Affimer Proteins as Versatile Tools for Target Identification, Inhibition and Characterisation in Gram-negative Bacteria

The rise of antibiotic-resistant bacteria poses a significant global health threat,
with Gram-negative bacteria, notably Pseudomonas aeruginosa, presenting
substantial challenges due to their resistance to last-resort antibiotics. The
emergence of resistance, coupled with high morbidity and mortality rates, gives
rise to an urgent need for novel antibiotic development.
Metallo-β-lactamases (MBLs) are enzymes found in Gram-negative bacteria
that render β-lactam antibiotics ineffective through hydrolysis of the β-lactam
bond. Among them, New Delhi metallo-β-lactamase 1 (NDM-1) is particularly
worrisome as it can hydrolyse nearly all β-lactam antibiotics, jeopardizing the
efficacy of our remaining arsenal of antibacterial therapies. The lack of
clinically available inhibitors against NDM-1 necessitates the development of
new MBL inhibitors.
This research investigates the potential of Affimer proteins as inhibitors and
characterisation tools of antibacterial drug targets in Gram-negative
bacteria. Affimer 18 exhibited strong inhibitory properties against NDM-1,
binding to NDM-1 with an affinity of 2 nM and inhibiting its activity across all
substrate classes with an IC50 value of <70 nM. Enzyme kinetics experiments
revealed Affimer 18 as a non-competitive (allosteric) inhibitor, supported by
structural characterisation of the Affimer/NDM-1 co-crystal structure. Further
investigation elucidated novel details of the NDM-1 catalytic mechanism and
identified core and substrate-specific residues necessary for NDM-1
hydrolysis.
In addition, Affimer proteins were employed to probe novel drug targets in P.
aeruginosa. Affimers were isolated against targets TolB and LptH; these
reagents reduced the cellular growth rate by 1.5-fold and increased antibiotic
susceptibility by 2-fold, indicating their potential as useful drug targets.
Moreover, these Affimer reagents were repurposed into a diagnostic system
for detecting P. aeruginosa cells.
Overall, this research highlights Affimers as valuable tools in antibacterial
resistance research and clinical diagnostics. The characterisation of a high-affinity, highly potent NDM-1 inhibitor provides insights for future drug discovery
efforts, while the exploration of novel drug targets in P. aeruginosa underscores
the versatility and potential applications of Affimer proteins.