Chemical tools to Detect and Inhibit IgA1 Proteases in Haemophilus influenzae

Non-encapsulated (non-typeable) Haemophilus influenzae is a major cause of mucosal infections such as otitis media, conjunctivitis, and exacerbations of chronic obstructive pulmonary disease. Rising antibiotic resistance has increased interest in anti-virulence strategies that reduce pathogenicity without exerting selective pressure for resistance. A key virulence factor of H. influenzae is immunoglobulin A1 protease (IgA1P), a secreted serine protease that promotes colonization by cleaving human IgA1 at the hinge region and enabling immune evasion. Despite its therapeutic promise, progress has been limited by the lack of chemical tools to probe IgA1P activity in its native biological environment. Here, we report the first activity-based probes that enable direct detection of active IgA1Ps in complex samples and H. influenzae clinical isolates. Competitive activity-based screening using these probes identified novel IgA1P inhibitors, and structure-activity relationship guided optimization yielded a potent lead IgA1P inhibitor. This inhibitor preserves IgA1 at the bacterial surface, demonstrating effective inhibition of IgA1P activity in its native context and chemical disruption of an IgA1P-dependent immune-evasion mechanism. Together, these chemical tools establish a versatile platform for elucidating IgA1Ps role in H. influenzae colonization and virulence, and for validating IgA1Ps as diagnostic markers and antivirulence targets.