Exploring IsdB/hemoglobin complex to develop new antimicrobial strategies against Staphylococcus aureus

Staphylococcus aureus has the ability to develop resistance to a great number of antibiotics used in clinics, posing significant challenges in clinical management. S. aureus relies on iron to grow and induce infections, therefore the bacterium developed different strategies to acquire iron from the host. In mammals, the main iron source is represented by hemic iron, which is extracted from cell-free hemoglobin (Hb) by two cell wall- anchored proteins named IsdB and IsdH, the former being the most relevant for virulence. In order to develop new antimicro- bial strategies for S. aureus based on iron starvation, we under- took a virtual screening-driven drug discovery campaign, aiming at targeting cell-free Hb released by hemolysins upon infection, at the IsdB-Hb interacting region, thus preventing heme extrac- tion. The most promising compound that we have identified, C35, binds Hb with a low micromolar dissociation constant. X- ray crystallography revealed that the compound binds Hb at a binding site among alpha-subunits, in a well-known allosteric site involved in a quaternary transition to a relaxed high-affinity con- formation. However, the compound was also able to directly interact with IsdB as observed by STD-NMR, thus revealing a second mechanism for inhibiting the formation of the IsdB-Hb complex and slowing down heme extraction. The development of more potent C35 derivatives will enable testing on S. aureus with the aim to find effective inhibitors of iron acquisition that might find application as antimicrobials or enhancers of antimicrobial therapy, thus contributing to contrast the insurgence of resistance in S. aureus. Project funded by: PRIN2020AE3LTA “Defeat antimicrobial resistance through iron starvation in Staphylococcus aureus (ERASE).”