DARPin-based therapeutics against S. aureus infection

Abstract Staphylococcus (S.) aureus is a pervasive gram-positive pathogen that is responsible for ~19,000 deaths in the US each year. The broad-spectrum-antibiotic used for the prevention or therapy of bacterial infections fostered the emergence of antibiotic resistant strains, such as the methicillin-resistant S. aureus (MRSA), which in turn increased the rate of therapy failure and mortality. The recent cases of community-acquired MRSA further exac- erbate the problem of S. aureus infections and underscore an urgent need for novel therapeutics for preventing and treating S. aureus infection. A distinguishing feature of all clinical S. aureus isolates is their ability to coagu- late blood through secretion of two coagulases that convert soluble fibrinogen into insoluble fibrin followed by agglutinating into these fibrin blood clots. Agglutinated fibrin creates a microenvironment that shields bacteria from antimicrobials and phagocytes, and promotes their growth. The overall objective of this study is to develop biologics able to block agglutination by inhibiting the interaction between S. aureus and fibrin(ogen). Clumping factor (ClfA), a MSCRAMM (microbial surface component recognizing adhesive matrix molecule) is critical in promoting agglutination. S. aureus mutants lacking functional ClfA cannot agglutinate, and as a result, display significant virulence defects in the mouse models of sepsis, septic arthritis and endocarditis. Thus, biologics able to block ClfA could represent an added therapeutic approach in the treatment of complicated MRSA infections. Unfortunately, monoclonal anti-ClfA antibodies developed thus far have failed in the clinic, likely due to their weak fibrinogen inhibitory activity (IC50 0.4-25 µM) despite high binding affinity (pM-nM) to ClfA. Using directed evolution and rational protein design, we recently engineered a panel of DARPin- (D) proteins with very potent fibrinogen inhibitory activity. Here, we propose to explore the clinical potential of these Ds, and 1) characterize the binding properties and neutralization capacity of these Ds against a panel of S. aureus isolates, 2) generate additional Ds with broad S. aureus reactivity, and 3) test the protective efficacy of Ds in a murine sepsis model. Successful completion of this work will yield novel therapeutic candidates for treating S. aureus infection and deepen our understanding of the role of ClfA in S. aureus biology.