Dealing with antibiotic resistance: antisense technology

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Project Summary/Abstract Bacterial infections are a leading cause of death, compromised health, and disability. Unfortunately, we are currently witnessing an increase in multiresistant infections and a decrease in the development of new antimicrobials. Consequently, the treatment costs are increasing, and a growing number of patients are succumbing to these infections. Furthermore, the increase in hard-to-treat or even untreatable bacteria also compromises medical procedures such as treatment of cancer and other chronic diseases, surgery, organ transplants, dental work, and care for premature infants. Compounding the problem, since SARS-CoV-2 coinfection with multidrug resistant bacteria has already been documented, the COVID-19 pandemic could accelerate the rise in antibiotic resistance by increasing patient exposure to antimicrobials. A solution to the antibiotic resistance problem could be the continuous development of new classes of antimicrobials. However, this route is slow and costly and needs to be complemented with other strategies. This proposal responds to this need and concentrates on searching strategies to extend the useful life of currently available drugs. The aminoglycoside 6′-N-acetyltransferase type Ib [AAC(6′)-Ib] is responsible for most cases of resistance to amikacin (Ak) and other aminoglycosides in Gram-negative pathogens. The dissemination of the aac(6′)-Ib gene among these pathogens erodes the efficacy of these antibiotics, which are an important component of the armamentarium against severe infections. The long-term goal of this research is to develop compounds that reduce Ak resistance to susceptibility levels and can be used as adjuvants to treat Ak-resistant infections. Specific aim 1 of this project proposes to optimize the structure of cell-penetrating peptides (CPP) bound to oligonucleotide analogs, known as external guide sequences (EGSs), that bind a complementary region of the aac(6′)-Ib mRNA and form a substrate for RNase P, which cleaves the mRNA preventing translation. The planned experiments consist of designing protease-resistant CPPs that maximize internalization and testing chimeric oligomers composed of deoxyribonucleotides and the newest generation of bridge nucleic acids. Specific Aim 2 will identify small molecule inhibitors of the AAC(6′)-Ib using combinatorial libraries and optimize them by structure-activity relationship analysis. This Specific aim also proposes to design water-soluble ionophores that in complex with zinc ions are strong inhibitors of the enzymatic inactivation of Ak. Specific aim 3 consists of testing the effect of Ak in association with combinations of the different compounds identified in the previous specific aims on Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii model strains using three-dimensional checkerboard assays, time-kill assays, and treatment of infections in the Galleria mellonella infection model. The most promising combinations will then be tested on about 100 genotypically well-defined K. pneumoniae, A. baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae clinical isolates.