Backbone-Determined Antiarrhythmic Structure-Activity Relationships for a Mirror Image, Oligomeric Depsipeptide Natural Product.

Cyclic oligomeric depsipeptides (COD) are a structural class within naturally occurring compounds with a wide range of biological activity. Verticilide is a COD (24-membered ring) that was identified by its inhibition of insect ryanodine receptor (RyR). We have since found that the enantiomer of verticilide (ent-verticilide, 1) is a potent inhibitor of mammalian RyR2, a cardiac calcium channel, and therefore a potential antiarrhythmic agent. Oddly, nat-verticilide does not inhibit RyR2. To further develop ent-verticilide as an antiarrhythmic, we explored potential SAR through systematic modification of the ester's functionality to both N-H and N-Me amides. The syntheses of these ent-verticilide-inspired analogs are detailed using a monomer-based platform enabled by enantioselective catalysis. Two analogs among 23 exhibited measurable reduction of calcium sparks in a functional assay of RyR2 activity. These findings illustrate the value of natural product-inspired therapeutic development, but the less-studied approach where the non-natural enantiomeric series harbors important SAR.

ent-Verticilide B1 Inhibits Type 2 Ryanodine Receptor Channels and is Antiarrhythmic in Casq2 -/- Mice.

Intracellular 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 RyR2 single channel and [3H]ryanodine binding assays, and in Casq2 -/- cardiomyocytes and mice, a gene-targeted model of SCD. ent-B1 inhibited RyR2 single channels 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 minutes and half-life of 45 minutes after intraperitoneal administration of 3 mg/kg in mice. In vivo, ent-B1 significantly reduced catecholamine-induced ventricular arrhythmias in Casq2 -/- mice in a dose-dependent manner. 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 STATEMENT: The 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.

Quantifying microplastic ingestion, degradation and excretion in insects using fluorescent plastics.

Plastic pollution is a growing threat to our natural environment. Plastic waste/pollution results from high emissions of both macro (>5 mm) and microplastics (MPs; <5 mm) as well as environmental fractioning of macroplastics into MPs. MPs have been shown to have a range of negative impacts on biota. Harmonized methods to accurately measure and count MPs from animal samples are limited, but what methods exist are not ideal for a controlled laboratory environment where plastic ingestion, degradation and elimination can be quantified and related to molecular, physiological and organismal traits. Here, we propose a complete method for isolating and quantifying fluorescent MPs by combining several previously reported approaches into one comprehensive workflow. We combine tissue dissection, organic material digestion, sample filtering and automated imaging techniques to show how fluorescently labelled MPs provided to insects (e.g. in their diet) in a laboratory setting can be isolated, identified and quantified. As a proof of concept, we fed crickets (Gryllodes sigillatus) a diet of 2.5% (w/w) fluorescently labelled plastics and isolated and quantified plastic particles within the gut and frass.

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

Ca 2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca 2+ 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, [ 3 H]ryanodine binding assays, and in Casq2 -/- cardiomyocytes and mice, a gene-targeted model of SCD. ent -B1 inhibited RyR2 single-channels and [ 3 H]ryanodine binding with low micromolar potency, and RyR2-mediated spontaneous Ca 2+ 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 statement: The 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.

The selective RyR2 inhibitor ent-verticilide suppresses atrial fibrillation susceptibility caused by Pitx2 deficiency.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and a major cause of stroke and morbidity. The strongest genetic risk factors for AF in humans are variants on chromosome 4q25, near the paired-like homeobox transcription factor 2 gene PITX2. Although mice deficient in Pitx2 (Pitx2+/-) have increased AF susceptibility, the mechanism remains controversial. Recent evidence has implicated hyperactivation of the cardiac ryanodine receptor (RyR2) in Pitx2 deficiency, which may be associated with AF susceptibility. We investigated pacing-induced AF susceptibility and spontaneous Ca2+ release events in Pitx2 haploinsufficient (+/-) mice and isolated atrial myocytes to test the hypothesis that hyperactivity of RyR2 increases susceptibility to AF, which can be prevented by a potent and selective RyR2 channel inhibitor, ent-verticilide. Compared with littermate wild-type Pitx2+/+, the frequency of Ca2+ sparks and spontaneous Ca2+ release events increased in permeabilized and intact atrial myocytes from Pitx2+/- mice. Atrial burst pacing consistently increased the incidence and duration of AF in Pitx2+/- mice. The RyR2 inhibitor ent-verticilide significantly reduced the frequency of spontaneous Ca2+ release in intact atrial myocytes and attenuated AF susceptibility with reduced AF incidence and duration. Our data demonstrate that RyR2 hyperactivity enhances SR Ca2+ leak and AF inducibility in Pitx2+/- mice via abnormal Ca2+ handling. Therapeutic targeting of hyperactive RyR2 in AF using ent-verticilide may be a viable mechanism-based approach to treat atrial arrhythmias caused by Pitx2 deficiency.

No accumulation of microplastics detected in western Canadian ringed seals (Pusa hispida).

Ringed seals (Pusa hispida) play a crucial role in Arctic food webs as important pelagic predators and represent an essential component of Inuvialuit culture and food security. Plastic pollution is recognized as a global threat of concern, and Arctic regions may act as sinks for anthropogenic debris. To date, mixed evidence exists concerning the propensity for Canadian Arctic marine mammals to ingest and retain plastic. Our study builds on existing literature by offering the first assessment of plastic ingestion in ringed seals harvested in the western Canadian Arctic. We detected no evidence of microplastic (particles ≥80 µm) retention in the stomachs of ten ringed seals from the Inuvialuit Settlement Region (ISR) in the Northwest Territories, Canada. These results are consistent with previous studies that have found that some marine mammals do not accumulate microplastics in evaluated regions.

