Molecular Pharmacology

TRPA1 is a non-selective cation channel that plays a crucial role in several pain and inflammatory conditions. Agents reducing membrane cholesterol decrease TRPA1 activation, but it remains unclear how cholesterol-lowering medications affect TRPA1 function. Given that TRPA1 is activated by a wide variety of chemicals, we explored whether statins have acute effects on this channel. We found that five commonly used statins activate human and mouse TRPA1 in a reversible and concentration-dependent manner. The effective concentrations were above the micromolar range, in the order: simvastatin {approx} lovastatin < fluvastatin < atorvastatin < pravastatin. Statin-induced activation was not correlated to changes in membrane order, nor mediated by N-terminal cysteine residues contributing to electrophilic compound agonism. Molecular docking calculations and the functional characterization of single-point mutants revealed two separate putative binding sites, one situated close to the kink of transmembrane segment 5 (TM5) and the other at the interface between TM4 and TM5. The mTRPA1 inhibitor A-967079 largely abrogated the response to the electrophilic agonist allyl isothiocyanate, but had weaker and varied effects across different statins and menthol. Mutation T877L strongly altered the effect of A-967079, also in an agonist-dependent manner, suggesting competitive binding between this antagonist and the non-electrophilic agonists. The identification of two distinct agonist binding sites may help explaining how TRPA1 is able to respond to a large variety of non-electrophilic compounds, while the finding of competitive interactions at one of these sites may help guide the development of agonist-specific antagonists of therapeutic relevance.

G protein-coupled receptors rely on dynamic conformational changes to coordinate G protein activation and recruitment of regulatory transducers such as G protein-coupled receptor kinases and {beta}-arrestins. The chemotactic receptor GPR183 has been implicated in a context-dependent role in hematological malignancies. Here, we investigated the impact of A338V mutation located within the C-terminal tail of GPR183. This mutation is associated with acute myeloid leukaemia. Using bioluminescence resonance energy transfer-based assays in HEK293A cells, we assessed receptor-proximal signaling events. The A338V variant displayed preserved agonist potency and comparable agonist-induced Gi activation relative to wild type, although constitutive activity towards Gi was modestly reduced. In contrast, recruitment of GRK2 and {beta}-arrestin2 was consistently impaired across multiple assay configurations. These differences were not attributable to altered receptor abundance, as the C-tail untagged mutant exhibited increased plasma membrane expression despite reduced regulatory transducer engagement. While intramolecular conformational biosensor measurements revealed subtle differences in global receptor conformation between WT and A338V, extensive molecular dynamics simulations supported the altered conformational sampling of the C-terminal tail in the A338V variant. Together, these data support a model in which the A338V substitution selectively alters C-terminal structural dynamics, impairing GRK2 and {beta}-arrestin2 recruitment while preserving G protein activation.

The calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor (GPCR) with an important role in calcium homeostasis, activating mutations of which cause hypocalcemia. Despite the receptors clinical importance and evidence suggesting {beta}-arrestin-1/2 can modulate CaSR signaling, the residues within CaSR required for {beta}-arrestin recruitment and the role of {beta}-arrestin in desensitization, trafficking and signaling are ill defined. We confirmed that {beta}-arrestin-1 and {beta}-arrestin-2 are recruited to CaSR upon receptor activation. Deletion of the distal cytoplasmic region of CaSR, which replicates several mutations identified in individuals with hypocalcemia, reduced {beta}-arrestin-1/2 recruitment and enhanced receptor signaling. Examination of the receptor cytoplasmic region identified three regions of serine and threonine residues that resemble phosphorylation codes identified in other GPCRs. Mutation of each of these residues to alanine demonstrated one region between amino acids 1003-1011 is important for both {beta}-arrestin-1 and {beta}-arrestin-2 recruitment, while disruption of a second region between residues 976-981 impaired {beta}-arrestin-2 recruitment. Alanine mutagenesis of these residues also reduced CaSR signaling and impaired receptor internalization suggesting an important role in receptor desensitization. Three variants in the ClinVar dataset reported in disorders of calcium homeostasis were identified and two, T1006M and T1008P, were shown to reduce {beta}-arrestin recruitment and enhance CaSR signaling. Further analysis of the T1006M variant showed it reduced CaSR internalization. Thus, we have identified two regions within the CaSR cytoplasmic region that are important for {beta}-arrestin recruitment, receptor desensitization and internalization. Mutation of residues within these regions may represent another mechanism for the pathogenesis of hypocalcemia.

