Phosphonothrixin (PPX), a phosphonate natural product known for its potent herbicidal activity, has attracted renewed interest because of the need for alternative herbicides. Its biological activity is confined to the (S)-enantiomer. Although the only reported enantioselective synthesis is complex, labor-intensive, and low yielding, direct chiral liquid chromatographic separation is likewise challenging because of its high polarity and lack of aromatic moieties, which limit interactions with commonly used chiral stationary phases (CSPs). In addition, phosphonic acid-containing compounds tend to chelate metal ions and adsorb to metal surfaces, further complicating chromatographic separation by causing pronounced peak tailing. Here, we describe a reliable and reproducible analytical and preparative method for the direct chiral separation of PPX using a zwitterionic sulfohexyl quinidine and quinine carbamate CSPs, as well as two synthetic phosphonate derivatives using a cellulose tris(3,5-dichlorophenylcarbamate) stationary phase as an alternative approach. The best separation of PPX was achieved with the zwitterionic sulfohexyl quinidine CSP (Rs = 2.4). The separated enantiomers were isolated, and their chemical and stereochemical identities were confirmed by mass spectrometry, optical rotation, and electronic circular dichroism spectroscopy. This approach enabled the successful isolation of enantiomerically pure (S)-(-)- and (R)-(+)-PPX.

Short QT syndrome (SQTS) is a genetically inherited autosomal dominant cardiac channelopathy associated with paroxysmal atrial and ventricular fibrillation, syncope, and sudden cardiac arrest. Although SQTS is a well-known arrhythmogenic disorder, no optimal therapies currently exist due to its rarity and the challenges associated with diagnosis. Rodent models and patient-derived induced pluripotent stem cell (iPSC) models have been used to evaluate potential drug effects, but physiological and genetic variability between species and individuals can limit their fidelity. To address these limitations, we generated an isogenic human iPSC (hiPSC) model by introducing the short QT syndrome type 3 (SQT3)-associated KCNJ2 mutation into a normal hiPSC line using the Prime Editing 7 (PE7) system and differentiating it into cardiomyocytes (CMs). Multi-electrode array (MEA) analysis confirmed shortened field potential duration (FPD), an in vitro correlate of the QT interval as an electrophysiological SQT3 phenotype, validating the model's relevance to SQT3. Quinidine produced a consistent FPD-prolonging effect in the isogenic hiPSC-CMs, recapitulating previous clinical observations in a small number of SQT1 patients and supporting the model's utility as a reliable in vitro surrogate for assessing therapeutic strategies. Additionally, chloroquine, a selected candidate that has not yet been clinically validated, was evaluated and found to prolong FPD and induce irregular spontaneous beats indicative of arrhythmia. Overall, these findings demonstrate the potential of isogenic hiPSC-based platforms for elucidating disease mechanisms in rare genetic arrhythmogenic disorders and advancing the identification of candidate therapeutic agents.

Orforglipron is an orally administered, small-molecule glucagon-like peptide-1 receptor agonist in clinical development for the treatment of type 2 diabetes and obesity. Orforglipron is a substrate of CYP3A4, organic anion transporting polypeptides (OATPs) 1B/1B3, and P-glycoprotein (P-gp); however, precipitants commonly used to define these mechanisms have not been fully characterized. The enzyme- and transporter-mediated DDI profile of orforglipron and that of CYP3A4/OATP1B/P-gp precipitants were characterized in six phase 1 clinical studies in healthy participants. Orforglipron pharmacokinetics were assessed with clarithromycin, carbamazepine, cyclosporine, and quinidine. Precipitant effects on CYP3A and OATP1B activity were measured using midazolam and coproporphyrin-I (CP-I). Orforglipron effects on CYP3A, P-gp, BCRP, and OATP1B were evaluated using midazolam, digoxin, rosuvastatin, simvastatin, atorvastatin, and CP-I. Mechanistic interrogation of the simvastatin interaction was also conducted. Clarithromycin, carbamazepine, and quinidine had minimal effect on CP-I pharmacokinetics, while the cyclosporine effect was substantial. Cyclosporine and quinidine weakly increased midazolam exposure. Orforglipron exposure increased in the presence of clarithromycin and cyclosporine and decreased with carbamazepine, demonstrating orforglipron exposure is affected by precipitants of CYP3A4 and OATP1B. Quinidine did not meaningfully change orforglipron exposure. Orforglipron had no clinically meaningful effect on the exposure of midazolam, digoxin, or atorvastatin. A weak increase in rosuvastatin exposure was consistent with BCRP inhibition, as CP-I data confirmed orforglipron does not affect OATP1B. Increase in simvastatin acid exposure was attributed to the unique disposition of simvastatin rather than enzyme or transporter inhibition. These findings address mechanistic precipitant knowledge gaps and provide a framework for managing potential orforglipron DDIs in clinical practice.

