Structure-Function Relationship Analysis of Peptidomimetic Anti-Infectives

Abstract

The escalating emergence and spread of antibiotic-resistant strains of bacteria became a growing threat to human health. As existing antibiotics lost efficacy against recently arising pathogens, the development of novel classes of antimicrobial agents became an urgent priority. Peptoids, which are one of the peptidomimetic materials consisting of an N-substituted glycine backbone, have received attention as antimicrobial applications. Previous research revealed that peptoid 1, a cationic amphipathic peptoid with a helical structure, exhibited broad-spectrum antibacterial activity, but its low selectivity between the bacterial and eukaryotic cell membranes remained to be improved. In this regard, recent studies have been focused to discover antimicrobial peptoids with enhanced therapeutic windows by conducting various modifications. In chapter 1, derivatives of peptoid 1 were designed to increase the cationic charge by substituting the NLys residue with a multi-cationic residue. The analogs with novel cationic residues generally showed improved selectivity due to the potency enhancement. Exceptionally, hit peptoid 3 demonstrated the same minimum inhibitory concentration (MIC) value for Escherichia coli, negligible hemolytic activity, and 2.5–6 fold increased LC50 values against eukaryotic cells compared to 1. Subsequently, the killing kinetic of hit peptoid 3 was evaluated and it was shown to act slower than control peptoid 1. From the difference observed in kinetics study, we tried to discover the working mechanism of 3 through various experiments, including membrane disruption, membrane depolarization, reactive oxygen species (ROS) generation analysis, and confocal microscopy. Finally, in vivo antibacterial activity evaluation of 3 was conducted, and the result showed concentration-dependent colony decrement of clinical Acinetobacter Baumannii. As a result, our study suggests that augmenting cationic charge in antimicrobial peptoids can provide a valid strategy to generate a selectivity-improved antibiotic drug candidate based on peptoid oligomers. Despite the supply of vaccines, chronic hepatitis B virus (HBV) infection and its complications pose a serious threat to human health, causing approximately 800,000 deaths annually. To combat this disease, various strategies have been attempted, including viral DNA replication inhibition, capsid assembly modulation, and virus entry inhibition. In this study, we focused on HBV entry inhibitors using the cyclosporin scaffold. Cyclosporines are a family of macrocyclic peptides composed of 11 amino acids and are orally bioavailable. The structure-permeability relationship of cyclosporine derivatives have been investigated to obtain insights for enhancing the oral administration rate. In chapter 2, the first-generation library of cyclosporin O derivatives were designed to increase the passive membrane permeability of the [Gln7, MeLeu11]1, which showed potent entry inhibition ability against HBV in previous work from our group. This library removed Gln7, which was thought to have a large polar surface area (PSA) and thus generate a high desolvation cost during membrane permeation. In addition, derivatives of this library include various peptoid submonomer substitutions at position 10. However, the peptide-peptoid hybrid CsO derivatives still lack passive membrane permeability, which arose from the flexible backbone structure in the hydrophobic environment due to the peptoid substitution at position 10. Among the first-generation library, cyclic peptide 9 showed 15-fold improved membrane permeability compared to the CsO (9.4×10-6 cm/s). We hypothesized the membrane permeability of cyclic peptide 9 arose from the introduction of a specific hydrogen bond donor (HBD) that could have exceptional stability by reducing the PSA of the molecule through intramolecular hydrogen bonding (IMHB) in the lipid environment. Based on 9, the second-generation library of cyclosporin O derivatives were designed to analyze the structure-permeability relationship (SPR) of macrocycles depending on the distance of the introduced HBD from the peptide backbone. The second-generation library was synthesized and purified, and their membrane permeability was evaluated through a parallel artificial membrane permeability assay (PAMPA). From the PAMPA experiment, there was difference in permeability among the types of amino acids substituted at the same position. Then, to verify the hypothesis that energetic stabilization by IMHB formation in a hydrophobic environment improves membrane permeability, 2D NMR analysis was conducted on VII, VIII, and IX, and based on the NOE cross peaks and 3JNH-Hα couplings, the distances and dihedral angles were obtained. This information was used as a restraint to reproduce the three-dimensional (3D) structure through molecular dynamics, and a structure that was energetically stable and had the least violation of constraints was selected as a representative conformation. By analyzing the regenerated structure, it was observed that the close proximity between the introduced HBD residue and the neighboring backbone carbonyl oxygen is available to attract each other through electrostatic interaction, thus reducing the PSA of the overall molecule at the highly membrane permeable VII and VIII. As a result, we generate a valid strategy for designing membrane permeable macrocycles by introducing HBD-containing amino acids into specific positions of the CsO scaffold. We believe that our research could be used as a basis for developing an orally administrable HBV entry inhibitor that can improve patient convenience.