There is growing interest in understanding how hormonal signaling pathways contribute to the pathophysiology of mood disorders, based on the premise that fluctuations in sex hormones influence mood, a relationship particularly evident in conditions such as premenstrual dysphoric disorder, prenatal depression, postpartum depression, and perimenopausal depression. Estrogen receptor alpha (ER) is predominantly localized in the nucleus, but can also be associated with the cell membrane, thus mediating a broad range of genomic and non-genomic effects through distinct intracellular pathways. By employing a combination of computational simulations and in vitro biochemical and cell-based assays, we systematically evaluated the potential binding and functional interactions of antidepressant compounds with ER. Our results provide compelling evidence that antidepressants may not only affect classical monoaminergic targets but also modulate hormone receptor activity, particularly that of ER. These findings are consistent with the hypothesis that ER plays an important role in mood regulation and highlight it as a potential therapeutic target. Moreover, this work raises the possibility that the clinical efficacy of certain antidepressants may, at least in part, derive from their capacity to influence estrogen receptor-mediated signaling. Significance statementClinical observations suggest a link between female sex hormones and mood, highlighted by the higher prevalence of depression in women and increased vulnerability to depression during hormonal fluctuations. Here, we report that structurally diverse conventional and rapid-acting antidepressants directly interact with estrogen receptor alpha (ER). This interaction is associated with rapid intracellular signaling in cellular models. These findings indicate that, alongside their conventional targets, antidepressants may also engage sex steroid receptor components and signaling. This work broadens our basic understanding of antidepressant pharmacology at the cellular level, offering an additional perspective that may inform future research into the biological mechanisms of mood disorders and suggest a framework for developing targeted therapies for hormone-associated depressive disorders.

Intracellular calcium signaling plays a vital role in regulating various cellular processes including gene regulation, motility, metabolism and cell death. Inositol 1,4,5-trisphosphate receptors (IP3R) on the Endoplasmic Reticulum (ER) are a major cation channel that regulates stimulus-induced calcium release from the ER. While several molecular players regulate activity of IP3R, its regulation by actin filaments were uncharacterized. Here we show that actin filaments polymerized by a specific actin nucleator INF2 facilitates agonist-induced IP3R activity. Our results demonstrate that INF2-mediated actin filaments regulate formation and/or stability of IP3R clusters on the ER that have been previously shown to be hotspots of ER calcium release. Using cell-biological and biochemical techniques we further show that INF2 physically interacts with IP3R isoforms, often at IP3R clusters. While INF2-IP3R interaction is independent of INF2-activity, the ability of INF2 to mediate IP3R clusters is dependent on its actin polymerization activity. Finally, we demonstrate that in addition to its calcium mobilization activity, INF2 on ER specifically regulates IP3R cluster positioning to mediate ER-mitochondrial contacts and facilitate ER to mitochondrial calcium transfer. Overall, these results reveal an actin-dependent step in regulation of IP3R activity both in terms of ER calcium release and modulation of ER-mitochondrial contacts. HighlightsO_LIINF2-mediated actin filaments potentiate agonist-induced IP3R-mediated ER calcium release without affecting the ER calcium stores per se. C_LIO_LIER-localization of INF2 is dispensable for its role on IP3R activity. Moreover INF2-mediated actin filaments affect the activity of all IP3R isoforms. C_LIO_LIINF2 interacts with IP3R in an activity and actin filament independent manner through its C-terminal region. C_LIO_LIINF2 regulates IP3R cluster formation in actin-filament dependent manner and thereby regulates IP3R activity. C_LIO_LIFurther we show that ER-localized INF2 specifically regulate IP3R cluster positioning thereby promoting ER to mitochondrial contact and calcium transfer. C_LI

