DEVELOPMENT OF IODONIUM-BASED GRAM-NEGATIVE ANTIBACTERIAL COMPOUNDS AND TARGET IDENTIFICATION USING A CHEMOPROTEOMICS APPROACH

Abstract

Antimicrobial resistance (AMR), especially among Gram-negative pathogens, is a major problem worldwide, and new classes of chemotherapeutics with novel mechanisms are urgently needed. Diphenyleneiodonium (DPIC) chloride is an antiseptic agent with excellent activity against Gram-positive bacteria and moderate inhibitory activity against Gram-negative bacteria. A number of new approaches towards developing therapeutics against Gram-negative pathogens are in urgent need. Although several mechanistic studies have been conducted, primarily in mammalian cells, the biological targets for DPIC remain to be elucidated in bacteria. In order to understand the mechanism of action, an iodonium probe containing an alkyne handle was next synthesized. This probe enables biorthogonal reactions and aids in identifying proteins that the probe and, by extension DPI has modified. Preliminary findings using activity-based protein profiling (ABPP) methods with bacterial lysate revealed that the iodonium compound modifies proteins with preliminary data suggesting that this modification was covalent and through a cysteine residue. Next, in order to find the biological targets of DPIC, LC-MS/MS-based competitive ABPP was carried out with E. coli. This analysis revealed that proteins with redox cofactors, as well as those involved in respiration were the most likely biological targets for DPIC. In order to validate these results, one of the target proteins, NuOF was cloned, purified, and was found to be covalently modified by the probe, likely through a cysteine residue. Having identified targets of DPI in bacteria, we next proceeded to perform a structure-activity relationship study with a goal of improving potency against Acinetobacter baumannii (A. baumannii), which is listed among the top priority pathogens for new drug development by the World Health Organization. Given the abundance of heterocyclic compounds in bioactive molecules, such as antibiotics, a series of heterocyclic iodonium analogs were synthesized to enhance the spectrum the antibacterial activity towards Gram-negative bacteria. Several compounds in this series were found to have excellent inhibitory activity against Gram-negative bacteria, including multi-drug resistant A. baumannii. The lead compound in this series was found to have bactericidal activity against A. baumannii, had a favourable selectivity index, and showed excellent activity in an animal model for infection. Using chemproteomics methods, the targets of the lead compound were identified. In summary, our study of the structure-activity relationship with iodonium salts led to the identification of a promising antibacterial candidate, while chemoproteomics with a newly developed probe provided insights into the unique mechanism of action for this class of antibacterials. In a separate approach to addressing AMR, we developed a strategy for the light-activated generation of fluoroquinolones. The use of optical control over the delivery of antibacterials, also known as antimicrobial photopharmacology, involves developing strategies for spatiotemporal control over the generation of a drug and minimizes environmental exposure to the drug. We designed and synthesized BODIPY-Levo, a caged derivative of levofloxacin. The prodrug does not generate the active antibiotic unless exposed to light (470 nm). The efficacy of this conjugate to inhibit bacterial growth in the presence of light is demonstrated. In summary, two independent small molecule-based approaches to address antimicrobial resistance were developed.