Discovery of Novel Small-Molecule Calcium Sensitizers for Cardiac Troponin C: A Combined Virtual and Experimental Screening Approach

Abstract

Heart failure is a leading cause of death throughout the world and is triggered by a disruption of the cardiac contractile machinery. This machinery is regulated in a calcium-dependent manner by the protein complex troponin. Calcium binds to the N-terminal domain of cardiac troponin C (cNTnC) setting into motion the cascade of events leading to muscle contraction. Because of the severity and prevalence of heart failure, there is a strong need to develop small-molecule therapeutics designed to increase the calcium sensitivity of cardiac troponin in order to treat this devastating condition. Molecules that are able to stabilize an open configuration of cNTnC and additionally facilitate the binding of the cardiac troponin I (cTnI) switch peptide have the potential to enable increased calcium sensitization and strengthened cardiac function. Here, we employed a high throughput virtual screening methodology built upon the ability of computational docking to reproduce known experimental results and to accurately recognize cNTnC conformations conducive to small molecule binding using a receiver operator characteristic curve analysis. This approach combined with concurrent stopped-flow kinetic experimental verification led to the identification of a number of sensitizers, which slowed the calcium off-rate. An initial hit, compound 4, was identified with medium affinity (84 ± 30 μM). Through refinement, a calcium sensitizing agent, compound 5, with an apparent affinity of 1.45 ± 0.09 μM was discovered. This molecule is one of the highest affinity calcium sensitizers known to date.

Gaussian accelerated molecular dynamics: Principles and applications

“…One of the current therapies 172 is to design small molecules that can stabilize an open structure of the TnC and facilitate binding of the TnC switch peptide. To identify potential small molecules for the treatment, Coldren et al 101 combined GaMD and high‐throughput virtual screening to predict binding conformations and affinities of small molecules in TnC. The simulations were compared with experiments for the TnC protein structures in complex with calcium sensitivity modulators.…”

“…In this review, we will present the principles and the most recent applications of GaMD. Robust GaMD has been established for advanced simulation studies of a wide range of biomolecular systems, especially the protein–nucleic acid interactions 63–65 such as the CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 gene‐editing system, 66,67 protein–protein/peptide interactions, 68–72 protein‐ligand binding, 55,56,73–76 protein folding, 77 protein enzymes, 52,58,78–92 membrane proteins (including GPCRs, 68,69,73,76,93–95 ion channels, 53,96 and γ‐secretase 97 ) and carbohydrates, 98–100 as well as drug design 101,102 …”

Actives-Based Receptor Selection Strongly Increases Success Rate in Structure-Based Drug Design and Leads to Identification of 22 Unique Potent Cancer Inhibitors

“…We have successfully demonstrated that knowledge of known actives significantly improves virtual screening. Previously, we had employed a similar strategy for a single target system, cardiac troponin with noteworthy success 63,64 . In this work, we verified the strength and generalizability of this approach over a diverse selection of target systems.…”

Molecular dynamics provides new insights into the mechanism of calcium signal transduction and interdomain interactions in cardiac troponin

“…This observation emphasizes the importance of protein dynamics to its function, although the observed timescale in hydrogen-deuterium exchange experiments is quite different from those accessible with MD simulations. Second, the knowledge of the structural details can guide the development of Ca 2+ sensitizers and other troponin-binding compounds [19][20][21] for heart disease therapy. TnC, as well as TnC-TnI interfaces, are generally targeted by cardio-tonic drugs [22,23], and even the putative agent of the 'French paradox' -trans-resveratrol is also a TnC N-lobe targeting compound [24].…”