Single-Domain Antibodies as Potent Inhibitors of Clinically Relevant β-Lactamases in Multidrug-Resistant Bacteria

ABSTRACT

Antimicrobial resistance (AMR) represents a critical threat to global health, largely driven by the dissemination of β-lactamases that inactivate frontline antibiotics. Among the most problematic are BlaMab-2 from Mycobacterium abscessus, KPC-2 and OXA-48 from Klebsiella pneumoniae, and VIM-2 from Pseudomonas aeruginosa, which together confer broad resistance to β-lactams and carbapenems. Current β-lactamase inhibitors face declining efficacy as resistance variants continue to emerge, underscoring the need for innovative strategies. Here, we explored single-domain antibodies (sd-Abs) as enzyme-directed inhibitors of β-lactamases. A library of sd-Abs was screened, and two candidates, B2 and B5, were characterized in vitro and in vivo. Both sd-Abs inhibited BlaMab-2 activity in E. coli expression systems, following a competitive inhibition mechanism, with B2 consistently displaying stronger potency (Ki ≈ 1.5 µM) than B5. Remarkably, B2 also demonstrated broad inhibitory activity against KPC-2, VIM-2, and OXA-48, while B5 showed an alternative inhibition profile, including uncompetitive characteristics against VIM-2 and OXA-48. Comparison with clinically deployed inhibitors revealed that the Ki values of B2 and B5 are of the same order of magnitude—or superior in some cases—highlighting their therapeutic promise. Our findings establish sd-Abs as a versatile platform for the inhibition of diverse β-lactamases, with B2 emerging as the most broadly effective candidate. By expanding the utility of existing β-lactams, sd-Abs could help restore antibiotic efficacy against multidrug-resistant pathogens. This study underscores the potential of antibody-based enzyme inhibitors as a new class of anti-resistance therapeutics.