We describe a mutation (E299V) in KCNJ2, the gene that encodes the strong inward rectifier K + channel protein (Kir2.1), in an 11-y-old boy. The unique short QT syndrome type-3 phenotype is associated with an extremely abbreviated QT interval (200 ms) on ECG and paroxysmal atrial fibrillation. Genetic screening identified an A896T substitution in a highly conserved region of KCNJ2 that resulted in a de novo mutation E299V. Whole-cell patch-clamp experiments showed that E299V presents an abnormally large outward I K1 at potentials above −55 mV (P < 0.001 versus wild type) due to a lack of inward rectification. Coexpression of wild-type and mutant channels to mimic the heterozygous condition still resulted in a large outward current. Coimmunoprecipitation and kinetic analysis showed that E299V and wild-type isoforms may heteromerize and that their interaction impairs function. The homomeric assembly of E299V mutant proteins actually results in gain of function. Computer simulations of ventricular excitation and propagation using both the homozygous and heterozygous conditions at three different levels of integration (single cell, 2D, and 3D) accurately reproduced the electrocardiographic phenotype of the proband, including an exceedingly short QT interval with merging of the QRS and the T wave, absence of ST segment, and peaked T waves. Numerical experiments predict that, in addition to the short QT interval, absence of inward rectification in the E299V mutation should result in atrial fibrillation. In addition, as predicted by simulations using a geometrically accurate three-dimensional ventricular model that included the His-Purkinje network, a slight reduction in ventricular excitability via 20% reduction of the sodium current should increase vulnerability to life-threatening ventricular tachyarrhythmia. cellular electrophysiology | computer models | genetics | ion channels | channelopathies T he short QT syndrome (SQTS) is an inherited arrhythmogenic disorder characterized by a remarkably abbreviated repolarization and a predisposition to supraventricular and ventricular arrhythmias in the absence of detectable structural heart disease (1). Mutations found in SQTS patients in the genes encoding potassium channels cause "gain of function," whereas mutations found in the alpha 1C subunit of the voltage-dependent L-type Ca + channel (CACNA1C), the beta 2b subunit of the voltage dependent Ca 2+ channel (CACNB2B) and the alpha2/delta subunit 1 of the voltage dependent Ca 2+ channel (CACNA2D1) cause "loss of function" (1, 2). In 2005, we reported a mutation (D172N) in the strong inward rectifier K + channel protein (Kir2.1), which is coded by KCNJ2, in an SQTS patient: Functional characterization revealed that D172N shows an increased outward component of the inward rectifier current I K1 (3). Computer simulation demonstrated that D172N leads to a shortening of the QT interval and predisposes the heart to develop reentrant arrhythmias. Here we report a different KCNJ2 mutation (E299V) identified in a child with a re...

Several neoclerodanes, such as salvinorin A (1) and herkinorin (3), have recently been shown to possess opioid receptor activity in vitro and in vivo. To explore the structure-affinity relationships of this interesting class of compounds, we have synthesized a series of analogues from 1 isolated from SalVia diVinorum. Here, we report the semisynthesis of neoclerodane diterpenes and their structure-affinity relationships at opioid receptors. This work will allow the further development of novel opioid receptor ligands.SalVia diVinorum is a plant from the mint family that has been used in the traditional spiritual practices by the Mazatec Indians of Oaxaca, Mexico. Chewing fresh leaves or smoking dried leaves will produce hallucinogenic-like experiences. 1 These experiences last for up to an hour and are reported to be potent and intense. [2][3][4][5] These effects, however, appear to be different than other hallucinogenic substances such as LSD or DOB. Recreational use of S. diVinourm is increasing in part due to its availability for purchase through the Internet. 6 The main active constituent isolated from the leaves of S. diVinorum is salvinorin A (1), a neoclerodane diterpene. 7,8 Compound 1 was found to be a potent and selective κ opioid receptor (κOR) agonist. 9-11 Interestingly, the pharmacology of 1 appears to be different than other κ agonists. 12 Recent work has shown that 1 decreases dopamine levels in the caudate putamen of mice and that this effect is blocked by the κOR antagonist nor-binaltorphimine. 13 A study has also shown that 1 dose dependently increases immobility in the forced swim test, indicating that 1 has depressivelike effects. 14 Furthermore, 1 disrupts climbing behavior on an inverted screen task. 15 This study showed that there are differences in the susceptibility of 1 and the standard κOR agonist U69,593 to antagonism by nor-binaltorphimine. This finding also indicates that 1 may bind in a manner that is qualitatively different than traditional κOR ligands.Recently, we described the synthesis of several analogues of 1 that were found to be opioid receptor ligands. 11 Among these compounds described were analogues 2-4. Propionate 2 was found to have approximately the same affinity for the κOR as 1 but was less potent as an agonist. This work also identified herkinorin (3), the first neoclerodane diterpene with µ opioid receptor affinity, and WH-1-32 (4), an analogue slightly more potent than 1 as a κOR agonist. 11 Recently, methoxymethyl analogue 5 16 and carbamate 6 17 were identified as having affinity for κORs similar to 1. Interestingly, 5 was found to be a full agonist more potent than 1, and 6 is a partial agonist at κORs. These observations illustrate that substitution at the C-2 position can have profound effects on opioid receptor affinity and activity.Recently, a model was proposed for the binding of 1 to the κOR. 18 This model was developed through the use of molecular modeling and mutagenesis studies. However, the binding pocket of salvinorin A will not be known until the ...