Desensitization and internalization of most G protein-coupled receptors (GPCRs) depend on phosphorylation by GPCR kinases (GRKs), promoting {beta}-arrestin recruitment. Atypical chemokine receptors (ACKRs), including ACKR3, are structurally related to classical chemokine receptors but do not activate heterotrimeric G proteins. ACKR3 signaling and trafficking have been proposed to depend on GRK5-mediated phosphorylation and {beta}-arrestin interaction. However, the respective roles of {beta}-arrestins, GRKs, and receptor phosphorylation in chemokine scavenging and in constitutive or ligand-induced trafficking remain debated. Using bioluminescence resonance energy transfer (BRET)-based biosensors and immunofluorescence imaging with fluorescently labeled receptors and chemokines, we examined ACKR3 interaction with {beta}-arrestin1/2 and assessed chemokine scavenging and receptor trafficking in {beta}-arrestin-deficient ({Delta}{beta}arr1/2) cells. We also evaluated the contribution of GRK-mediated phosphorylation. {beta}-arrestins supported agonist-independent receptor internalization but were dispensable for chemokine-induced internalization and chemokine scavenging. In contrast, GRKs were required for ligand-promoted endocytosis, with either GRK2/3 or GRK5/6 being sufficient. Mutation of ACKR3 phosphorylation sites impaired {beta}-arrestin recruitment but did not completely block internalization and scavenging, whereas complete C-terminal truncation abolished both processes. Consistently, kinase-dead GRK2 rescued ACKR3 endocytosis in {Delta}GRK2/3/5/6 cells, indicating a scaffolding role partially independent of kinase activity. Moreover, G{beta}{gamma} was not required for GRK2-mediated ACKR3 endocytosis, as a PH-domain-deleted GRK2 mutant restored internalization in {Delta}GRK2/3/5/6 cells, and G{beta}{gamma} sequestration by {beta}ARKct-CAAX did not inhibit this process consistent with the notion that ACKR3 does not promote G protein activation. Thus, ligand-promoted ACKR3 internalization and chemokine scavenging occur independently of {beta}-arrestins but requires GRKs. One-sentence summaryGRKs are essential for ACKR3 endocytosis and chemokine scavenging, whereas {beta}-arrestins and receptor phosphorylation are dispensable.

Targeting the kappa opioid receptor (KOR) system has emerged as a potential alternative to current analgesics, however, advancing the therapeutic development of KOR requires further elucidation of its intracellular signaling events and modulators. Among these intracellular modulators, Regulators of G protein signaling (RGS) proteins act as key modulators of GPCR signaling to shape nociceptive circuits and influence pain processing. Despite this, the molecular diversity of RGS proteins that shape KOR signaling and its behavioral consequences remains largely unexplored. Here we report that RGS6, a member of the R7 RGS family, is highly expressed in nociceptive areas and modulates multiple modalities of KOR-dependent anti-nociception and nocifensive behaviors. Using global single and double knockout mouse models we show that this anti-nociceptive phenotype was highly specific to RGS6 within the R7 RGS family. Further we demonstrate that the R7 RGS family displays a lack of functional redundancy in regulation of KOR signaling and behaviors. Using peripherally restricted KOR agonists, we found that KOR-RGS6 anti-nociceptive signaling displays sex differences in a site-specific manner, as females but not males displayed enhanced anti-nociceptive and blunted nocifensive behaviors. Our findings suggest that RGS6 is a highly specific modulator of KOR-dependent anti-nociceptive signaling and plays an essential role in modulating nociceptive circuits, potentially aiding in the development of novel analgesic drugs and therapeutics.

Progressive cardiomyopathy is the leading cause of death in Duchenne muscular dystrophy (DMD). Dysregulation of calcium handling has been implicated in cardiomyopathy progression in DMD. Here we describe a therapeutic approach to improve calcium homeostasis in a mouse model of DMD using the novel therapeutic NDC-1171, which is a positive allosteric modulator of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump. We synthesized NDC-1171 and treated 4-week-old D2.mdx mice (n=9) via oral gavage. A group of D2.mdx mice (n=9) and a group of DBA/2J mice (n=9; background strain) received a vehicle on the same schedule. We used ultrasound to assess left ventricular function, followed by a treadmill exhaustion test and a 4-paw grip strength test to assess skeletal muscle function. NDC-1171 attenuated cardiac functional decline in D2.mdx mice. At 16 weeks of age, left ventricular ejection fraction (LVEF) was significantly preserved in mice treated with NDC-1171 (57.7{square}{+/-}{square}0.5%) compared to mice treated with a vehicle (50.7{square}{+/-}{square}0.9%, p{square}<{square}0.05), though remained lower than background strain controls (62.4{square}{+/-}{square}0.6%). In contrast, functional behavior testing revealed no significant improvement in skeletal muscle function with treatment. These data suggest that treatment with the SERCA pump modulator NDC-1171 helps preserve cardiac function in a murine model of DMD, even as skeletal muscle function was impaired. Future work will be needed to determine if the benefits of this novel SERCA activator translate to large animal and clinical studies, but these initial results are promising and could help guide development of future treatments for pediatric patients with muscular dystrophy.

