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.

Generality-Driven Catalyst Development: A Universal Catalyst for Enantioselective Nitroalkene Reduction.

Cracking the selectivity-generality paradox is among the most pressing challenges in asymmetric catalysis. This obstacle prevents the immediate and successful translation of new methods to diverse small molecules. This is particularly rate-limiting for therapeutic development, where availability and structural diversity are often critical components of successful campaigns. Here we describe the union of generality-driven enantioselective catalysis and the preparation of diverse peptidomimetics. A single new organocatalyst provides high selectivity and substrate generality that is matched only by a combination of metal and organocatalysts. Within organocatalysis, this discovery breaks a 16-year monolithic paradigm, uncovering a powerful new scaffold for enantioselective reduction with behavior that suggests the recognition of a nitroethylene minimal catalaphile.

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 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.

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.

In Vivo Pharmacokinetic and Pharmacodynamic Properties of the Antiarrhythmic Molecule ent-Verticilide.

The unnatural verticilide enantiomer (ent-verticilide) is a selective and potent inhibitor of cardiac ryanodine receptor (RyR2) calcium release channels and exhibits antiarrhythmic activity in a murine model of catecholaminergic polymorphic ventricular tachycardia (CPVT). To determine verticilide's pharmacokinetic and pharmacodynamic properties in vivo, we developed a bioassay to measure nat- and ent-verticilide in murine plasma and correlated plasma concentrations with antiarrhythmic efficacy in a mouse model of CPVT. nat-Verticilide rapidly degraded in plasma in vitro, showing >95% degradation within 5 minutes, whereas ent-verticilide showed <1% degradation over 6 hours. Plasma was collected from mice following intraperitoneal administration of ent-verticilide at two doses (3 mg/kg, 30 mg/kg). Peak C max and area under the plasma-concentration time curve (AUC) scaled proportionally to dose, and the half-life was 6.9 hours for the 3-mg/kg dose and 6.4 hours for the 30-mg/kg dose. Antiarrhythmic efficacy was examined using a catecholamine challenge protocol at time points ranging from 5 to 1440 minutes after intraperitoneal dosing. ent-Verticilide inhibited ventricular arrhythmias as early as 7 minutes after administration in a concentration-dependent manner, with an estimated potency (IC50) of 266 ng/ml (312 nM) and an estimated maximum inhibitory effect of 93.5%. Unlike the US Food and Drug Administration-approved pan-RyR blocker dantrolene, the RyR2-selective blocker ent-verticilide (30 mg/kg) did not reduce skeletal muscle strength in vivo. We conclude that ent-verticilide has favorable pharmacokinetic properties and reduces ventricular arrhythmias with an estimated potency in the nanomolar range, warranting further drug development. SIGNIFICANCE STATEMENT: ent-Verticilide has therapeutic potential to treat cardiac arrhythmias, but little is known about its pharmacological profile in vivo. The primary purpose of this study is to determine the systemic exposure and pharmacokinetics of ent-verticilide in mice and estimate its efficacy and potency in vivo. The current work suggests ent-verticilide has favorable pharmacokinetic properties and reduces ventricular arrhythmias with an estimated potency in the nanomolar range, warranting further drug development.

Enantioselective Synthesis of cis- and trans-Cycloheptyl ß-Fluoro Amines by Sequential aza-Henry Addition/Ring-Closing Metathesis.

The synthesis of 7-membered carbocyclic ß-fluoroamines is accomplished by a combination of the enantioselective aza-Henry reaction of aliphatic N-Boc imines and ring-closing metathesis. Use of reductive denitration gives both diastereomers of the ß-fluoro amine carbocycle, each with high enantiomeric excess.

Secondary Orbital Effect Involving Fluorine is Responsible for Substrate-Controlled Diastereodivergence in the Catalyzed syn-aza-Henry Reaction of α-Fluoronitroalkanes.

The fluorine atom is a powerful, yet enigmatic influence on chemical reactions. True to form, fluorine was recently discovered to effect diastereodivergence in an enantioselective aza-Henry reaction, resulting in a very rare case of syn-ß-amino nitroalkane products. More bewildering was the observation of an apparent hierarchy of substituents within this substrate-controlled behavior: Ph>F>alkyl. These cases have now been examined comprehensively by computational methods, including both non-fluorinated and α-fluoro nitronate additions to aldimines catalyzed by a chiral bis(amidine) [BAM] proton complex. This study revealed the network of non-covalent interactions that dictate anti- (α-aryl) versus syn-selectivity (α-alkyl) using α-fluoronitronate nucleophiles, and an underlying secondary orbital interaction between fluorine and the activated azomethine.

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.

