First Insight on Small Molecules as Cardiac Calsequestrin Stabilizers

Abstract

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by mutations of cardiac calsequestrin (CASQ2) that impair its characteristic ability of Ca²⁺-induced polymerization-depolymerization. However, stabilizing the CASQ2 polymer by pharmacological agents to treat CPVT has not been reported so far. Here, we tested whether small molecules can stabilize CASQ2 polymers. We synthesized 24 glycinate/alaninate/acetate α-pyranone analogs and conducted the CASQ2 depolymerization assay. Most of the molecules of this class of compounds inhibited the depolymerization of the protein upon Ca²⁺ chelation by ethylene glycol tetraacetic acid. Structure-activity relationship studies revealed that the compounds with the 4-fluoro-phenyl group at the C-6 position of the pyranone ring and open-chain primary amine at C-4 are the most active of the class. This is the first report of an α-pyranone class of compounds with the ability to stabilize CASQ2 polymers and opens up the possibility to target Ca²⁺-release disorders via modulation of CASQ2 polymerization.

Figure 1

Structures of synthesized pyranone derivatives.

Figure 2

ORTEP diagram drawn with 50% thermal ellipsoidal probability with atom numbering scheme of compound 3b (CCDC No: 150756).

Scheme 1. Synthesis of 4,6-Disubstituted-2H-pyranone-3-carbonyl-glycinate/alaninate/acetate (3a–x)

Reagents and conditions: (i) glycine/alanine ester, HATU, DIPEA, DCM, rt, 3–4 h; (ii) aliphatic amine, ethanol, reflux, 2–6 h.

Figure 3

Colored heatmap visualizing the results of EGTA-mediated resolubilization/depolymerization assay. Concentration values are scaled by row. Compound code is indicated in the x axis. Compounds 3e, 3u, and 3v have the highest potentials (>50% at 100 nM) to inhibit depolymerization of CASQ2.

Figure 4

Compound 3u is bound at the interface of CASQ2 dimer (1SJI). (A) Electrostatic surface image of the binding site. The ester group is projected out of the protein surface. (B) Binding mode of 3u in CASQ2 dimer. The amine group forms H-bonding interaction with the first monomer, and Ph–F is involved in electrostatic interaction with the second monomer. The molecule forms several hydrophobic interactions with Ala297, Lys301, and Tyr298 of the first and Leu65, Val64, and Glu66 of the second monomer holding the two monomers together. The model was created using Maestro. The ligand is represented using ball and stick, and the interacting residues are represented in line notation. The protein structure is represented in ribbon.

Figure 5

Structure–activity relationship of synthesized pyranone derivatives.