Structure-Activity Relationships for the N-Me- Versus N-H-Amide Modification to Macrocyclic ent-Verticilide Antiarrhythmics.

The synthesis of all N-Me and N-H analogues of ent-verticilide is described, enabling a structure-activity relationship study based on cardiac ryanodine receptor (RyR2) calcium ion channel inhibition. The use of permeabilized cardiomyocytes allowed us to correlate the degree of N-methylation with activity without concern for changes in passive membrane permeability that these modifications can cause. A key hypothesis was that the minimal pharmacophore may be repeated in this cyclic oligomeric octadepsipeptide (a 24-membered macrocycle), opening the possibility that target engagement will not necessarily be lost with a single N-Me → N-H modification. The effect in the corresponding 18-membered ring oligomer (ent-verticilide B1) was also investigated. We report here that a high degree of N-methyl amide content is critical for activity in the ent-verticilide series but not entirely so for the ent-verticilide B1 series.

Resolving Bromonitromethane Sourcing by Synthesis: Preparation at the Decagram Scale.

The accessibility of bromonitromethane has declined in recent years, limiting its viability as a reagent for chemical synthesis. The reinvestigation and optimization of a variety of preparations, and the development of safe operating principles, are described. The reproducible protocol described here leverages the effectiveness of hydroxide for nitromethane bromination while respecting its incompatibility with the product it forms. This careful balance was achieved at scales up to 56 g, resulting in a reproducible procedure that provides straightforward, sustainable, and affordable access to this critical reagent.

Microplastics around an Arctic seabird colony: Particle community composition varies across environmental matrices.

Plastic pollution is a contaminant of global concern, as it is present even in remote ecosystems - like the Arctic. Arctic seabirds are vulnerable to ingesting plastic pollution, and these ingested particles are shed in the form of microplastics via guano. This suggests that Arctic seabird guano may act as a vector for the movement of microplastics into and around northern ecosystems. While contaminant-laden guano deposition patterns create a clear concentration gradient of chemicals around seabird colonies, this has not yet been investigated with plastic pollution. Here we tested whether a contaminant gradient of plastic pollution exists around a seabird colony that is primarily comprised of northern fulmars (Fulmarus glacialis) in the Canadian Arctic. Atmospheric deposition, surface water, and surface sediment samples were collected below the cliff-side of the colony and at increasing intervals of 1 km from the colony. Fulmars were also collected when foraging away from their colony. Microplastics and other anthropogenic microparticles were identified in all three environmental matrices as well as fulmar guano. Fibers were the most common shape in fulmar guano, atmospheric deposition and surface sediment, and fragments were the most common shape in surface water. We did not find a gradient of microplastic concentrations in environmental matrices related to distance from the colony. Combined, these results suggest that fulmars are not the primary source of microplastic around this colony. Further research is warranted to understand sources of microplastics to the areas around the colonies, including to what extent seabirds transport and concentrate microplastics in Arctic ecosystems, and whether concentrations proximate to large colonies may be species dependent.

Breeding seabirds as vectors of microplastics from sea to land: Evidence from colonies in Arctic Canada.

The presence and persistence of microplastics in the environment is increasingly recognized, however, how they are distributed throughout environmental systems requires further understanding. Seabirds have been identified as vectors of chemical contaminants from marine to terrestrial environments, and studies have recently identified seabirds as possible vectors of plastic pollution in the marine environment. However, their role in the distribution of microplastic pollution in the Arctic has yet to be explored. We examined two species of seabirds known to ingest plastics: northern fulmars (Fulmarus glacialis; n = 27) and thick-billed murres (Uria lomvia; n = 30) as potential vectors for the transport of microplastics in and around breeding colonies. Our results indicated anthropogenic particles in the faecal precursors of both species. Twenty-four anthropogenic particles were found in the fulmar faecal precursor samples (M = 0.89, SD = 1.09; 23 fibres and one fragment), and 10 anthropogenic particles were found in the murre faecal precursor samples (M = 0.33, SD = 0.92; 5 fibres, 4 fragments, and one foam). Through the use of bird population surveys and the quantification of anthropogenic particles found in the faecal precursors of sampled seabirds from the same colony, we estimate that fulmars and murres may deposit between 3.3 (CIboot 1.9 × 106-4.9 × 106) and 45.5 (CIboot 9.1 × 106-91.9 × 106) million anthropogenic particles, respectively, per year into the environment during their breeding period at these colonies. These estimates indicate that migratory seabirds could be contributing to the distribution and local hotspots of microplastics in Arctic environments, however, they are still likely a relatively small source of plastic pollution in terms of mass in the environment and may not contribute as much as other reported sources such as atmospheric deposition in the Arctic.

No plastics detected in seal (Phocidae) stomachs harvested in the eastern Canadian Arctic.

Through collaboration with Inuit hunters, we examined the stomach contents of 142 seals (ringed seals [Phoca hispida; n = 135], bearded seals [Erignathus barbatus; n = 6], and one harbour seal [Phoca vitualina; n = 1]) hunted between 2007 and 2019 from communities around Nunavut to assess whether seals in the eastern Canadian Arctic ingest and retain plastics in their stomachs. The seals in this study ranged from juveniles to adults of up to 30 years of age, and 55% of the seals were males. We found no evidence of plastic ingestion in any of the seals suggesting that seals in Nunavut are not accumulating plastics (>425 µm) in their stomachs. These data provide important baseline information for future plastic pollution monitoring programs in the Arctic.