Deconvolution of Physicochemical Properties Contributing to Passive Diffusion of Depsipeptides

项目总结

PROJECT SUMMARY Calcium in cardiomyocytes is released via an intracellular calcium channel, ryanodine receptor 2 (RyR2). In hearts where there are mutations in RyR2, spontaneous Ca2+ leakages can occur resulting in cardiac arrhythmias. Small molecule therapeutics such as flecainide, tetracaine, and dantrolene have low specificity, low membrane permeability, low solubility, poor selectivity, low potency, or toxicity. Therapeutic selectivity between RyR isoforms (RyR1, RyR2, RyR3) is lacking, heightening our interest in developing ent-verticilide as an anti- arrhythmic agent. This proposal focuses on the discovery and development of new therapeutics as antiarrhythmic agents. The proposed work is founded on our discovery of potent and selective inhibition of RyR2-mediated calcium flux by ent-verticilide. Through a cross-disciplinary collaboration, it was discovered that ent-verticilide is a selective inhibitor of RyR2-mediated calcium release, including a preliminary study of efficacy in vivo. As a 24 membered cyclic depsipeptide with a molecular weight of 853 Da, ent-verticilide falls outside of the category of a traditional small molecule drug. While “Beyond Rule of 5” compounds with in vivo activity are growing in number, an understanding of their pharmacokinetics (PK) has lagged, thereby requiring new chemical tools and creative tactics to advance the field. We will investigate the permeability of this unnatural product and its analogues by development of a structure-activity relationship profile focused on both permeability and increased efficacy. We aim to systematically design and synthesize structural analogues of ent-verticilide with varying degrees of N-methylation. Following the synthesis, we will study passive permeability and collect structural data to inform SAR, providing additional perspective for feedback to Specific Aim 1. By methodicalstructural change to ent-verticilide, we will create an SAR-based feedback loop between permeability, activity, structure, and conformation. A strength of this approach is the combination of rigorous tools to study passive membrane permeability, and cardiomyocyte-based functional studies using both permeabilized and non-permeabilized cells to achieve an overall hypothesis-driven approach to discover how analogues of ent-verticilide travel through cellular membranes and ultimately target RyR2. Another strength is our positioning to prepare diverse analogues that include ring-chain variants likely to exhibit contrasting permeability. With increased understanding of the mechanism of action, we hypothesize that we can design analogues of ent-verticilide with improved potency and selectivity, thereby providing a potential therapeutic against fatal ventricular arrhythmias.