The Development of a Pipeline for the Identification and Validation of Small-Molecule RelA Inhibitors for Use as Anti-biofilm Drugs

Abstract

Biofilm infections have no effective medical treatments and can only be disrupted via physical means. This means that any biofilm infection that is not addressable surgically can never be eliminated and can only be managed as a chronic disease. Therefore, there is an urgent need for the development of new classes of drugs that can target the metabolic mechanisms within biofilms which render them recalcitrant to traditional antibiotics. This antibiotic recalcitrance of bacterial biofilms can be attributed largely to the formation of persister cells within the biofilm structure. These biofilm persister cells can be resistant to up to 1000 times the minimal inhibitory concentrations of many antibiotics as compared to their planktonic envirovars; they are thought to be the prokaryotic equivalent of metazoan stem cells. Their metabolic resistance has been demonstrated to be an active process induced by the stringent response that is triggered by the ribosomally-associated enzyme RelA in response to amino acid starvation. This 84-kD pyrophosphokinase produces the "magic spot" alarmones, collectively called (p)ppGpp. These alarmones act by directly regulating transcription by binding to RNA polymerase. These transcriptional changes lead to a major shift in cellular function to both upregulate oxidative stress-combating enzymes and down regulate major cellular functions associated with growth and replication. These changes in gene expression produce the quiescent persister cells. In this work, we describe a hybrid in silico-laboratory pipeline for identifying and validating small-molecule inhibitors of RelA for use in the combinatorial treatment of bacterial biofilms as re-potentiators of classical antibiotics.