The NLRP3 inflammasome pathway is central to host defense, but dysregulated activation of inflammasomes promotes diseases associated with metabolic syndrome (diabetes, obesity, CVD, MASLD), neurodegenerative diseases (Alzheimers and Parkinsons), autoinflammatory conditions (CAPS, gout), and respiratory illnesses (asthma/COPD, and COVID-19). Therapeutic modulation of NLRP3 is challenging as it requires selective blockade of detrimental inflammasome activation without broadly suppressing innate immunity. Here, we used a phenotypic screen in THP-1 ASC-GFP monocytes to identify FDA-approved drugs that can block LPS-induced priming of NLRP3 inflammasome or inhibit NLRP3 assembly (ASC speck formation) without disrupting upstream priming. Various classes of drugs, such as antidepressants (Fluoxetine, Duloxetine), antihypertensives (Irbesartan, amlodipine, nebivolol), antidiabetics (Rosiglitazone), {beta}-adrenergic agonists (Salmeterol), antimalarials (Mefloquine), antifungals (Azoles, ciclopirox), and antivirals (Saquinavir, Remdesivir), were identified as potent blockers of either priming or assembly of NLRP3 inflammasome. Hits were validated in several biochemical assays, including effect on release of proinflammatory cytokines, autophagy, lysosomal biogenesis, LPS binding, NF-kB nuclear localization, mitochondrial membrane potential, mitochondrial ROS, and biophysical properties of the cell membrane. A subset of identified drugs was tested in murine studies to probe effects on NLRP3 inflammasome assembly/activation and LPS-induced sepsis. Mice treated with ASC puncta blockers showed markedly reduced proinflammatory cytokines in peritoneal lavage and plasma. Mice treated with LPS-priming blockers showed a sex-specific increase in survival rate in the mouse model of LPS-induced mortality, validating the in vitro screen. Further studies in primary human cells and in vivo disease models are needed to assess the repurposing and therapeutic relevance of identified drugs.

Background: GLP-1 receptor agonists (GLP1-RAs) are an established treatment for type 2 diabetes mellitus (T2DM) and obesity. Their widespread use is set to increase through both indication expansion and patent expiry. As well as efficacy, it is crucial to understand the safety of this drug class to enable optimal use. Here we demonstrate how a genetic approach can augment signal-detection and post-market authorization surveillance. Methods: We used single nucleotide polymorphisms (SNPs) in GLP1R to recapitulate the effect of agonism with GLP1RAs on circulating glucose, glycated hemoglobin (HbA1c), body mass index (BMI) and risk of type 2 diabetes (T2DM) using Mendelian randomisation. We then tested if the adverse effect highlighted by medicines regulators of pancreatitis and the emerging effect of sarcopenia were causally related to GLP1R agonism, using this approach. Analyses were conducted in UK biobank and replicated in FinnGen and All of Us, results being combined using meta-analysis. Analyses were further stratified by a priori risk factors of age and alcohol consumption. Results: Genetically proxied GLP-1R agonism was associated with a reduction in glucose (exp({beta}) = 0.95 95% CI [0.94, 0.97]), HbA1c (exp({beta}) = 0.94 95% CI [0.92, 0.95]), and BMI (exp({beta})=0.98 95% CI [0.97, 0.99]); and a reduced risk of T2DM (OR = 0.82 95% CI [0.79 to 0.86]). Risk of acute and chronic pancreatitis was however increased (OR = 1.10 95% CI [1.01 to 1.20] and OR = 1.05 95% CI [0.95, 1.17], respectively), which varied as a function of age with risk most pronounced in those aged 50-59 years-old (OR = 1.79 95% CI [1.43, 2.24], OR = 1.57 95% CI [1.16, 2.12]) and in drinkers (OR = 1.32 95% CI [1.12, 1.54], OR = 1.36 95% CI [1.12, 1.65]). Risk of sarcopenia also increased (OR 1.34; 95% CI 1.05,1,71). Conclusions: Genetically proxied agonism with GLP-1RAs recapitulated the pharmacological effects of GLP1-1RAs on glycaemic traits, BMI and T2DM risk. This approach supports a causal effect of GLP-1RAs on the well reported adverse effects of pancreatitis and further indicates age and alcohol consumption as risk modifying effects. The less well reported but emerging effect of sarcopenia appears to also be casually related to agonism at GLP-1R. These analyses suggest a genetic approach could be used as an adjunct to signal detection studies to enhance safety regulation as well as personalisation of the use of these drugs.