Preparation of N-Aryl Amides by Epimerization-Free Umpolung Amide Synthesis.

Amide synthesis is one of the most widely practiced chemical reactions, owing to its use in drug development and peptide synthesis. Despite the importance of these applications, the attendant effort to eliminate waste associated with these protocols has met with limited success, and pernicious α-epimerization is most often minimized but not eliminated when targeting challenging amides (e.g., N-aryl amides). This effort has focused on what is essentially a single paradigm in amide formation wherein an electrophilic acyl donor reacts with a nucleophilic amine. Umpolung amide synthesis (UmAS) emerged from α-halo nitroalkane reactions with amines and has since been developed into a method for the synthesis of enantiopure amides using entirely catalytic, enantioselective synthesis. However, its inability to forge N-aryl amides has been a longstanding problem, one limiting its application more broadly in drug development where α-chiral N-aryl amides are increasingly common. We report here the reaction of α-fluoronitroalkanes and N-aryl hydroxyl amines for the direct synthesis of N-aryl amides using a simple Brønsted base as the promoter. No other activating agents are required, and experiments guided by mechanistic hypotheses outline a mechanism based on the UmAS paradigm and confirm that the N-aryl amide, not the N-aryl hydroxamic acid, is the direct product. Ultimately, select chiral α-amino-N-aryl amides were prepared with complete conservation of enantioenrichment, in contrast to a parallel demonstration of their ability to epimerize using the conventional amide synthesis alternative.

Enantioselective iodolactonization to prepare ε-lactone rings using hypervalent iodine.

Despite the rapid growth of enantioselective halolactonization reactions in recent years, most are effective only when forming smaller (6,5,4-membered) rings. Seven-membered ε-lactones, are rarely formed with high selectivity, and never without conformational bias. We describe the first highly enantioselective 7-exo-trig iodolactonizations of conformationally unbiased ε-unsaturated carboxylic acids, effected by an unusual combination of a bifunctional BAM catalyst, I2, and I(iii) reagent (PhI(OAc)2:PIDA).

Exercise Causes Arrhythmogenic Remodeling of Intracellular Calcium Dynamics in Plakophilin-2-Deficient Hearts.

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 Ca2+ (Ca2+i) homeostasis. Here, we study the relation between excercise, catecholaminergic stimulation, Ca2+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 Ca2+ load, increased the frequency and amplitude of spontaneous ryanodine receptor (ryanodine receptor 2)-mediated Ca2+ 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 Ca2+i transient amplitude showed a differential response to ß-adrenergic blockade that depended on the purported ability of the blockers to reach intracellular receptors. Additional experiments showed significant reduction of isoproterenol-induced Ca2+i sparks and ventricular arrhythmias in PKP2cKO hearts exposed to an experimental blocker of ryanodine receptor 2 channels. CONCLUSIONS: Exercise disproportionately affects Ca2+i homeostasis in PKP2-deficient hearts in a manner facilitated by stimulation of intracellular ß-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 ß-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.

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.

Fluorine-induced diastereodivergence discovered in an equally rare enantioselective syn-aza-Henry reaction.

Attention to the aza-Henry reaction, particularly over the past two decades, has resulted in a wide range of effective catalysts for the enantio- and diastereoselective versions, driven by the versatility of the ß-amino nitroalkane products as precursors to secondary amines and vic-diamines. Despite this broad effort, syn-diastereoselective variants are exceedingly rare. We have discovered a subset of α-fluoro nitroalkane additions that are characterized by an unusual crossover in diastereoselection, often delivering the products with high selectivities. We report here a rigorous comparative analysis of non-fluorinated and α-fluoro nitroalkanes in their additions to azomethines. Both homogeneous and heterogeneous catalysis were applied to probe the possibility that this phenomenon might be more widely operative in the enantioselective additions of fluorine-substituted carbon nucleophiles. A complete correlation within four categories is described that uncovered a clear trend, while revealing a dramatic and distinct reversal of diastereoselection that would normally go undetected.

Ring Size as an Independent Variable in Cyclooligomeric Depsipeptide Antiarrhythmic Activity.

Hit-to-lead studies employ a variety of strategies to optimize binding to a target of interest. When a structure for the target is available, hypothesis-driven structure-activity relationships (SAR) are a powerful strategy for refining the pharmacophore to achieve robust binding and selectivity characteristics necessary to identify a lead compound. Recrafting the three-dimensional space occupied by a small molecule, optimization of hydrogen bond contacts, and enhancing local attractive interactions are traditional approaches in medicinal chemistry. Ring size, however, is rarely able to be leveraged as an independent variable because most hits lack the symmetry required for such a study. Our discovery that the cyclic oligomeric depsipeptide ent-verticilide inhibits mammalian cardiac ryanodine receptor calcium release channels with submicromolar potency provided an opportunity to explore ring size as a variable, independent of other structural or functional group changes. We report here that ring size can be a critical independent variable, suggesting that modest conformational changes alone can dramatically affect potency.

