Manganese-Catalyzed Borylation of Unactivated Alkyl Chlorides.

The use of low-cost manganese(II) bromide (MnBr2) and tetramethylethylenediamine (TMEDA) catalyzes the cross coupling of (bis)pinacolatodiboron with a wide range of alkyl halides, demonstrating the first manganese-catalyzed coupling with alkyl electrophiles. This method allows access to primary, secondary, and tertiary boronic esters from the parent chlorides, which were previously inaccessible as coupling partners. The reaction proceeds in high yield with as little as 1000 ppm catalyst loading, while 5 mol % can provide high yields in as little as 30 min. Finally, radical-clock experiments revealed that at 0 °C direct borylation outcompetes alternative radical processes, thereby providing synthetically useful, temperature-controlled reaction outcomes.

Iron-catalyzed borylation of alkyl electrophiles.

The use of low-cost iron(III) acetoacetate (Fe(acac)3) and tetramethylethylenediamine (TMEDA) enables the direct cross-coupling of alkyl halides with bis(pinacolato)diboron. This approach allows for the borylation of activated or unactivated primary, secondary, and tertiary bromides. Moreover, even the borylation of benzylic or allylic chlorides, tosylates, and mesylates are possible. The reactions proceed under mild conditions at room temperature and show broad functional-group compatibility and "robustness" as measured by a modified Glorius robustness screen.

Blocking Scn10a channels in heart reduces late sodium current and is antiarrhythmic.

RATIONALE: Although the sodium channel locus SCN10A has been implicated by genome-wide association studies as a modulator of cardiac electrophysiology, the role of its gene product Nav1.8 as a modulator of cardiac ion currents is unknown. OBJECTIVE: We determined the electrophysiological and pharmacological properties of Nav1.8 in heterologous cell systems and assessed the antiarrhythmic effect of Nav1.8 block on isolated mouse and rabbit ventricular cardiomyocytes. METHODS AND RESULTS: We first demonstrated that Scn10a transcripts are identified in mouse heart and that the blocker A-803467 is highly specific for Nav1.8 current over that of Nav1.5, the canonical cardiac sodium channel encoded by SCN5A. We then showed that low concentrations of A-803467 selectively block "late" sodium current and shorten action potentials in mouse and rabbit cardiomyocytes. Exaggerated late sodium current is known to mediate arrhythmogenic early afterdepolarizations in heart, and these were similarly suppressed by low concentrations of A-803467. CONCLUSIONS: Scn10a expression contributes to late sodium current in heart and represents a new target for antiarrhythmic intervention.

Informatic and functional approaches to identifying a regulatory region for the cardiac sodium channel.

RATIONALE: Although multiple lines of evidence suggest that variable expression of the cardiac sodium channel gene SCN5A plays a role in susceptibility to arrhythmia, little is known about its transcriptional regulation. OBJECTIVE: We used in silico and in vitro experiments to identify possible noncoding sequences important for transcriptional regulation of SCN5A. The results were extended to mice in which a putative regulatory region was deleted. METHODS AND RESULTS: We identified 92 noncoding regions highly conserved (>70%) between human and mouse SCN5A orthologs. Three conserved noncoding sequences (CNS) showed significant (>5-fold) activity in luciferase assays. Further in vitro studies indicated one, CNS28 in intron 1, as a potential regulatory region. Using recombinase-mediated cassette exchange (RMCE), we generated mice in which a 435-base pair region encompassing CNS28 was removed. Animals homozygous for the deletion showed significant increases in SCN5A transcripts, Na(V)1.5 protein abundance, and sodium current measured in isolated ventricular myocytes. ECGs revealed a significantly shorter QRS (10.7±0.2 ms in controls versus 9.7±0.2 ms in knockouts), indicating more rapid ventricular conduction. In vitro analysis of CNS28 identified a short 3' segment within this region required for regulatory activity and including an E-box motif. Deletion of this segment reduced reporter activity to 3.6%±0.3% of baseline in CHO cells and 16%±3% in myocytes (both P<0.05), and mutation of individual sites in the E-box restored activity to 62%±4% and 57%±2% of baseline in CHO cells and myocytes, respectively (both P<0.05). CONCLUSIONS: These findings establish that regulation of cardiac sodium channel expression modulates channel function in vivo, and identify a noncoding region underlying this regulation.