The neurosteroid allopregnanolone is a positive allosteric modulator of GABA(A) receptors, which has proved beneficial in the treatment of major depressive disorder and epilepsies. It also has a role in treating the mood swings that are associated with fluctuations in its level during the menstrual cycle. Nonetheless, a subset of women do not tolerate high levels of allopregnanolone. Iso-allopregnanolone, a negative allosteric modulator, as well as synthetic steroid antagonists are used to treat such conditions. However, steroid-based medications are difficult to deliver and their specificity of action can be unclear. Recently introduced novel nonsteroidal agents that, like iso-allopregnanolone, can reverse the action of positive allosteric modulators without changing the positive action of GABA, might provide an alternative. We surveyed a number of them on human 1{beta}3{delta} GABAARs using a [3H]muscimol binding assay. A 6-membered ring spiro-hydantoin, DKD99, allosterically reversed the positive allosteric action of allopregnanolone over a wide concentration range (6 to 1,000 nM). DKD99 shifted allopregnanolones modulation curve 10-fold to the right. Furthermore, it has a much lower affinity when exerting similar actions on 1{beta}3{gamma}2 receptors. Agents such as this have utility for elucidating underlying mechanisms and may offer an alternative pathway for the development of nonsteroidal therapies against the positive allosteric modulatory actions of neurosteroids.

The KCa2.2 and KCa3.1 channels are fundamental regulator of cellular K+ concentration, and promising target to treat diseases such as spinocerebellar ataxia and cancer. To fully exploit their therapeutic potential, and to continue studying their pathophysiological role, it is crucial to develop selective modulators for each of these two channels. Here we present a computational study to identify the molecular determinants behind the selectivity of two recently reported KCa2.2 modulators. We leveraged a protocol combining in silico mutagenesis, molecular dynamics simulations, and protein-ligand docking to analyse the pockets targeted by these ligands. We identified a Ser353/Pro245 substitution to be the main driver of the distinct pocket shapes in KCa2.2 and KCa3.1 channels, ultimately defining modulator selectivity. This approach provides novel insights into the structural differences of this binding site across potassium channel subtypes, shedding light on the selectivity determinants of modulators targeting this pocket.

