Mechanistic studies of antimicrobial oligoimidazoliums (OIMS)

• Main Author: Koh, Chong Hui
• Other Authors: Chan Bee Eng, Mary
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2024
• Subjects: Other; Antimicrobial polymer
• Online Access: https://hdl.handle.net/10356/181484
• Institution: Nanyang Technological University

Antimicrobial resistance (AMR) has become a major problem aggravated by the overuse of antibiotics and the slow pace of development for new classes of antibiotics. Resistance to classical antibiotics which target essential processes in rapidly growing bacteria, is already widespread and seems inevitable. Exploitation of bacterial membrane biology is one of the few remaining frontiers for development of new generations of antimicrobial agents. Antimicrobial peptides (AMPs) and polymers (AMPos) have since attracted interest and become a prominent class of membrane-active antibacterial compounds. They are generally positively charged and typically kill bacteria which have negatively charged membrane via membrane perturbation or disruption. Their non-specific mode of killing through electrostatic interactions also leads to bacterial inability to evolve resistance, while provide a solution to AMR pressing issue. While AMPos show several privileges over AMPs such as low production cost and higher flexibility in design, AMPos are usually not as potent or selective as classical antibiotics. Our research group has previously designed and synthesized a series of novel main-chain imidazolium-derived cationic polymers (PIMs) which are AMPos. Among these candidates, PIM1 which is a perfect alternating copolymer of main-chain imidazolium and butyl linker, has strong potency across broad-spectrum Gam-positive and Gram-negative bacteria with low toxicity to mammalian cells. We discovered that PIM1 has minimal physical membrane disruption activity and utilizes proton motive force (PMF) for intracellular uptake, and subsequently binds to deoxyribonucleic acid (DNA) that lead to bacterial death. These suggest that PIM1 does not share the common mechanism of action by conventional AMPos that kill bacteria by physical membrane disruption via electrostatic charge interaction. Since the cationic charge is not a prominent factor for the mechanism of action of PIM1, we proposed that the main-chain imidazolium rings of PIM1 could undergo hydrogen deprotonation at the carbon-2 position to form polycarbon acids. This is supported by previous studies which showed that the hydrogen at the carbon-2 position (denoted as C(2)-H) of monomeric imidazolium rings is readily deprotonated in water and subsequently formed carbon acids. The formation of carbon acids could decrease the cationic charge density and increase the hydrophobicity of PIM1, which contributes to a different mechanism of membrane action. We hypothesized that carbon acids enhance the membrane translocation process of PIM1 to enter the bacterial cytosol, allowing efficient binding to DNA for bacterial killing. Therefore, we developed oligoimidazoliums (OIMs) with precise repeating units to study the relative importance of carbon acidity for antibacterial efficacy. We compared the potency of the parental OIMs (structurally similar to PIM1 with six or eight repeating unit) against two groups of OIMs: a non-carbon acid group as the hydrogen at the carbon-2 position of the imidazolium ring was replaced by substituents, while another group retains its carbon acidity but with substitutions at the carbon-4 position of the imidazolium ring. We showed that OIMs that are carbon acids, i.e. those containing dissociable C(2)-H, have good antibacterial properties, as opposed to OIMs which are not carbon acids. Carbon acids also have higher bacterial cytosolic uptake than non-carbon acid, collaborating that the formation of carbon acids assist the membrane translocation of OIMs into the cytosol for bacterial killing. We also demonstrated that membrane potential component (ΔΨ) of PMF is in play to assist the uptake of OIMs into the cytosol of methicillin-resistant Staphylococcus aureus, and mutations that lead to decreased PMF level decreased the potency of OIM carbon acids. We also found that the carbon acidity enhances the cytosolic uptake and rapid killing of OIMs at low PMF conditions. Novel parental OIMs with incorporated degradable bonds showed good potency and biocompatibility in murine infection models. These OIM carbon acids also retain their potency in complex formulations containing confounding components such as proteins (milk) and salts, which impair the efficacy of other cationic antimicrobial polymers in many real-world applications. With a deep understanding on the killing mechanism of this class of main-chain oligoimidazoliums, we could further optimize the structural design of OIMs to improve their biomedical applications and develop OIMs into a new potential therapeutic drug.