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Calming the Hyperactive Heart


By: Carol A. Rouzer, VICB Communications
Published:  March 19, 2019

 

The unnatural enantiomer of a fungal natural product blocks the ryanodine receptor 2, preventing some forms of heart arrhythmia.

 

The ryanodine receptor is an intracellular Ca2+ release channel that plays a role in the physiology of excitable cells, such as neurons and myocytes. The three isoforms of the receptor, RyR1, RyR2, and RyR3, are differentially expressed in tissues throughout the body where they carry out distinct functions. RyR2 is particularly important in the heart because of its role in excitation-contraction coupling. Dysfunction of RyR2 can lead to serious and potentially fatal cardiac arrhythmias in a condition known as catecholaminergic polymorphic ventricular tachycardia (CPVT). For this reason, selective inhibitors of RyR2 are a topic of considerable research interest. Now, Vanderbilt Institute of Chemical Biology member Jeffrey Johnston, his collaborator Björn Knollmann (Department of Medicine, VUMC), and their laboratories announce the discovery that the enantiomer of a fungal natural product is a potent and selective inhibitor of RyR2 [S. M. Batiste, D. J. Blackwell, D. O. Kryshtal, et al. Proc. Natl. Acad. Sci. U.S.A. (2019) published online February 21, doi: 10.1073/pnas.1816685116].

 

During normal cardiomyocyte excitation-contraction coupling, RyR2 functions to release Ca2+ from the sarcoplasmic reticulum (SR) in response to an initial flow of Ca2+ into the cell through electrically stimulated L-type Ca2+ channels in the plasma membrane (sarcolemma) (Figure 1). The Ca2+ released by RyR2 is necessary to stimulate contraction by the nearby myofilaments. Dysfunction occurs either as a result of mutation of RyR2 or one of its associated modulatory proteins or through disease-associated damage that causes leakage of Ca2+ through the channel. This inappropriately timed flow of Ca2+ triggers the activation of the Na+/Ca2+ exchange transporter (NCX) on the cell surface, leading to depolarization and an associated contraction during diastole, when the cell should be in the relaxed state. These delayed after depolarizations (DADs) lead to arrhythmia, a situation that is exacerbated in the presence of catecholamines. A number of RyR2 inhibitors or putative inhibitors, including flecainide, dantrolene, and tetracaine (Figure 2) are available, but none of these provides the potency and selectivity required for use as a research tool compound or as a first line therapy for CPVT. This led the researchers to focus on verticilide, a fungal cyclooligomeric depsipeptide natural product known to block the insect ryanodine receptor.

 

FIGURE 1. Function of RyR2 in cardiomyocytes. RyR2 (shown in gray) is located in the sarcoplasmic reticulum (SR) membrane in regions where the SR is very closely associated with the plasma membrane (sarcolemma, SL). The two structures are separated by the very narrow dyadic cleft. An action potential opens the L-type Ca2+ channel (LCC) in the SL, leading to an influx of Ca2+ into the dyadic cleft. The Ca2+ triggers Ca2+-induced Ca2+ release (CICR) by RyR2, leading to a flood of Ca2+ into the dyadic cleft. This Ca2+ diffuses out into the cytoplasm where it reaches the myofilaments, stimulating contraction. Then re-uptake of the Ca2+ into the SR by the sarcoplasmic reticulum ATPase (SERCA) and expulsion from the cell by the Na+/Ca2+ exchange protein (NCX) returns the system to its resting state. Figure reproduced under the Creative Commons Attribution 4.0 International License from L. M. Blayney & F. A. Lai, (2009) Pharmacol. Therapeut., 123, 151.

 

 

 

FIGURE 2. Compounds that block one or more of the ryanodine receptors. Figure reproduced with permission from S. M. Batiste, D. J. Blackwell, D. O. Kryshtal, et al. Proc. Natl. Acad. Sci. U.S.A. (2019) published online February 21, doi: 10.1073/pnas.1816685116. Copyright 2019.