Background: Cysteamine is the only disease-modifying therapy for nephropathic cystinosis and has shown promise in mitochondrial disorders, but its clinical utility is limited by poor tolerability due to high peak concentrations with existing formulations. TTI-0102 is a novel natural controlled-release cysteamine prodrug designed to provide sustained cysteamine exposure with improved tolerability. Methods: A multi-center, randomized, single-blind, placebo-controlled Phase 2 trial enrolled 9 patients with MELAS syndrome caused by mtDNA m.3243A>G mutation (>50% heteroplasmy) and moderate disease severity (NMDAS score 15-45). Patients received placebo (n=3) or TTI-0102 at 2.75 g/day for one week then 5.5 g/day (n=6, equivalent to 2.5 g/day cysteamine base). Pharmacokinetic parameters, safety, and pharmacodynamic biomarkers including pyruvate, taurine, pantothenic acid, tryptophan, GSH/GSSG, lactate, GDF-15, and FGF-21 were assessed. Clinical efficacy was evaluated using the Modified Fatigue Impact Scale (MFIS) and 12-minute walk test. Results: TTI-0102 demonstrated expected gastrointestinal side effects (nausea, vomiting, diarrhea) consistent with the cysteamine class, with dropout occurring in patients 50 kg receiving fixed 5.5 g/day dosing. Weight-based dosing at 60 {+/-} 5 mg/kg TTI-0102 (~26 mg/kg cysteamine base equivalent) achieved sustained 24-hour cysteamine exposure with half the daily dose and peak concentrations lower than expected by dose proportionality, compared to approved formulations (Procysbi: 56 mg/kg, peak 2.5 mg/L vs. TTI-0102: 26 mg/kg, peak ~2 mg/L). TTI-0102 significantly elevated pantothenic acid (plateauing at 2 weeks) and taurine levels, providing mitochondrial cofactor support and antioxidant effects. Statistically significant pharmacodynamic effects included increased plasma pyruvate (p=0.03) without lactate elevation, suggesting enhanced glycolytic flux, and decreased tryptophan (p<0.01), potentially reducing oxidative stress from neurotoxic kynurenine pathway metabolites. Interestingly, increase in plasma pyruvate and decrease in tryptophan were negligible at doses up to 40 mg/kg/day, optimal at 60 mg/kg/day, and slightly less at 65 mg/kg/day. GSH/GSSG measurements were confounded by sample stability issues. GDF-15, FGF-21, and 12-minute walk distance showed no treatment-related changes. Most notably, MFIS total scores demonstrated significant improvement in TTI-0102-treated patients at 60 mg/kg/day average dose compared to placebo (p=0.04). Polynomial regression revealed therapeutic onset at ~4 weeks, maximal benefit at ~12 weeks, and subsequent plateau. Conclusions: This Phase 2 trial provides proof-of-concept that TTI-0102 is safe and well-tolerated in MELAS patients while treated with less than 65 mg/kg/day, with efficacy signals in fatigue reduction, a cardinal symptom affecting 71-100% of mitochondrial disease patients. The drug tri-faceted mechanism through sustained cysteamine, taurine, and pantothenic acid delivery addresses oxidative stress, mitochondrial energy metabolism, and cofactor deficiency. Significant MFIS improvement coupled with favorable modulation of pyruvate and tryptophan supports advancing TTI-0102 to larger Phase 2b/3 trials in mitochondrial disease employing weight-based dosing (60 {+/-} 5 mg/kg), validated patient-reported outcomes, and minimum 12-week treatment duration. The same mechanism of cysteamine/cystine thiol-disulfide exchange in lysosomes that may benefit mitochondrial diseases also supports cystinosis treatment. An investigator-initiated study in cystinosis will evaluate whether once-daily TTI-0102 at 60 {+/-} 5 mg/kg can maintain therapeutic WBC cystine levels, potentially offering improved adherence and quality of life compared to current twice-daily or four-times-daily regimens, and this weight-adjusted dosing strategy and pharmacodynamic biomarkers identified in the MELAS study are going to be used to inform the design of the planned Phase 2 study in Leigh syndrome, another mitochondrial disorder, in collaboration with the Childrens Hospital of Philadelphia (CHOP), with particular attention to dose optimization and biomarker-based assessment of pharmacological activity. Acknowledgement: We are very thankful to the patients and the clinical teams of Radboud University Nijmegen Medical Centre (Netherlands) and Centre Hospitalier Universitaire d'Angers (France) for their participation in this operationally challenging study.

Background: Mocravimod is an oral sphingosine-1-phosphate (S1P) receptor modulator. This Phase 1 multiple-ascending-dose study evaluated its safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) in healthy volunteers. Methods: In this double-blind, randomized, placebo-controlled, parallel-group trial, 60 healthy male volunteers were enrolled in five cohorts. Mocravimod was administered once daily at 0.3, 0.6, 1.2, or 3.0 mg for 14 days, or at 2.0 mg for 28 days. Safety assessments included adverse events (AEs), laboratory tests, vital signs, electrocardiography, and Holter monitoring. PK of mocravimod and its active metabolite, mocravimod-phosphate, and PD effects on absolute lymphocyte count (ALC) and leukocyte subsets were assessed. Results: Fifty-nine of 60 participants completed the study. One participant in the 3.0 mg cohort discontinued treatment because of asymptomatic transaminase elevation. No deaths or serious AEs occurred. AEs were mostly mild or moderate, transient, and showed no clear dose relationship. Mocravimod produced dose-dependent reductions in ALC from 0.6 mg onward, with maximum geometric mean reductions of 65%, 74%, 83%, and 77% at 0.6, 1.2, 2.0, and 3.0 mg, respectively. ALC values recovered to above the lower limit of normal during follow-up in all cohorts. Holter monitoring showed an initial placebo-corrected reduction in heart rate of approximately 10-15 beats/min at doses of 1.2-3.0 mg, which attenuated with continued dosing. One participant in the 3.0 mg cohort had a recurrent daytime second-degree atrioventricular block (Mobitz I/Wenckebach), reported as a mild non-dose-limiting AE. No QT prolongation was observed. Exposure to mocravimod and mocravimod-phosphate increased approximately dose-proportionally. Steady state was reached by Day 14 (Day 28 in the 2.0 mg cohort), accumulation was approximately five- to sevenfold, terminal half-lives were approximately 100-40 hours for both analytes, and parent-to-metabolite exposure ratios were close to 1. Conclusions: Once-daily mocravimod up to 3.0 mg for 14 days and 2.0 mg for 28 days was generally well tolerated and showed predictable S1P-modulator class effects on lymphocyte counts and heart rate, with PK properties supporting once-daily dosing and further clinical development.

