterial activity against methicillin-resistant Staphylococcus aureus, and chemical genetic studies implicated efflux as a mechanism for resistance in Escherichia coli. Additionally, we highlight the in vitro absorption, distribution, metabolism, and excretion and in vivo pharmacokinetic profiles of compound 55 to further demonstrate the potential utility of this small-molecule inhibitor.

Breaking a pathogen's iron will: Inhibiting siderophore production as an antimicrobial strategy

“…Inhibitors of the PPTase enzyme will prevent the attachment of the TAMRA-CoA to the peptide, preventing the FRET signal [28, 29]. A subsequent high throughput screen of over 300,000 compounds yielded the identification of ML267 (Figure 4B) as an inhibitor of Sfp-PPTase, with approximately 30-fold selectivity over the bacterial fatty acid synthesis PPTase and greater than 500-fold relative to the human orthologue [31, 32]. ML267 is an allosteric, reversible inhibitor with a high nanomolar IC 50 value 3 that is bactericidal against Gram positive pathogens including B. subtilis and methicillin-sensitive and -resistant strains of Staphylococcus aureus (MRSA).…”

“…B. ML267 inhibitor of Sfp-PPTase identified by high throughput screening using the FRET assay using probes in part A. [31, 32] C. BODIPY-TMR fluorescence polarization probe developed to assay PPTase inhibitors. [33] D. The two most inhibitory compounds of the PPTase from M. tuberculosis are shown, determined when using the fluorescence polarization assay where the probe is the FRET acceptor in part A.…”

Legionellashows a diverse secondary metabolism dependent on a broad spectrum Sfp-type phosphopantetheinyl transferase

“…To rule out the possibility that any PPTase was missed in this analysis, we used 2-pyridinyl- N -(4-aryl)-piperazine-1-carbothioamides (4–6), specific inhibitors of bacterial Sfp-like PPTases (Foley et al, 2014), to shut off legioliulin production. Legioliulin production and growth are closely linked.…”

“…The enzyme was able to activate the NRPS IndC from P. luminescens , even though no NRPS product is known for any Legionella strain so far confirming this function. To investigate the effect on suppression of the Sfp-type PPTase, we grew L. parisiensis in the presence of Sfp-type PPTase inhibitors (Foley et al, 2014) and showed that legioliulin production, in addition to cell viability, is halted (Table S4; Fig. 5).…”

“…Putative PPTase inhibitors 4–6 (Foley et al, 2014) were then added in different concentrations, and the cells were allowed to grow for 24 h. For visualization of legioliulin production in L. parisiensis , the cells were illuminated with long-wave UV-light. The MIC of the PPTase inhibitors were tested in triplicate on L. parisiensis, L. pneumophila and L. longbeachae using the OD 600 value.…”