DFT-Based Stereochemical Rationales for the Bifunctional Brønsted Acid/Base-Catalyzed Diastereodivergent and Enantioselective aza-Henry Reactions of α-Nitro Esters.

A pair of chiral bis(amidine) [BAM] proton complexes provide reagent (catalyst)-controlled, highly diastereo- and enantioselective direct aza-Henry reactions leading to α-alkyl-substituted α,ß-diamino esters. A C2-symmetric ligand provides high anti-selectivity, while a nonsymmetric congener exhibits syn-selectivity in this example of diastereodivergent, enantioselective catalysis. A detailed computational analysis is reported for the first time, one that supports distinct models for selectivity resulting from the more hindered binding cavity of the C1-symmetric ligand. Binding in this congested pocket accommodates four hydrogen bond contacts among ligands and substrates, ultimately favoring a pre-syn arrangement highlighted by pyridinium-azomethine activation and quinolinium-nitronate activation. The complementary transition states reveal a wide range of alternatives. Comparing the C1- and C2-symmetric catalysts highlights distinct electrophile binding orientations despite their common hydrogen bond donor-acceptor features. Among the factors driving unusual high syn-diastereoselection are favorable dispersion forces that leverage the anthracenyl substituent of the C1-symmetric ligand.

The Formation of Impossible Rings in Macrocyclooligomerizations for Cyclodepsipeptide Synthesis: The 18-from-12 Paradox.

A reinvestigation into the macrocyclooligomerization (MCO) of a tetradepsipeptide is reported, uncovering a paradox in which the MCO of depsipeptide monomers can produce "impossible" ring sizes: a 12-atom chain produced the expected 24-membered ring, alongside unexpected 18- and 30-membered cyclic oligomeric depsipeptides (CODs). We report an alternative preparation of authentic 18- and 36-membered macrocycles for this case using a stepwise synthesis that provides definitive analytical characterization for each ring size. Our investigation yields a recharacterization and reassignment of two macrocycles originally reported in this MCO series, along with updated yields and isothermal titration calorimetry data after implementation of new critical protocols for purification and subsequent analysis. Initial studies to probe this mechanistic conundrum are described.

RYR2 Channel Inhibition Is the Principal Mechanism of Flecainide Action in CPVT.

RATIONALE: The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive RyR2 (cardiac ryanodine receptor) mediated calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro, reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide's efficacy in CPVT. OBJECTIVE: To determine whether RyR2 block independently contributes to flecainide's efficacy for suppressing spontaneous sarcoplasmic reticulum Ca release and for preventing ventricular tachycardia in vivo. METHODS AND RESULTS: We synthesized N-methylated flecainide analogues (QX-flecainide and N-methyl flecainide) and showed that N-methylation reduces flecainide's inhibitory potency on RyR2 channels incorporated into artificial lipid bilayers. N-methylation did not alter flecainide's inhibitory activity on human cardiac sodium channels expressed in HEK293T cells. Antiarrhythmic efficacy was tested utilizing a Casq2 (cardiac calsequestrin) knockout (Casq2-/-) CPVT mouse model. In membrane-permeabilized Casq2-/- cardiomyocytes-lacking intact sarcolemma and devoid of sodium channel contribution-flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2-/- cardiomyocytes pretreated with tetrodotoxin to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous sarcoplasmic reticulum Ca release, while QX-flecainide and N-methyl flecainide did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2-/- mice, whereas N-methyl flecainide had no significant effect on arrhythmia burden, despite comparable sodium channel block. CONCLUSIONS: Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone did not prevent ventricular tachycardia. Hence, RyR2 channel inhibition likely constitutes the principal mechanism of antiarrhythmic action of flecainide in CPVT.

Substituted Imidazoline Synthesis: A Diastereo- and Enantioselective aza-Henry Route to a Human Proteasome Modulator.

The first enantio- and diastereoselective synthesis of Tepe's human proteasome modulator is described. Routes to this and other highly substituted chiral imidazolines generally produce racemic material. Key to the route disclosed here is a gram-scale anti-selective aza-Henry reaction of an α-alkyl α-nitro ester nucleophile, catalyzed by a Bis(Amidine) [BAM] chiral proton complex, delivering the key intermediate in high yield as a single stereoisomer. The adduct is reduced to the amino ester and converted to an imidazoline.