BackgroundThe discontinuation of Fasiglifam (TAK-875), a GPR40/FFAR1 full agonist, during Phase 3 clinical trials due to hepatotoxicity led to widespread abandonment of GPR40 as a viable therapeutic target for type 2 diabetes mellitus (T2DM). However, mechanistic evidence suggests that Fasiglifams hepatotoxicity arises from mitochondrial liability driven by high lipophilicity (aLogP = 5.31), rather than from on-target GPR40 signaling. We hypothesized that target-level failure was incorrectly inferred from compound-level safety concerns, and that superior candidates exist within publicly available databases. MethodsWe queried ChEMBL Release 36 (28 GB SQLite, 74 tables) for all compounds with documented GPR40/FFAR1 activity (UniProt: O14842). Compounds were filtered by EC50 [&le;] 10 nM in nM units with standard relation "=". Drug-likeness was assessed using Lipinskis Rule of Five (Ro5), aLogP, molecular weight (MW), hydrogen bond donors/acceptors (HBD/HBA), and polar surface area (PSA). A parallel analysis of Therapeutic Target Database (TTD v10.1.01, 4,298 targets) provided clinical context. A real-world evidence (RWE) patient stratification framework was constructed using EMR data from tens of millions of patients with >10 years of longitudinal follow-up. ResultsOf 2,637 GPR40-active compounds in ChEMBL 36, 526 (19.9%) demonstrated EC50 < 100 nM and 102 (3.9%) demonstrated EC50 < 10 nM. Eight compounds met stringent drug-likeness criteria (Ro5 violations = 0, aLogP < 5.0, EC50 [&le;] 1 nM). The lead compound (CHEMBL4859651) exhibited EC50 = 0.04 nM (8.75-fold more potent than Fasiglifam), MW = 297 Da (43% lower), and aLogP = 4.30 (19% lower), with zero Ro5 violations. Mean MW of the eight candidates was 317 {+/-} 28 Da versus 524 Da for Fasiglifam. A parallel GCK analysis identified a protein-protein interaction target (CHEMBL3885579, GCK-GKRP interface) harboring 40 exclusive compounds as an orthogonal strategy for partial GCK activation. ConclusionsSystematic cheminformatic analysis reveals that compounds with substantially superior activity and drug-likeness profiles relative to Fasiglifam exist within ChEMBL 36. Fasiglifams hepatotoxicity is attributable to compound-specific physicochemical properties, not GPR40-mediated toxicity. RWE patient stratification may further mitigate hepatotoxicity risk for next-generation GPR40 agonists. These findings argue for systematic reappraisal of GPR40 as a viable therapeutic target for T2DM.