Voluntary muscle contraction is triggered by the neurotransmitter acetylcholine binding its receptors on the postsynaptic membrane of the neuromuscular junction, opening ion channels that allow cation influx and initiate depolarization[1-3]. Mutations in muscle acetylcholine receptors disrupt this process by either impairing (fast-channel) or prolonging (slow-channel) channel openings[1,4]. These defects cause congenital myasthenic syndromes (CMS), characterized by severe muscle weakness that is often present at birth and, in some cases, progresses to paralysis and death[5,6]. The structural mechanisms underlying these pathogenic defects and their pharmacological correction remain unknown. Here, using cryogenic electron microscopy, chemical biology and electrophysiology, we determined the structures and functional consequences of representative CMS mutant receptors with and without drugs. In fast-channel disease-associated mutants, we discovered a cryptic allosteric site targeted by positive modulators that restore gating in a mutation-specific manner. In receptor mutants associated with slow-channel disease, quinidine, fluoxetine and reboxetine act as pore blockers; notably, the antidepressant reboxetine selectively blocks desensitized receptors in a mutation-independent fashion, suggesting repurposing potential. Mechanistically, fast-channel mutations uncouple agonist binding from gating, whereas slow-channel mutations stabilize an abnormally widened, desensitized-like pore. These findings reveal unifying principles of CMS pathogenesis and provide a framework for precision therapies.

Edoxaban, a direct factor Xa inhibitor and known P-glycoprotein (P-gp) substrate, exhibits nonlinear increases in systemic exposure after oral administration and significant interaction with quinidine, a P-gp inhibitor. However, it remains unclear how P-gp in each organ contributes to these phenomena. We developed a physiologically based pharmacokinetic (PK) model to describe PK of edoxaban after oral (0.05-60 mg) and intravenous administration (30 mg), as well as its interaction with quinidine following edoxaban intravenously. Parameters were estimated using the Cluster Gauss-Newton method through top-down and middle-out approaches. Accurate description of renal clearance, which required incorporation of a hypothetical basolateral uptake process, underscoring the importance of unidentified renal transporters involved in edoxaban urinary secretion. Nonlinearity of dose-normalized area under blood concentration-time curve was highly sensitive to KmP-gp, with intestinal unbound concentrations exceeding KmP-gp at clinically relevant doses, consistent with intestinal P-gp saturation as the major driver of nonlinearity. In intravenous edoxaban-oral quinidine drug-drug interaction analysis, quinidine markedly reduced renal clearance and modestly increased hepatic availability, reflecting inhibition of renal and, to a lesser extent, hepatic P-gp. In conclusion, an edoxaban physiologically based PK model incorporating renal uptake transport enabled physiologically consistent description of renal clearance. Saturation of intestinal P-gp was identified as the primary contributor to nonlinear PK, while inhibition of renal P-gp was the main mechanism underlying the edoxaban intravenously administered with quinidine. These findings provide mechanistic insight into transporter contributions to edoxaban disposition and drug-drug interactions and highlight the importance of tissue-specific transporter processes in nonlinear pharmacokinetics. SIGNIFICANCE STATEMENT: Incorporating renal uptake transport is essential for mechanistically describing edoxaban renal clearance in physiologically based pharmacokinetic (PK) modeling. The model identifies intestinal P-glycoprotein saturation as the primary driver of nonlinear PK and renal P-glycoprotein inhibition as the major mechanism of the intraveous edoxaban-quinidine drug-drug interaction. These findings offer mechanistic insight into transporter contributions to edoxaban disposition and drug-drug interactions and emphasize the role of tissue-specific transport processes in nonlinear PK.

Pathogenic KCNT1 variants such as F346L and R961H engender distinct clinical phenotypes and therapeutic responses. We investigated their electrophysiological properties and sensitivity to the potassium-channel blocker quinidine in an in vitro expression system. Whole-cell patch-clamp recordings from CHO cells expressing wild-type (WT) or variant KCNT1 constructs showed that both F346L and R961H conferred gain-of-function phenotypes, with current densities significantly greater than WT. Quinidine suppressed R961H-mediated currents across multiple tested concentrations, whereas F346L currents did not show consistent quinidine-mediated suppression. AlphaFold 3-based structural mapping provided spatial context for this divergent pharmacological response. These in vitro findings paralleled the clinical courses of the respective patients: seizures decreased in the individual carrying R961H but remained unchanged in the patient with F346L.

