Biology subjects

Schwarz, J. A.

Publications and source records attributed to Schwarz, J. A..

4 recordsLinked to original sources

bioRxiv2023

ent-Verticilide B1 inhibits type 2 ryanodine receptor channels and is antiarrhythmic in Casq2-/- mice

Ca2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca2+ handling causes ventricular arrhythmias. We previously discovered the RyR2-selective inhibitor ent-(+)-verticilide (ent-1), a 24-membered cyclooligomeric depsipeptide that is the enantiomeric form of a natural product (nat-(-)-verticilide). Here, we examined its 18-membered ring-size oligomer (ent-verticilide B1; "ent-B1") in single RyR2 channel assays, [3H]ryanodine binding assays, and in Casq2-/- cardiomyocytes and mice, a gene-targeted model of SCD. ent-B1 inhibited RyR2 single-channels and [3H]ryanodine binding with low micromolar potency, and RyR2-mediated spontaneous Ca2+ release in Casq2-/- cardiomyocytes with sub-micromolar potency. ent-B1 was a partial RyR2 inhibitor, with maximal inhibitory efficacy of less than 50%. ent-B1 was stable in plasma, with a peak plasma concentration of 1460 ng/ml at 10 min and half-life of 45 min after intraperitoneal administration of 3 mg/kg in mice. Both 3 mg/kg and 30 mg/kg ent-B1 significantly reduced catecholamine-induced ventricular arrhythmia in Casq2-/- mice. Hence, we have identified a novel chemical entity - ent-B1 - that preserves the mechanism of action of a hit compound and shows therapeutic efficacy. These findings strengthen RyR2 as an antiarrhythmic drug target and highlight the potential of investigating the mirror-image isomers of natural products to discover new therapeutics. Significance statementThe cardiac ryanodine receptor (RyR2) is an untapped target in the stagnant field of antiarrhythmic drug development. We have confirmed RyR2 as an antiarrhythmic target in a mouse model of sudden cardiac death and shown the therapeutic efficacy of a second enantiomeric natural product.

pharmacology and toxicology↗

bioRxiv2023

RyR2 Binding of an Antiarrhythmic Cyclic Depsipeptide Mapped Using Confocal Fluorescence Lifetime Detection of FRET

Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca2+-release channels contributes to heart failure and arrhythmias. Reducing RyR2 activity, particularly during cardiac relaxation (diastole), is a desirable therapeutic goal. We previously reported that the unnatural enantiomer (ent) of an insect-RyR activator, verticilide, inhibits porcine and mouse RyR2 at diastolic (nanomolar) Ca2+ and has in vivo efficacy against atrial and ventricular arrhythmia. To determine the ent-verticilide structural mode of action on RyR2 and guide its further development via medicinal chemistry structure-activity relationship studies, here we used fluorescence lifetime (FLT)-measurements of Forster resonance energy transfer (FRET) in HEK293 cells expressing human RyR2. For these studies, we used an RyR-specific FRET molecular-toolkit and computational methods for trilateration (i.e., using distances to locate a point of interest). Multi-exponential analysis of FLT-FRET measurements between four donor-labeled FKBP12.6 variants and acceptor-labeled ent-verticilide, yielded distance relationships placing the acceptor probe at two candidate loci within the RyR2 cryo-EM map. One locus is within the Ry12 domain (at the corner periphery of the RyR2 tetrameric complex). The other locus is sandwiched at the interface between helical domain 1 and the SPRY3 domain. These findings document RyR2-target engagement by ent-verticilide, reveal new insight into the mechanism of action of this new class of RyR2-targeting drug candidate, and can serve as input in future computational determinations of the ent-verticilide binding site on RyR2 that will inform structure-activity studies for lead optimization.

biophysics↗

bioRxiv2021

Cardiac RyR N-terminal region biosensors for FRET-based high-throughput screening

The N-terminal region (NTR) of the ryanodine receptor (RyR) calcium channels is critical to the regulation of Ca2+ release during excitation-contraction coupling. NTR hosts numerous mutations linked to skeletal and cardiac myopathies (RyR1 and RyR2, respectively), highlighting its potential as therapeutic target. Here, we labeled the NTR of mouse RyR2 at subdomains A, B, and C with donor and acceptor pairs for fluorescence resonance energy transfer (FRET), obtaining two biosensors. Using fluorescence lifetime (FLT)-detection of intramolecular FRET, we developed high-throughput screening (HTS) assays with the biosensors to identify small-molecule modulators of RyR. We screened a 1280-compound validation library and identified several hits. Hits with saturable FRET dose-response profiles, and previously unreported effects on RyR activity, were further tested using [3H]ryanodine binding to isolated sarcoplasmic reticulum vesicles, to measure their effects on full-length RyR opening in its natural membrane environment. We identified three novel inhibitors of both RyR1 and RyR2, and two RyR1-selective inhibitors at nanomolar Ca2+. These compounds may function as inhibitors of leaky RyRs in muscle. Two of these hits activated RyR1 only at micromolar Ca2+, highlighting them as potential activators of excitation-contraction coupling. These results indicate that large-scale HTS using this platform can lead to compounds with potential for therapeutic development.

biophysics↗

bioRxiv2021

Hydraulic diversity stabilizes productivity in a large-scale subtropical tree biodiversity experiment

Extreme climatic events threaten forests and their climate mitigation potential globally. Understanding the drivers promoting ecosystem stability is therefore considered crucial to mitigate adverse climate change effects on forests. Here, we use structural equation models to explain how tree species richness, asynchronous species dynamics and diversity in hydraulic traits affect the stability of forest productivity along an experimentally manipulated biodiversity gradient ranging from 1 to 24 tree species. Tree species richness improved stability by increasing species asynchrony. That is, at higher species richness, inter-annual variation in productivity among tree species buffered the community against stress-related productivity declines. This effect was mediated by the diversity of species hydraulic traits regarding drought tolerance and stomatal control, but not by the community-weighted means of these traits. The identified mechanisms by which tree species richness stabilizes forest productivity emphasize the importance of hydraulically diverse, mixed-species forests to adapt to climate change.

ecology↗