Infectious diseases remain a major public health threat, with limited therapeutic options available for several pathogens, including multidrug-resistant Pseudomonas aeruginosa. To address this challenge, we have developed a novel approach using bifunctional molecules (chimeras) that recruit complement protein C3 and activate the immune system to eliminate infections. C3 is a highly abundant plasma protein (5–13 µM) and a central component of all three major pathways of the innate immune system: the classical pathway, alternative pathway, and lectin pathway. Deposition of C3 on the bacterial surface initiates a complement activation cascade, leading to bacterial clearance via multiple mechanisms, including membrane lysis by the membrane attack complex and immune cell recruitment through opsonization.
To identify C3 binders, we screened several DNA-encoded libraries and discovered hits with low micromolar affinity for C3. The hits were validated using surface plasmon resonance (SPR) and STD-NMR. Incorporating proprietary lysine-targeting handles into the hit structures enabled chemoproteomic analysis, which localized the binding site near Lys678 of C3. Subsequent medicinal chemistry optimization yielded binders with varying affinities. Conjugating these C3 binders to bacterial-targeting peptides produced bifunctional molecules (chimeras) capable of inducing C3 deposition on the P. aeruginosa membrane and inhibiting bacterial growth at sub-micromolar concentrations. This effect was shown to be dependent on both complement-active serum and the C3-binding moiety. Our lead chimera demonstrates favorable pharmacological properties, including good aqueous solubility, plasma stability, moderate microsomal stability, no hemolysis at 2 µM, and no cytotoxicity in mammalian cells at 10 µM. Pharmacokinetic studies in mice reveal an in vivo half-life of approximately 5 hours and efficient accumulation in lung tissue. In murine lung infection models using both carbapenem-sensitive (ATCC27853) and -resistant (AR #0246) P. aeruginosa strains, treatment with our lead compound at 29 µmol/kg (BID) resulted in a robust 2-log reduction in bacterial load. The complement-recruiting chimera platform is now being expanded to target other pathogens and holds promise in the fight against antimicrobial resistance.