Plant chemistry and toxicology are complex with a rich history. Much about plant chemistry remains unknown, and our understanding relies on animal research combined with documented human experiences. The dynamic science of pharmacognosy, which highlights the therapeutic value of plants, has elucidated several major classes of organic molecules found in plants, of which alkaloids represent one. Others include phenols and phenylpropanoids; terpenes and resins; glycosides; and proteins, peptides, and lectins. Plant alkaloids represent a diverse array of secondary plant metabolites exemplified by familiar compounds such as nicotine, caffeine, cocaine, mescaline, ephedrine, and strychnine. Chemically classified as amines, plant alkaloids are defined by their function as bases generally containing one or more nitrogen atoms within a heterocyclic structure.  They typically exhibit strong pharmacologic activity and serve as a constituent of these plants’ chemical arsenals. A given plant species may contain one, a few, or many types of alkaloids. Certain plant families are particularly rich in these phytochemicals, such as the Papaveraceae (poppy) and Solanaceae (nightshades). Most in their pure forms are nonvolatile, colorless crystalline solids with a bitter taste. Plant alkaloids illustrate the overlap between the toxic and the therapeutic: one may appreciate the cytoprotective effects of the vinca alkaloids and the genotoxic effects of the pyrrolizidine alkaloids; the disruption of cholinergic neurotransmission by hyoscyamine and the therapeutic potential of pro-cholinergic galantamine in neurodegenerative disorders such as Alzheimer disease; the lethal potential of the “deadly nightshades” contrasted with the light shed on novel life-promoting therapeutic agents.  Within this same group which we find coniine, the piperidine alkaloid and so-called “killer of Socrates” that claimed the philosopher’s life by respiratory arrest, we have the indole alkaloids with therapeutic potential in obstructive lung disease.  In certain cases, one alkaloid may serve as the antidote for the toxicity of another, as atropine and physostigmine reverse each other’s toxicities. Since the discovery of morphine by modern chemistry, through repurposing and metabolic engineering, plant alkaloids have been exploited for their immense medicinal properties in a wealth of pharmacologic applications. These include the treatment of pain (e.g., morphine, cocaine), fever (e.g., quinine), malignancy (e.g., vinblastine, berberine), asthma (e.g., ephedrine), dysrhythmias (e.g., quinidine), hyperglycemia (e.g., piperine), hypertension (e.g., reserpine), and bacterial infections (e.g., ciprofloxacin). This activity will incorporate the available data to highlight the less desirable effects of these metabolites, the exposure to which may lead to disease.

Many drugs that block voltage-gated K[+] channels encoded by the human Ether-à-go-go-Related Gene (hERG) can cause long QT syndrome and life-threatening cardiac arrhythmias, yet the molecular mechanisms that determine this risk remain unclear. A process that may counteract arrhythmogenic hERG block, termed facilitation, is common to many clinically-approved hERG blockers including nifekalant, amiodarone, promethazine, imipramine, nortriptyline, haloperidol, verapamil, carvedilol, metoprolol, propranolol, quinidine, fluoxetine, and chlorpheniramine. Facilitation is an increase in hERG current, under certain conditions, due to these blockers. Here, we propose that an agonism-while-blocking mechanism undergirds facilitation. We focus on nifekalant, a Class III antiarrhythmic drug and exemplar hERG blocker that induces facilitation. We tested the hypothesis that nifekalant opens hERG channel gates while blocking, and that unblock of these open-yet-blocked channels results in supranormal hERG current. We develop rate-theory kinetic models to identify features of agonism-while-blocking that produce facilitation. We generate atomistic models that predict that nifekalant blocks the hERG conduction path while modulating the intracellular conduction gate. Voltage-clamp measurements reveal that agonism-while-blocking undergirds nifekalant block and facilitation. We speculate that this agonism-while-blocking mechanism contributes to the relative safety of hERG blockers that induce facilitation.

KCNT1 channels are important targets in a variety of conditions, especially childhood epilepsies associated with activating mutations. Growing interest in this target has driven the discovery of KCNT1 channel modulators to serve as probes and leads for the development of novel therapeutics. Early pharmacological modulators such as quinidine, bepridil, and clofilium found through drug repurposing were generally limited by poor selectivity, low potency, and safety concerns, restricting their clinical potential. Structure-guided virtual screening, high-throughput screening, and systematic optimization have identified a suite of new KCNT1 modulators with more favorable pharmacological properties than existing in vitro probes identified through drug repurposing. This review provides an overview of the peer-reviewed literature describing chemotypes with structural features that enhance potency and selectivity compared to earlier KCNT1 modulators. Also discussed are structure-activity relationship (SAR) studies that have enabled the rational optimization of molecules to enhance overall drug-like properties. Together, these insights provide a foundation for the rational design of next generation KCNT1-targeted therapies. Progress in this field has been tremendous, and the chemical diversity of small molecule KCNT1 inhibitors has grown rapidly. While KCNT1 activators are rarer, progress has been made in this area as well.

Precise evaluation of plasma protein binding (PPB) (fu) is critical for predicting the pharmacokinetic behaviour of new chemical entities.In this study, we describe a rapid equilibrium dialysis (RED)-based flux dialysis approach for estimating PPB (fu) utilising pre-equilibration diffusion data.The empirical membrane permeability (Pmem) of the RED system was established using reference compounds spanning a wide range of binding affinities, including quinidine, imipramine, fluconazole and sertraline.The averaged Pmem value was subsequently applied to determine fu for test compounds such as efavirenz, mifepristone and warfarin, representing diverse physicochemical and binding properties.The current approach demonstrates immediate applicability for low- to moderately bound compounds, where reliable flux data can be obtained within a shorter timeframe.While early-phase diffusion provides useful kinetic information, accurate fu estimation for highly bound compounds requires extended sampling to obtain reliable flux measurements.Fu values obtained from the flux-based model showed close agreement with literature-reported equilibrium dialysis data, supporting the robustness and predictive validity of the approach.The results demonstrate proof-of-concept feasibility of a flux-based approach using RED device, while highlighting limitations related to signal sensitivity and assumptions associated with empirical permeability.