Striking In vivo phenotype of a disease-associated human SCN5A mutation producing minimal changes in vitro.

BACKGROUND: The D1275N SCN5A mutation has been associated with a range of unusual phenotypes, including conduction disease and dilated cardiomyopathy, as well as atrial and ventricular tachyarrhythmias. However, when D1275N is studied in heterologous expression systems, most studies show near-normal sodium channel function. Thus, the relationship of the variant to the clinical phenotypes remains uncertain. METHODS AND RESULTS: We identified D1275N in a patient with atrial flutter, atrial standstill, conduction disease, and sinus node dysfunction. There was no major difference in biophysical properties between wild-type and D1275N channels expressed in Chinese hamster ovary cells or tsA201 cells in the absence or presence of ß1 subunits. To determine D1275N function in vivo, the Scn5a locus was modified to knock out the mouse gene, and the full-length wild-type (H) or D1275N (DN) human SCN5A cDNAs were then inserted at the modified locus by recombinase mediated cassette exchange. Mice carrying the DN allele displayed slow conduction, heart block, atrial fibrillation, ventricular tachycardia, and a dilated cardiomyopathy phenotype, with no significant fibrosis or myocyte disarray on histological examination. The DN allele conferred gene-dose-dependent increases in SCN5A mRNA abundance but reduced sodium channel protein abundance and peak sodium current amplitudes (H/H, 41.0±2.9 pA/pF at -30 mV; DN/H, 19.2±3.1 pA/pF, P<0.001 vs. H/H; DN/DN, 9.3±1.1 pA/pF, P<0.001 versus H/H). CONCLUSIONS: Although D1275N produces near-normal currents in multiple heterologous expression experiments, our data establish this variant as a pathological mutation that generates conduction slowing, arrhythmias, and a dilated cardiomyopathy phenotype by reducing cardiac sodium current.

Systematic profiling of conditional degron tag technologies for target validation studies.

Conditional degron tags (CDTs) are a powerful tool for target validation that combines the kinetics and reversible action of pharmacological agents with the generalizability of genetic manipulation. However, successful design of a CDT fusion protein often requires a prolonged, ad hoc cycle of construct design, failure, and re-design. To address this limitation, we report here a system to rapidly compare the activity of five unique CDTs: AID/AID2, IKZF3d, dTAG, HaloTag, and SMASh. We demonstrate the utility of this system against 16 unique protein targets. We find that expression and degradation are highly dependent on the specific CDT, the construct design, and the target. None of the CDTs leads to efficient expression and/or degradation across all targets; however, our systematic approach enables the identification of at least one optimal CDT fusion for each target. To enable the adoption of CDT strategies more broadly, we have made these reagents, and a detailed protocol, available as a community resource.

Targeted Covalent Inhibition of Plasmodium FK506 Binding Protein 35.

FK506-binding protein 35, FKBP35, has been implicated as an essential malarial enzyme. Rapamycin and FK506 exhibit antiplasmodium activity in cultured parasites. However, due to the highly conserved nature of the binding pockets of FKBPs and the immunosuppressive properties of these drugs, there is a need for compounds that selectively inhibit FKBP35 and lack the undesired side effects. In contrast to human FKBPs, FKBP35 contains a cysteine, C106, adjacent to the rapamycin binding pocket, providing an opportunity to develop targeted covalent inhibitors of Plasmodium FKBP35. Here, we synthesize inhibitors of FKBP35, show that they directly bind FKBP35 in a model cellular setting, selectively covalently modify C106, and exhibit antiplasmodium activity in blood-stage cultured parasites.

Contrasting Nav1.8 Activity in Scn10a-/- Ventricular Myocytes and the Intact Heart.

