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

Abstract

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 the in silico search 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 the Corynebacterium ammoniagenes FADS. Then, it was used to find new FADS ligands from a compound library of 14,000 molecules. A small set of 17 in silico filtered 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, and Streptococcus pneumoniae, where the last 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.

Mining the Flavoproteome of Brucella ovis, the Brucellosis Causing Agent in Ovis aries

“…This list could be potential targets for the search for antimicrobials. Some of them are already being explored as targets of inhibitors in other bacteria, as for example, UDP-N-acetylmuramate dehydrogenase ( 102 , 103 ), riboflavin synthase ( 104 ), bifunctional riboflavin kinase/FAD synthase ( 25 , 26 ), or chorismate synthase ( 105 ). In agreement, a comparative metabolomics study in B. melitensis also pointed to synthase as an attractive target ( 106 ).…”

Purification and characterization of recombinant FAD synthetase from Neurospora crassa

“…Based on the recent analysis of the FADS isoforms isolated from humans resulting as an emergency protein used for the patients suffering from the deficiencies caused by FADS [ 37 ]. Virtual screening protocols with respect to certain ligands interacting with FADS from prokaryotes ( Corynebacterium ammoniagenes ) resulted in therapeutic strategy, screening for antimicrobial resistant pathogens causing tuberculosis and pneumonia indicate the potentiality of FADS as drug target [ 38 , 39 ]. Therefore the recombinant NcFADS could provide an opportunity to go for the detailed structural analysis using X-ray crystallography or multi-dimensional NMR, also synthesis of the side-directed mutants at the active site can further help in understanding the kinetic and structural aspects of the enzyme.…”

The promise of endogenous and exogenous riboflavin in anti-infection

“…However, the characteristics of FAD synthase in S. pneumoniae are different from those in other bacteria [ 65 ]. In addition, several FAD synthase inhibitors were shown to inhibit the growth of M. tuberculosis and S. pneumoniae [ 66 ].…”