Rational molecular targeting of the inter-subunit interaction between human cardiac troponin hcTnC and hcTnI using switch peptide-competitive biogenic medicines

Highlights

• Biogenic competitors are used to competitively disrupt human cardiac hcTnC–hcTnI interaction.

• A multistep screening protocol is performed against a biogenic compound library to identify competitors.

• Small-molecule collismycin and compound e are identified as potent competitors.

• Both competitors and hcTnI switch peptide share a common binding site of hcTnC.

Abstract

The human cardiac troponin (hcTn) has been implicated in diverse cardiovascular diseases (CDs). The protein function is regulated by the inter-subunit interaction between the N-terminal domain of hcTnC and the C-terminal switch peptide of hcTnI; disruption of the interaction has been recognized as a potential therapeutic strategy for CDs. Here, we report use of biogenic medicines as small-molecule competitors to directly disrupt the protein–protein interaction by competitively targeting the core binding site (CBS) of hcTnC NTD domain. A multistep virtual screening protocol is performed against a biogenic compound library to identify competitor candidates and competition assay is employed to verify the screening results. Consequently, two compounds Collismycin and Compound e are identified as strong competitors (CC₅₀ < 10 μM) with hcTnI for hcTnC CBS site, while other tested compounds are found to have moderate (CC₅₀ = 10–100 μM), low (CC₅₀ > 100 μM) or no (CC₅₀ = N.D.) potency. The competitor ligands are anchored at the core groove of hcTnC CBS site through aromatic and hydrophobic interactions, while few peripheral hydrogen bonds are formed to further confer specificity for domain–compound recognition. These molecular-level findings would benefit from further in vitro and in vivo studies at cellular and animal levels, which can help to practice the ultimate therapeutic purpose.

Introduction

The human cardiac troponin (hcTn) is a calcium-sensitive protein that plays a crucial role in mediation of the intermolecular interaction between actin and myosin (Katrukha, 2013), which is a complex of three regulatory subunits hcTnC, hcTnT and hcTnI with different structures and functions (Sharma et al. (2004)). Calcium binding triggers a complicated conformational change that leads to the recognition and interaction of hcTn with actin and tropomyosin and anchors the hcTn into thin filament (Davis and Tikunova, 2008). hcTn is closely associated with the pathogenesis of cardiovascular diseases (CDs), which has been developed as a good cardiac biomarker and potential therapeutic target of diverse CDs (Hachey et al., 2017). Although the elevated level of all the three hcTn subunits can be used in disease diagnosis, only the hcTnC has been considered as the druggable site of calcium-sensitizing agents (Potluri et al., 2004). Crystallographic analysis solved the three-dimensional structure of hcTn complex (White et al., 1987), which contains a regulatory head and an IT arm as well as a flexible liner between them (Fig. 1). The regulatory head consists of a calcium-bound N-terminal domain (NTD) of hcTnC and a switch peptide of hcTnI. The switch peptide is at the C-terminal tail of hcTnI and packs against a switch peptide-binding pocket (SPBP) on the NTD surface of hcTnC. The SPBP pocket also contains a core binding site (CBS) that has a core groove to accommodate the peptide in the pocket. The peptide exhibits self-binding behavior (Yang et al., 2015a; (2015b)) that serves as a molecular switch to regulate the biological function of hcTn tri-complex system. Recent study demonstrated that the calcium plays an essential role in regulation of the intermolecular interaction of hcTnI switch peptide with hcTnC NTD domain; lack of calcium causes a considerable unfolding in the domain and largely stabilizes the interaction (Yu et al., 2019).

Direct targeting of the intermolecular interaction of hcTnI switch peptide with hcTnC NTD domain has been exploited as a new and promising strategy for the treatment of CDs. For example, Xu et al. (2017) have successfully designed peptide inhibitors to target the interaction as potential therapeutics for heart failure based on previously described self-binding peptides (Bai et al., 2017). Instead, we herein attempted employing small-molecule compounds to disrupt the inter-subunit hcTnC–hcTnI interaction by competitively targeting the CBS site of hcTnC NTD domain. Previously, integrative screening strategy was described to successfully discover competitor compounds for druggable protein–peptide complex systems (Shen et al., 2019; Xu et al., 2019). With the strategy we were able to perform high-throughput virtual screening against a structurally diverse biogenic compound library to identify potential competitors with hcTnI switch peptide for hcTnC CBS site. Their competitive potency on the intermolecular interaction between hcTnC NTD domain and hcTnI switch peptide were also investigated systematically at molecular level.