Fragment-Based Drug Discovery of Inhibitors of Phosphopantetheine Adenylyltransferase from Gram-Negative Bacteria

Abstract

The discovery and development of new antibiotics capable of curing infections due to multidrug-resistant and pandrug-resistant Gram-negative bacteria are a major challenge with fundamental importance to our global healthcare system. Part of our broad program at Novartis to address this urgent, unmet need includes the search for new agents that inhibit novel bacterial targets. Here we report the discovery and hit-to-lead optimization of new inhibitors of phosphopantetheine adenylyltransferase (PPAT) from Gram-negative bacteria. Utilizing a fragment-based screening approach, we discovered a number of unique scaffolds capable of interacting with the pantetheine site of E. coli PPAT and inhibiting enzymatic activity, including triazolopyrimidinone 6. Structure-based optimization resulted in the identification of two lead compounds as selective, small molecule inhibitors of bacterial PPAT: triazolopyrimidinone 53 and azabenzimidazole 54 efficiently inhibited E. coli and P. aeruginosa PPAT and displayed modest cellular potency against the efflux-deficient E. coli Δ tolC mutant strain.

Using a fragment-based approach to identify novel chemical scaffolds targeting the dihydrofolate reductase (DHFR) fromMycobacterium tuberculosis

“…About 37 statistically positive hits (Supplementary figure 2), which represent about 2.8% of the library with significant chemical diversity, were identified when screened at a fragment concentration of 10 mM. This hit rate is similar to that obtained in several other FBDD campaigns 27,44,24 . The remaining molecules of the library did not significantly stabilise the MtDHFR or displayed a negative ΔTm, indicating a stabilisation of the unfolded state of MtDHFR, consequently, these molecules were also not considered for further analysis.…”

Vitamin in the Crosshairs: Targeting Pantothenate and Coenzyme A Biosynthesis for New Antituberculosis Agents

“…Hydrogen bonding interactions with the indicated active site residues are shown as black dashed lines. (C) Inhibitors of E. coli and P. aeruginosa PPAT displaying sub-micromolar activity (Moreau et al, 2018). (D) Selective inhibitors of MtPPAT identified through in silico screening, which are predicted to prevent substrate binding at the active site (Podshivalov et al, 2017).…”

“…While no inhibitors have been reported for MtPPAT using structure-based approaches, inhibitors have been described for EcPPAT, its homologue from E. coli (Zhao et al, 2003;Miller et al, 2010;Moreau et al, 2018;Skepper et al, 2018;Liyanage et al, 2019;Wang et al, 2020). The two homologues share 44% identity and 77% similarity in their amino acid sequences, and both exist as hexamers in their active form (Timofeev et al, 2010).…”

“…While several differences are present between the two structures, such as the number of subunits involved in ligand binding, evaluation of EcPPAT inhibitors can guide future design of compounds to inhibit MtPPAT. Recently, a fragment-based strategy facilitated by crystallography revealed several highly potent compounds (83 and 84, Figure 8C) with nanomolar activity against EcPPAT and P. aeruginosa PPAT (PaPPAT), and moderate cellular activity against the efflux-deficient E. coli DtolC mutant (Moreau et al, 2018;Skepper et al, 2018). Inhibitor development studies of other bacterial PPAT enzymes, including those from S. aureus, S. pneumoniae, and H. pylori have also been performed, but none of these have led to the development of clinical candidates (De Jonge et al, 2013;Cheng et al, 2013).…”

Addressing Antimicrobial Resistance through New Medicinal and Synthetic Chemistry Strategies

“…90 A research group from Novartis has identified inhibitors of gram-negative bacterial PPAT using an FBLG approach. 91 All fragment hits bind at the 4′-phosphopantetheine site of Escherichia coli PPAT. Lead compounds 23a and 23b ( Fig.…”