Computational Design and Lab-Based Investigation of a Novel Small-Molecule Inhibitor That Targets CadA Metal Efflux Pump Activity in Hospital Methicillin-Resistant Staphylococcus Aureus: A Research Protocol

Abstract

Introduction: Heavy metal exposure has been previously reported to decrease bacterial growth. During the growing antibiotic crisis in healthcare settings, metals may reduce bacterial burden in hospital environments. Despite proving successful, this has driven the evolution of metal-resistant strains like methicillin-resistant Staphylococcus aureus (MRSA). By expelling cadmium and zinc through surface efflux pumps such as CadA, MRSA is able to thrive in metal-rich environments. This protocol investigates the inhibitory activity of Scinapsin, a novel small-molecule inhibitor designed to bind the catalytic site of CadA. Scinapsin could be applied to clinical settings to combat metal resistance in MRSA populations amidst the COVID-19 pandemic. Methods: Computational biochemistry is used to characterize the structure of the S. aureus CadA protein. Small-molecule library screening generated a hit compound, which after modification to improve binding affinity produced the final Scinapsin structure. Several MSRA strains were screened for the presence of CadA from which the most favourable strain for the experiment is chosen (i.e. prevalence in hospitals, CadA expression levels). For the experimental protocol, MRSA strains with metal resistance are incubated with increasing concentrations of Scinapsin, either in the presence or absence of zinc and cadmium (at 50% of predetermined MIC). Samples are then diluted and plated to allow for CFU counting. Results: Scinapsin is anticipated to have an inhibitory effect on MRSA growth in the presence of metals at 50% MIC, confirming the successful inactivation of CadA function. A saturation point may also occur at higher concentrations of Scinapsin where no further growth inhibition is achieved. Discussion: Docking analysis has confirmed the theoretical feasibility for Scinapsin to act as a CadA-specific inhibitor. In an in vitro setting like the one presented, Scinapsin should allow for excess zinc and cadmium to accumulate in the cytoplasm and ultimately cause cell death. Further experiments could aim to confirm the proposed biological mechanism of antibacterial activity. Conclusion: Scinapsin holds promise in reversing metal resistance in hospital MRSA populations and may pave the way for other small-molecule antibacterial drugs. Future research is needed to determine safe levels of Scinapsin exposure for humans and how this inhibitor affects other hospital microbes.