Studies on macrocyclic peptide scaffolds with conformation, membrane permeability, and antiviral activity

Abstract

A protein-protein interaction (PPI) is a vital process among biomolecules to regulate biological systems such as signaling transductions from extra- to intracellular proteins or receptor-hormone recognitions. PPIs are mediated by the large surface area of the desired biomolecules such as proteins, receptors, DNA, or RNA. Thus, it has been considered the most challenging target by traditional modalities, such as small molecules due to the large surface area. To complement their cons, a molecular size has been extended beyond rule-of-five (e.g., Harvoni), or different molecular entities such as biologics (e.g., Humira) are released in therapeutic markets. However, targeting intracellular PPIs is still difficult, and alternative molecular entities are being explored to meet this unmet need. Macrocycles have emerged as a potent molecular class for tackling challenging targets such as intracellular PPIs. Thus, a deep understanding of macrocycles with excellent physicochemical properties and biological activities is required. This work demonstrates macrocyclic peptides with conformation, physicochemical properties, and antiviral activity and analyzes their relationships. In chapter 2, a fundamental investigation of a base macrocyclic scaffold, cyclosporin O (CsO), was described such as structural and physicochemical properties. CsO has been underexplored due to the less biological activity, although it is a unique member with no MeBmt1 residue in the cyclosporine family. Here, we focused on the structural features of CsO, which are the same as that of cyclosporin A (CsA), rather than the biological activity to adopt excellent physicochemical properties. We evaluated conformation, flexibility, lipophilicity, and membrane permeability, and explained their relationships. The conformational flexibility rationalized the different membrane permeability and protein binding between CsA and CsO. The discrepancy in in vivo pharmacokinetic profiles obtained for CsA and CsO was explained by the blood-to-plasma partitioning ratio and cyclophilin A binding. CsO exhibited good oral bioavailability (F = 12%) despite its large size even without any formulation, whereas F value is not determined for CsA where self-emulsifying formulation was not adopted. Our observations suggested that CsO can be a promising scaffold to reach membrane permeable and oral bioavailable macrocycles. In chapter 3, the conformational flexibility, called the chameleonic behavior, has been investigated as a decisive conformational property to reconciling membrane permeability, solubility, and protein binding of large macrocycles. However, there is no rational guideline to exploit the chameleonic behavior on macrocycles. We designed four series of CsO derivatives with side chain and backbone modifications to determine design principles controlling the chameleonic behavior in the macrocycles. The population ratio obtained from NMR spectra was used to describe the chameleonic behavior, and CsO derivatives showed the different extents depending on the structural modifications; it was rationalized by the conformation and alternative H-bond networks. We identified the structural components that can control the chameleonic behavior of macrocycles. The impact of the chameleonic behavior on lipophilicity and membrane permeability was evaluated, and their graph showed distinctive relationships regarding the chameleonic behavior. It suggested that the chameleonic behavior can complement some degree of polarity, and moderate flexibility is required to maximize membrane permeability.Our results will provide general guidelines that can control the chameleonic behavior to reach the optimal membrane permeability of macrocycles. In chapter 4, we generated a novel CsO library by incorporating peptoid side chains for biological applications. Recently, it was reported that CsA blocked sodium-taurocholate cotransporting polypeptide (NTCP), which is an entry of hepatitis B virus (HBV) invasion on hepatocytes, and thus, the CsO library screened as HBV entry inhibitors. In vitro HBV entry inhibition assay was performed at a single concentration, and IC50 values were obtained for four derivatives that exhibited comparable inhibitory activity, as shown in CsA. Among them, the compound 21 showed the greatest potency (IC50 = 0.36 ± 0.1 μM) with minimal cytotoxicity (LC50 > 200 μM).We prove the biological applicability of our CsO scaffold and will utilize it for screening various targets. In chapter 5, we designed a macrocyclic peptide library consisting of the sequence adopted from the PreS1 domain of HBV large surface protein to inhibit HBV invasion. PreS1 macrocyclic derivatives were rationally modified by focusing on the pharmacophore domain and the proteolytic stability because it consists of natural amino acids. In vitro HBV entry inhibitory activity and whole blood stability were evaluated. Peptide 4 exhibited potent inhibitory activity and improved proteolytic stability than the parent linear peptide. A longer half-life of 1.9 ± 0.3 h was confirmed under intravenous injection in in vivo pharmacokinetic studies, and significant reduction of intracellular HBV DNA and covalently closed circular DNA (cccDNA) were observed in in vivo infectious mouse model with a humanized liver.