In silico discovery and biological validation of ligands of FAD synthase, a promising new antimicrobial target

Author summary Developing cures for antimicrobial-resistant microorganisms is a pressing necessity. Addressing this problem requires the discovery of novel therapeutic targets -for example, bacterial proteins with no human homologues- and the development of cost-effective drug screening protocols. In this work, we tackled the problem on both sides. We developed an efficient and successful integrative computational protocol for screening inhibitory-molecules for unexplored targets. We used it to discover five novel inhibitors of flavin-adenine dinucleotide synthase (FADS), a promising protein target of pathogens causingtuberculosisandpneumonia. New treatments for diseases caused by antimicrobial-resistant microorganisms can be developed by identifying unexplored therapeutic targets and by designing efficient drug screening protocols. In this study, we have screened a library of compounds to find ligands for the flavin-adenine dinucleotide synthase (FADS) -a potential target for drug design against tuberculosis and pneumonia- by implementing a new and efficient virtual screening protocol. The protocol has been developed for thein silicosearch of ligands of unexplored therapeutic targets, for which limited information about ligands or ligand-receptor structures is available. It implements an integrative funnel-like strategy with filtering layers that increase in computational accuracy. The protocol starts with a pharmacophore-based virtual screening strategy that uses ligand-free receptor conformations from molecular dynamics (MD) simulations. Then, it performs a molecular docking stage using several docking programs and an exponential consensus ranking strategy. The last filter, samples the conformations of compounds bound to the target using MD simulations. The MD conformations are scored using several traditional scoring functions in combination with a newly-proposed score that takes into account the fluctuations of the molecule with a Morse-based potential. The protocol was optimized and validated using a compound library with known ligands of theCorynebacterium ammoniagenesFADS. Then, it was used to find new FADS ligands from a compound library of 14,000 molecules. A small set of 17in silicofiltered molecules were tested experimentally. We identified five inhibitors of the activity of the flavin adenylyl transferase module of the FADS, and some of them were able to inhibit growth of three bacterial species:C. ammoniagenes,Mycobacterium tuberculosis, andStreptococcus pneumoniae, where the two are human pathogens. Overall, the results show that the integrative VS protocol is a cost-effective solution for the discovery of ligands of unexplored therapeutic targets.