BACKGROUND: Genome-wide association studies have implicated variants in SCN10A, which encodes Nav1.8, as modulators of cardiac conduction. Follow-up work has indicated the SCN10A sequence includes an intronic enhancer for SCN5A. Yet the role of the Nav1.8 protein in the myocardium itself is still unclear. To investigate this, we use homozygous knockout mice (Scn10a-/-) generated by disruption of exons 4 and 5, leaving the Scn5a enhancer intact. METHODS AND RESULTS: We previously reported that pharmacologic blockade of Nav1.8 in wild-type animals blunts action potential prolongation by ATX-II at slow drive rates (≤1 Hz). Here we present evidence of the same blunting in Scn10a-/- compared to wild-type ventricular myocytes, supporting the conclusion that Nav1.8 contributes to late sodium current at slow rates. In contrast to earlier studies, we found no differences in electrocardiographic parameters between genotypes. Low-dose ATX-II exposure in lightly anesthetized animals and Langendorff-perfused hearts prolonged QTc and generated arrhythmias to the same extent in wild-type and Scn10a-/-. RNA sequencing failed to identify full-length Scn10a transcripts in wild-type or knockout isolated ventricular myocytes. However, loss of late current in Scn10a-/- myocytes was replicated independently in a blinded set of experiments. CONCLUSIONS: While Scn10a transcripts are not detectible in ventricular cardiomyocytes, gene deletion results in reproducible loss of late sodium current under extreme experimental conditions. However, there are no identifiable consequences of this Scn10a deletion in the intact mouse heart at usual rates. These findings argue that common variants in SCN10A that affect ventricular conduction do so by modulating SCN5A.

SCN10A/Nav1.8 modulation of peak and late sodium currents in patients with early onset atrial fibrillation.

AIMS: To test the hypothesis that vulnerability to atrial fibrillation (AF) is associated with rare coding sequence variation in the SCN10A gene, which encodes the voltage-gated sodium channel isoform NaV1.8 found primarily in peripheral nerves and to identify potentially disease-related mechanisms in high-priority rare variants using in-vitro electrophysiology. METHODS AND RESULTS: We re-sequenced SCN10A in 274 patients with early onset AF from the Vanderbilt AF Registry to identify rare coding variants. Engineered variants were transiently expressed in ND7/23 cells and whole-cell voltage clamp experiments were conducted to elucidate their functional properties. Resequencing SCN10A identified 18 heterozygous rare coding variants (minor allele frequency ≤1%) in 18 (6.6%) AF probands. Four probands were carriers of two rare variants each and 14 were carriers of one coding variant. Based on evidence of co-segregation, initial assessment of functional importance, and presence in ≥1 AF proband, three variants (417delK, A1886V, and the compound variant Y158D-R814H) were selected for functional studies. The 417delK variant displayed near absent current while A1886V and Y158D-R814H exhibited enhanced peak and late (INa-L) sodium currents; both Y158D and R818H individually contributed to this phenotype. CONCLUSION: Rare SCN10A variants encoding Nav1.8 were identified in 6.6% of patients with early onset AF. In-vitro electrophysiological studies demonstrated profoundly altered function in 3/3 high-priority variants. Collectively, these data strongly support the hypothesis that rare SCN10A variants may contribute to AF susceptibility.

Increased late sodium current contributes to long QT-related arrhythmia susceptibility in female mice.

AIMS: Female gender is a risk factor for long QT-related arrhythmias, but the underlying mechanisms remain uncertain. Here, we tested the hypothesis that gender-dependent function of the post-depolarization 'late' sodium current (I(Na-L)) contributes. METHODS AND RESULTS: Studies were conducted in mice in which the canonical cardiac sodium channel Scn5a locus was disrupted, and expression of human wild-type SCN5A cDNA substituted. Baseline QT intervals were similar in male and female mice, but exposure to the sodium channel opener anemone toxin ATX-II elicited polymorphic ventricular tachycardia in 0/9 males vs. 6/9 females. Ventricular I(Na-L) and action potential durations were increased in myocytes isolated from female mice compared with those from males before and especially after treatment with ATX-II. Further, ATX-II elicited potentially arrhythmogenic early afterdepolarizations in myocytes from 0/5 male mice and 3/5 female mice. CONCLUSION: These data identify variable late I(Na) as a modulator of gender-dependent arrhythmia susceptibility.