Two new neoclerodane diterpenes, salvinicins A (4) and B (5), were isolated from the dried leaves of Salvia divinorum. The structures of these compounds were elucidated by spectroscopic techniques, including 1 H and 13 C NMR, NOESY, HMQC, and HMBC. The absolute stereochemistry of these compounds was assigned on the basis of single crystal X-ray crystallographic analysis of salvinicin A (4) and a 3,4-dichlorobenzoate derivative of salvinorin B.The genus Salvia is one of the most widespread members of the Lamiaceae (formerly Labiatae) family and is featured prominently in the pharmacopeias of many countries throughout the world. 1 Among these is Salvia divinorum Epling & Játiva, a sage native to Oaxaca, Mexico. An infusion prepared from four or five pairs of fresh or dried leaves is used by the Mazatec Indians to stop diarrhea, relieve headache and rheumatism, and to treat a 'semi-magical' disease known as panzón de barrego or swollen belly. 2 S. divinorum is also used in traditional spiritual practices of the Mazatecs to produce "mystical" or hallucinogenic experiences. 2The active ingredient in S. divinorum is salvinorin A (1a) (Figure 1) Currently, S. divinorum is unregulated in most countries and available throughout the world for purchase over the internet. However, it is listed as a controlled substance in Denmark, Australia, and Italy. Obtaining S. divinorum is easy in countries where it is unregulated, and it represents a cheap, easy solution for those who wish to experiment with drugs and perception altering substances. To date, U.S. laws for controlled substances do not ban the sale of S. divinorum or its active components. This has resulted in various on-line botanical companies advertising and selling S. divinorum as a legal alternative to other regulated plant hallucinogens. Therefore, it is predictable that its misuse will increase.As a hallucinogen, 1a is structurally unique. It bears no similarity to classical hallucinogens, such as LSD (2). Furthermore, it has no similarity to other opioid ligands, such as morphine (3). Given its potential for abuse, as well as, its unique pharmacological properties, we 18-20 and others 21-23 have begun to study the chemistry and pharmacology associated with constituents from S. divinorum.Previous phytochemical investigation of S. divinorum resulted in the identification of several neoclerodane diterpenes present in the leaves, salvinorins A -F and divinatorins A -C. 5,6, 8,24,25 Here, we report the identification of two new neoclerodane diterpenes present in commercially available S. divinorum leaves.Commercially available dried leaves of S. divinorum were extracted with acetone, and the acetone extract was subjected to repeated flash column chromatographies using a variety of solvent mixtures to afford salvinicins A (4) (65 mg) and B (5) (14 mg). Compound 4 gave a pseudomolecular ion peak at m/z 551.2080 ([M+Na] + ) in the HRESIMS suggesting a molecular formula of C 25 H 36 O 12 and an index of unsaturation of 8. Its IR spectrum showed absorption bands f...

Introduction: The triple-combination (TC) cystic fibrosis transmembrane conductance regulator (CFTR) modulator regimen elexacaftor, tezacaftor, and ivacaftor was shown to be safe and efficacious in phase 3 trials of people with cystic fibrosis (pwCF) C 12 years of age with C 1 F508del-CFTR allele. Here, a simulation study predicted ivacaftor, tezacaftor, and elexacaftor exposures and impacts on CFTR modulation following transition from ivacaftor [a cytochrome P450 3A (CYP3A) substrate], lumacaftor (a CYP3A inducer)/ivacaftor, or tezacaftor/ivacaftor to TC. Methods: Physiologically based pharmacokinetic (PBPK) modeling was used to evaluate plasma exposures during transition from monoor dual-combination CFTR modulator regimens to TC. PBPK models were parameterized using data from human hepatocytes to account for CYP3A induction by lumacaftor and validated to match clinical data from healthy volunteers and pwCF. Using dosing regimens for pwCF C 12 years of age, simulations were performed for ivacaftor, lumacaftor/ivacaftor, and tezacaftor/ivacaftor dosing for 14 days followed by immediate transition to elexacaftor/tezacaftor/ivacaftor dosing for 14 days. Drug exposures during transitions were compared with respective half-maximal effective concentrations (EC 50) estimated from efficacy endpoint data from clinical studies. Results: In simulations of immediate transition from ivacaftor or tezacaftor/ivacaftor to TC, the preceding treatment had no impact on ivacaftor, tezacaftor, or elexacaftor exposures. In simulations of immediate transition from lumacaftor/ivacaftor to TC, ivacaftor exposure decreased to 64% of maximum effective concentration (EC), due to reduction in ivacaftor dose and residual CYP3A4 induction, then returned to 90-95% of maximum EC. Lumacaftor-mediated CYP3A induction resolved within approximately 2 weeks. In all simulations, ivacaftor, tezacaftor, and elexacaftor exposures approached steady state within 2 weeks following transition and, at all times, ivacaftor and C 1 CFTR corrector remained above EC 50. Conclusion: PBPK modeling indicates that immediate transition to the elexacaftor/tezacaftor/ivacaftor regimen from an ivacaftor, lumacaftor/ivacaftor, or tezacaftor/ivacaftor Digital Features To view digital features for this article go to