 

 

Cycloodepsipeptides are polymers of hydroxy acids and amino acids that have cyclized to form a large ring (Figure 3). Many natural products in this class possess interesting biological activities, and as one might expect, those isolated from biological sources are chiral molecules that are synthesized in a single enantiomeric form. In the case of verticilide, the natural product is nat-(‒)-verticilide (nat-1) (Figure 2). The Johnston lab had developed a chemical synthesis of this molecule from linear dipeptide and tetradepsipeptide precursors using macrocyclooligomerization [S. M. Batiste & J. N. Johnston, Proc. Natl. Acad. Sci. U.S.A., (2016) 113, 14893]. The beauty of this approach was that it also enabled synthesis of the enantiomer of nat-1, ent-(+)-verticilide (ent-1), and it provided opportunities for chemical modification of the compounds. For this particular study, they synthesized nat-1, ent-1, and precursors of both molecules that lacked the methyl groups on the amide nitrogen atoms.

 

 

FIGURE 3. Depsipeptides are polymers of amino acids and hydroxy acids linked by peptide (red) and ester (blue) linkages. A cyclodepsipeptide results from the cyclization reaction of a linear depsipeptide.


 

The Knollmann lab investigators tested the effects of all four compounds on RyR2-mediated Ca2+ release in cardiomyocytes from calsequestrin knockout (Casq2-/-) mice. Calsequestrin is a Ca2+ binding protein that modulates RyR2 function in cardiomyocytes. Mice lacking this protein serve as a validated model for CPVT. The researchers monitored Ca2+ release in Casq2-/- cardiomyocytes by preincubating them with the fluorescent Ca2+ chelator Fura-2 AM followed by treatment with the catecholamine isoproterenol to promote leakage. Sporadic bursts of Ca2+ from leaking RyR2 produced fluorescent "Ca2+ sparks" that could be registered microscopically. Treatment of the cells with the four compounds of interest led to no effect except in the case of ent-1, which substantially reduced detected sparks. Further work demonstrated that ent-1 had no effect on baseline free cytoxolic Ca2+ levels, the interaction of RyR2 with its regulatory binding partners, or phosphorylation of RyR2. Ent-1 had no effect on RyR1, so it was more selective than tetracaine, which inhibits the function of both proteins.
     

The researchers next carried out dose-response studies to determine the ability of ent-1 to inhibit [3H]ryanodine binding to RyR2 in SR membrane vesicles. Ryanodine, a diterpenoid from the Ryania speciosa plant, binds to the open form of RyR2. Hence, ryanodine binding can be used to assay RyR2 activity. Ent-1 inhibited [3H]ryanodine binding to RyR2 with relatively high potency (IC50 of 0.1 μm) but poor efficacy (maximum inhibition of 20%) in this assay.
     

In an expansion of their studies, the investigators next explored the effects of ent-1 on Ca2+ sparks in cardiomyocytes from wild-type mice and mice bearing the R4496C mutation of the RyR2 receptor, which causes CPVT in humans. In both cases, ent-1 reduced both the frequency and amplitude of the sparks. Thus, ent-1 differed from dantrolene and tetracaine, which reduce spark frequency but do not affect amplitude and from flecainide, which reduces spark amplitude while actually increasing frequency. Thus ent-1 was more effective than the other three compounds in this model system, and it was also more potent. Notably, its efficacy in intact cells was substantially higher than that observed in the binding assay.
     

Electrophysiological studies demonstrated that ent-1 reduced the frequency and amplitude of DADs in Casq2-/- cardiomyocytes cultured in the presence of isoproterenol. Again, ent-1 reduced both the frequency and amplitude of DADs without affecting the normal cardiomyocyte action potential. These encouraging findings led the researchers to test the effects of ent-1 in Casq2-/- mice in vivo. The compound had no effect on the baseline heart rate or electrocardiograms of subject mice; however, it reduced the peak heart rate observed following administration of isoproterenol, and it also decreased the frequency of isoproterenol-induced ventricular ectopic beats and tachycardia.
     

Together, the results demonstrate that ent-1 is a promising lead compound for the study of RyR2 function, and potentially for treatment of CPVT patients. A clear advantage for future research is that a synthetic scheme is available that will allow testing of the effects of structural modifications of this interesting scaffold. Thus far, we know that having the correct enantiomer is critical and that the N-methyl groups are required. As a test of one linear precursor demonstrated no activity, we also know that the compound must retain its cyclic structure. These observations suggest a specific ligand-receptor type of interaction that can be explored and exploited further.

 

 

 

ViewPNAS article: Unnatural verticilide enantiomer inhibits type 2 ryanodine receptor-mediated calcium leak and is antiarrhythmic

 

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