Efficient Inhibition of Bacterial Biofilm Through Interference of Protein-Protein Interaction of Master Regulator Proteins: a Proof of Concept Study with SinR- SinI Complex of Bacillus subtilis.

Biofilm-associated microbial growth is a major cause of environmental, industrial, and public health concern. Therefore, there is a pressing need to discover and develop efficient antibiofilm strategies. Regulatory proteins vital for biofilm formation might be ideal targets for developing novel antibiofilm therapeutics. Their activities often depend on protein-protein interactions. Therefore, such targets present unique opportunities and challenges to drug discovery. In Bacillus subtilis, a model organism for studying biofilms, SinR acts as the master regulator of the biofilm formation cascade. Under favourable growth conditions, it represses the epsA-O and tapA-sipW-tasA operons, which encode for essential structural components of biofilms. Under unfavourable growth conditions, SinI, an agonist protein, inactivates SinR by forming a heterotrimeric complex. This results in derepression of epsA-O and tapA-sipW-tasA operons and leads to the phenotypic switch from planktonic to biofilm-associated form. We hypothesized that inhibiting SinR-SinI interaction might warrant repression of epsA-O and tapA-sipW-tasA operons and inhibit biofilm formation. To evaluate this hypothesis, we carried out a drug repurposing study for identifying potential inhibitors of SinI. Cefoperazone and itraconazole were identified as potential inhibitors with virtual screening. The stability of their interaction with SinI was assessed in extended MD performed over 100 ns. Both cefoperazone and itraconazole showed stable interaction. In in vitro studies, cefoperazone hindered the interaction of purified recombinant SinI and SinR. In the whole cell-based biofilm inhibition assays also cefoperazone was found to efficiently inhibited biofilm formation. These results provide proof of concept for targeting protein-protein interaction of master regulators as potential target for discovery and development of antibiofilm therapeutics. We propose that similar drug repurposing studies targeting key regulators of biofilm formation cascade could be an efficient approach for discovering novel anti-biofilm therapeutics against priority pathogens.