V. Illuminating the dark side of natural products
5.3 Conclusion
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or Ca2+ channel block, we measured the ECG QRS duration (prolonged by Na+ channel blockers) and the PR interval (prolonged by Ca2+ channel blockers). ent-21 had no significant effect on QRS (Figure 52A) or PR interval (Figure 52B), indicating that Na+ or Ca2+ channel block by ent-21 does not contribute to its antiarrhythmic activity in vivo.
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release mediated arrhythmias;178 however, new small-molecule tools are needed to further understand the role of Ca2+ flux regulators and their relevance for preventing arrhythmias, especially since flecainide is contraindicated in patients with structural heart disease and heart failure.202 In the brain, inhibition of RyR2-mediated Ca2+ leak has proven beneficial in preventing or delaying the onset of neuropathological symptoms in a variety of diseases. Our current investigation led us to examine the effects of a known insect RyR modulator, nat-(-)-verticilide, two synthetic precursors, and their mirror image isomers (ent). Whereas natural verticilide had no effect on mammalian RyR2, remarkably, we found that its enantiomer significantly inhibited RyR2-mediated Ca2+ leak by a distinct mechanism of action compared to other RyR2 inhibitors (Figure 53).
ent-(+)-Verticilide significantly attenuated spontaneous Ca2+ release in myocytes isolated from both a CPVT mouse model and wild-type mice. Importantly, nat-21, 52, 53, and their respective enantiomers did not bind to Ca2+. The combined reduction of Ca2+ spark frequency, amplitude, and mass resulted in a drastic reduction of Ca2+ leak in the presence of ent-(+)- verticilide, which is a different mode of action than the prototype compounds tetracaine and flecainide. Additionally, [3H]ryanodine binding experiments revealed that ent-(+)-verticilide selectively acts on RyR2, and not RyR1. This finding is significant because current RyR probes (e.g. flecainide and tetracaine) block all RyR subtypes, making them inadequate tools to study the role of RyR2-mediated Ca2+ leak in various diseases. ent-(+)-Verticilide inhibited spontaneous Ca2+ release in intact cardiomyocytes (as opposed to only permeabilized), and therefore
202 Echt, D. S.; Liebson, P. R.; Mitchell, L. B.; Peters, R. W.; Obias-Manno, D.; Barker, A. H.; Arensberg, D.; Baker, A.; Friedman, L.; Greene, H. L.; et al. N. Engl. J. Med. 1991, 324, 781.
Figure 53. Summary of our discovery of a novel inhibitor of RyR2-mediated Ca2+ leak
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demonstrated ability to effectively permeate a cell membrane. Furthermore, the activity observed in vitro translated to in vivo studies, where ent-(+)-verticilide significantly reduced isoproterenol- induced PVCs in Casq2-/- mice. These data suggest that we have discovered a novel potent inhibitor of intracellular Ca2+ leak, and a potentially promising lead compound for drug discovery platforms aimed at developing therapeutics for heart failure, atrial fibrillation, and CPVT, and neurological diseases such as Alzheimer’s disease, seizures, and neurodegenerative disorders.
However, a definite molecular mechanism for inhibition of spontaneous Ca2+ release by ent-(+)-verticilide remains unknown, as verticilide does not resemble any other known Ca2+ release inhibitors. The observed decrease in spontaneous Ca2+ release by ent-(+)-verticilide may be due to direct binding to RyR2, or it may indirectly regulate RyR2 opening via accessory proteins.
Previous studies have shown that calmodulin (CaM),203 Ca2+-CaM Kinase II,204 and FK 506 proteins,205 among others,206 are all important regulators of RyR2 function. It is possible that ent- (+)-verticilide suppresses Ca2+ leak by indirectly inhibiting RyR2 via one of these mechanisms.
Future studies are needed to establish the precise molecular mechanism(s) responsible for ent-(+)- verticilide-induced Ca2+ spark suppression.
At this time, however, our report highlights a rare and exciting discovery of an unnatural enantiomer that potently suppresses RyR2-mediated Ca2+ leak, while the natural product is completely inactive. Due to synthetic barriers, there are few instances where the biological activity of an unnatural enantiomer can be studied in comparison to its naturally occurring analogue184,185,186,207,208,209,210,211,212,213 Within these instances, the unnatural enantiomer is generally found to be comparably potent to the natural product,186,209-213 and there are a select few examples where the unnatural enantiomer is greater than 3x more potent than the natural product.184,185 However, our discovery is unique since, to the best of our knowledge, there are no examples where the unnatural enantiomer of a natural product is highly potent in a biological system while the natural enantiomer is completely inactive. Our ability to access the unnatural – or “dark” –enantiomer and discovery of its activity raises the question whether similar dark chemical space holds generally untapped potential. In the case of verticilide, the unnatural enantiomer induces a pharmacological behavior similar, but mechanistically orthogonal to existing Ca2+ release inhibitors flecainide and tetracaine, while the natural product is inactive. This discovery not only confirms the importance of developing straightforward synthetic methods toward natural products and their derivatives, but also contributes to the ongoing excitement to explore ‘dark chemical space’182,187 for chemical biology.
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