The impact of chemical modifications of antisense oligonucleotides on CTX-M-15 β-lactamase target affinity and expression of ceftriaxone-resistant

Antimicrobial resistance is a current public health challenge. In line with the new One Health policy and the World Health Organization priority pathogen list, this study explores antisense oligonucleotides targeting ß-lactamase CTX-M-15, responsible for one of the most prevalent mechanisms of resistance to third-generation cephalosporins in enterobacteria. As an alternative to common PNA chemistry, 22 different combinations of chemical modifications were designed to address the main hurdles of intrabacterial oligonucleotide efficient delivery and stability. Chemical modifications included nucleolipid conjugation and backbone chemical modifications (PTO, 2'OMe, 2'MOE, and locked nucleic acid). Self-assembly properties, biophysical and thermodynamic characterization were further considered, along with cell-free translation assays of ß-lactamase. The therapeutic potential was screened through minimal inhibitory concentration to ceftriaxone. Our results demonstrated reproducible micellar self-assembly for all nucleolipid conjugates. Whereas nucleolipid conjugation in terms of thermodynamics can decrease the binding affinity for some chemistries, further inducing still efficient but lower ß-lactamase inhibition, it remained essential for in cellulo effects of decreasing antibiotic resistance. While most chemical modifications turned out to be efficient, the most potent chemistry was MOE PTO gapmer, providing the lowest binding affinity and highest ß-lactamase inhibition. This work demonstrated the interest of nucleolipid conjugation along with backbone modifications to address the challenge of antimicrobial resistance.