Abigail N. Smith

Although alkylated 5-membered heteroarenes are common in pharmaceuticals, there are limited methods to construct them by cross- electrophile coupling. In this report, a systematic study of 5-membered heteroaryl bromides sheds light on an unappreciated reason for this challenge: their relative reactivity spans almost three orders of magnitude and includes examples in the range of aryl iodides and aryl chlorides. By taking this reactivity into consideration, successful conditions were found using PyBCam^CN for primary alkyl halides and ^4,4'-MeOBpy complexes for secondary alkyl halides (42 examples, 60% average yield).

Ryanodine receptor 2 (RyR2) hyperactivity is frequently observed in structural heart disease (SHD), commonly caused by ischemic heart disease. This aberrant Ca ²⁺ release promotes irregular electrical activity and life‐threatening arrhythmias. Our previous work demonstrated that the pan‐ryanodine receptor (RyR) inhibitor dantrolene can reverse arrhythmogenic substrates, but its lack of RyR2 selectivity limits its therapeutic potential. The unnatural verticilide enantiomer ( ent ‐verticilide) was identified as a selective RyR2 inhibitor, showing promising specificity without activating skeletal muscle RyR1. This study evaluated the antiarrhythmic potential of a selective RyR2 antagonist, ent ‐(+)‐verticilide ( ent ‐Vert), in human ventricular slices. Right and left ventricular slices were prepared from human hearts not designated for transplantation. Pseudo‐electrocardiograms (ECG) tracked premature ventricular contraction (PVC) incidence, and optical mapping identified arrhythmogenic substrates. Baseline optical recordings were obtained before treatment with isoproterenol (Iso) (250 nM) and caffeine (200 µM) to induce Ca ^{2 +} leak and arrhythmogenic activity, followed by sequential ent ‐Vert application (1 and 3 µM). The effects of ent ‐Vert alone were tested with baseline recordings followed by a single 3 µM dose. Iso and caffeine increased PVC incidence in both right (RV) and left ventricular (LV) slices, but ent ‐Vert (1 and 3 µM) significantly reduced it. Iso and caffeine also shortened action potential duration (APD) in both slice types, but ent ‐Vert, after Iso and caffeine or alone (under control conditions), did not significantly change APD. In conclusion, ent ‐Vert suppressed arrhythmic triggers and reduced arrhythmogenic substrates in human ventricular slices, highlighting its potential as a targeted antiarrhythmic therapy for SHD. image Key points: RyR2 hyperactivity is a major driver of arrhythmias in structural heart disease, producing abnormal Ca ^{2 +} release and premature ventricular contractions. The RyR2‐selective inhibitor ent ‐Vert was evaluated in human RV and LV slices to determine its antiarrhythmic potential. β‐Adrenergic and RyR2 activation with Iso + caffeine induced Ca ^{2 +} leak, ectopic beats and shortened APD. Sequential ent ‐Vert application (1 and 3 µM) significantly suppressed ectopy incidence in a dose‐dependent manner. ent ‐Vert did not alter APD or conduction velocity under control or stimulated conditions, demonstrating that it suppresses arrhythmic triggers without disrupting normal cardiac electrophysiology.

The number of peptide-like scaffolds found in late-stage drug development is increasing, but a critical unanswered question in the field is whether substituents (side chains) or the backbone drive passive permeability. The backbone is scrutinized in this study. Five series of macrocyclic peptidic compounds were prepared, and their passive permeability was determined (PAMPA, Caco-2), to delineate structure–permeability relationships. Each series was based on the cell-permeable antiarrhythmic compound ent-verticilide, a cyclic oligomeric depsipeptide (COD) containing repeating ester/N-Me amide didepsipeptide monomers. One key finding is that native lipophilic ester functionality can impart a favorable level of permeability, but ester content alone is not the final determinant – the analog with highest P_app was discovered by a single ester-to-N-H amide replacement. Furthermore, the relative composition of esters and N-Me amides in a series had more nuanced permeability behavior. Overall, a systematic approach to structure–permeability correlations suggests that a combinatorial-like investigation of functionality in peptidic or peptide-like compounds could better identify leads with optimal passive permeability, perhaps prior to modification of side chains.

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 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.

Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca²⁺-release channels contributes to heart failure and arrhythmias. Reducing the 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 Förster 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). Multiexponential 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.

Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca ²⁺ -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) Ca ²⁺ 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 Förster 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.

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.

Background: Exercise training, and catecholaminergic stimulation, increase the incidence of arrhythmic events in patients affected with arrhythmogenic right ventricular cardiomyopathy correlated with plakophilin-2 (PKP2) mutations. Separate data show that reduced abundance of PKP2 leads to dysregulation of intracellular Ca ²⁺ (Ca ²⁺ _i ) homeostasis. Here, we study the relation between excercise, catecholaminergic stimulation, Ca ²⁺ _i homeostasis, and arrhythmogenesis in PKP2-deficient murine hearts. Methods: Experiments were performed in myocytes from a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout murine line (PKP2cKO). For training, mice underwent 75 minutes of treadmill running once per day, 5 days each week for 6 weeks. We used multiple approaches including imaging, high-resolution mass spectrometry, electrocardiography, and pharmacological challenges to study the functional properties of cells/hearts in vitro and in vivo. Results: In myocytes from PKP2cKO animals, training increased sarcoplasmic reticulum Ca ²⁺ load, increased the frequency and amplitude of spontaneous ryanodine receptor (ryanodine receptor 2)–mediated Ca ²⁺ release events (sparks), and changed the time course of sarcomeric shortening. Phosphoproteomics analysis revealed that training led to hyperphosphorylation of phospholamban in residues 16 and 17, suggesting a catecholaminergic component. Isoproterenol-induced increase in Ca ²⁺ _i transient amplitude showed a differential response to β-adrenergic blockade that depended on the purported ability of the blockers to reach intracellular receptors. Additional experiments showed significant reduction of isoproterenol-induced Ca ²⁺ _i sparks and ventricular arrhythmias in PKP2cKO hearts exposed to an experimental blocker of ryanodine receptor 2 channels. Conclusions: Exercise disproportionately affects Ca ²⁺ _i homeostasis in PKP2-deficient hearts in a manner facilitated by stimulation of intracellular β-adrenergic receptors and hyperphosphorylation of phospholamban. These cellular changes create a proarrhythmogenic state that can be mitigated by ryanodine receptor 2 blockade. Our data unveil an arrhythmogenic mechanism for exercise-induced or catecholaminergic life-threatening arrhythmias in the setting of PKP2 deficit. We suggest that membrane-permeable β-blockers are potentially more efficient for patients with arrhythmogenic right ventricular cardiomyopathy, highlight the potential for ryanodine receptor 2 channel blockers as treatment for the control of heart rhythm in the population at risk, and propose that PKP2-dependent and phospholamban-dependent arrhythmogenic right ventricular cardiomyopathy–related arrhythmias have a common mechanism.

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.