Development of a Novel Class of Multifunctional Virulence-Attenuating Antibiotics.

Abstract

Resistance to traditional antibiotics arises largely because killing bacteria or halting their reproduction induces a selective pressure on mutants able to survive treatment. Virulence-attenuating antibiotics attempt to reduce resistance induction by instead targeting the mechanisms bacteria use to enhance survival within a host (“virulence factors”). Our collaborators in the Sun group at the University of Missouri – Columbia identified streptokinase (SK) as a virulence factor that confers Group A Streptococcus (GAS) the ability to infect human hosts. SK potently activates the human fibrinolytic enzyme plasmin, which allows GAS to evade the host’s response of clotting around sites of infection. High-throughput screening (HTS) for compounds able to attenuate GAS-SK production at the transcriptional level identified 3-allyl-2-(butylthio)-3H-spiro[benzo[h]quinazoline-5,1'-cyclohexan]-4(6H)-one as a moderately potent lead compound. Through extensive structure-activity relationship (SAR) studies, we were able to identify an analog, 3-allyl-2-(ethylthio)-9-methoxy-5,5-dimethyl-5,6-dihydrobenzo[h]quinazolin-4(3H)-one, with 35-fold higher potency (IC50 = 1.3 µM). We also identified several substitution patterns that confer enhanced metabolic stability to the molecular scaffold. In tandem with the SAR expansion effort, the identification of the macromolecular target(s) of this class of compounds was also pursued. To this end, we developed several classes of chemical probes designed to specifically interact with target protein(s), while offering a mechanism for the visualization or selective purification of the protein-probe complex. Though these probes have yet to convincingly identify a possible target, the use of more sensitive target identification assays, including quantitative proteomics and phage display, is currently being investigated. Finally, RNA microarray studies suggested that compounds from this series also inhibit biofilm formation. Biofilm-embedded colonies secrete a sticky extracellular matrix that effectively sequesters them from the immune response and antibiotic treatment. A second SAR study explored the extent to which our compounds can be used to control biofilms in the clinically-relevant Staphylococcus species. This study successfully identified a number of potent analogs, including several that exerted protective effects in murine models of S. aureus infection. One compound, 9-methoxy-3,5,5-trimethyl-2-((2,2,2-trifluoroethyl)thio)-5,6-dihydrobenzo[h]quinazolin-4(3H)-one, was found to be both reasonably active (IC50 = 3.7 µM) and metabolically stable (microsomal t1/2 = 19.6 min), increasing its potential for achieving efficacy in vivo.