The atypical receptor ACKR3 works together with the canonical chemokine receptor CXCR4 to drive cell migration along gradients of their shared agonist CXCL12. CXCR4 promotes chemotaxis by activating canonical G protein pathways and recruiting {beta}-arrestins. ACKR3 indirectly regulates CXCR4-mediated chemotaxis by scavenging CXCL12. Unlike canonical chemokine receptors, ACKR3 does not couple to G proteins and instead is 100% biased towards {beta}-arrestins. CXCR4 activation by CXCL12 is exquisitely sensitive to subtle changes in both receptor and ligand. By contrast, ACKR3 is activation-prone: it recruits {beta}-arrestins in response to many ligands and is much less sensitive to mutations, suggesting distinct activation mechanisms compared to CXCR4. To explore the basis of these differences, we compared the dynamics of ACKR3 and CXCR4 complexes with chemokines using molecular dynamic (MD) simulations. Ten-microsecond atomistic MD simulations revealed that CXCR4 adopts a stable active state when bound to WT CXCL12 but transitions to an inactive state when in complex with the antagonist variant, [P2G]CXCL12. By comparison, ACKR3 exhibits a variable transmembrane (TM) 6 state distribution and persistently "active" TM7 when complexed with either WT CXCL12 or [P2G]CXCL12, the latter retaining substantial agonistic activity at ACKR3. We further identified ligand-mediated residue interaction networks in the TM core that regulate TM6 and TM7 activation in CXCR4 but are absent or disrupted in ACKR3, resulting in less constrained receptor dynamics. These findings were validated by BRET-based assays with CXCL12 and ACKR3 mutants. Together, the data suggests that the unique conformational dynamics of ACKR3 govern its activation propensity, its ligand promiscuity, and its atypical effector coupling.

Nicotinic acetylcholine receptors (nAChRs) belong to the pentameric ligand-gated ion channel superfamily (pLGICs). Among them, the neuronal homomeric 7 nAChR is highly permeable to calcium and plays critical roles in synaptic transmission, cell signaling, and inflammation modulation. The biogenesis of 7 nAChRs is enhanced by the chaperone proteins RIC-3 and NACHO. Previously, we reported a motif in the 5-HT3A receptor, another pLGIC, involved in RIC-3 modulation. Residues in this motif are conserved and also found within the L1-MX segment of the 7 nACh subunit. We therefore explored the regulatory roles of these conserved residues in the biogenesis of 7 nAChRs using multiple approaches, including heterologous expression in Xenopus laevis oocytes, mutagenesis, pull-down assays, cell-surface labeling, and two-electrode voltage-clamp (TEVC) recordings. We find that synthetic 7 L1-MX peptide interacts with both RIC-3 and NACHO. In particular, conserved residues W330, R332, and L336 in the L1-MX positively regulates the assembly of 7 oligomers and the biogenesis of 7nAChR. In presence of residues W330, R332, and L336, NACHO promotes an assembly of an 7 pentamer which is resistant to strong denaturing conditions. NACHO-promoted 7 pentamer is also resistant to Endo H enzyme. Sensitivity of the pentamer to moderate temperatures (37 {degrees}C, 45 {degrees}C, and 50 {degrees}C) suggests that NACHO stabilizes the pentamer via non-covalent interactions. In contrast, Ala replacements at these residues disrupt the biogenesis and abolish 7 current. NACHO and RIC-3 co-expression yields partial rescue of functional expression for some Ala replacement constructs. SUMMARYThis work identifies regulatory roles of conserved residues W330, R332, and L336 in the biogenesis of 7 nAChR. This discovery positions MX subdomain as a promising target for future drug development that can minimize adverse effects.

BackgroundTriple-negative breast cancer (TNBC) presents significant therapeutic limitations due to its aggressive heterogeneity and the rapid emergence of adaptive resistance to apoptosis-based regimens. Addressing these challenges requires polypharmacological strategies capable of modulating multiple signalling networks simultaneously. While the Cannabis sativa phytocomplex offers a vast chemical space for multi-target intervention, the quantitative pharmacological basis of its synergistic interactions remains largely uncharacterised. PurposeThis study aimed to deconstruct the synergistic landscape of high-purity phytocannabinoids (CBD, CBG, CBD-A) in combination with the sesquiterpene {beta}-caryophyllene (BCP) against TNBC, using MDA-MB-231 as a primary model and Hs578T as a validation line. MethodsGrowth Rate (GR) inhibition metrics and the SynergyFinder+ framework were used to map pharmacological interactions across four reference models. Subcellular dynamics and phenotypic transitions were characterised by high-resolution label-free holotomographic microscopy combined with live-cell kinetic imaging and single-cell fate mapping. ResultsTwo highly potent synergistic clusters were identified for CBD-CBG-BCP combinations, with ZIP, HSA, and Bliss synergy scores exceeding 65. CBD-A exhibited minimal interaction potential and was excluded from ternary studies. GR-based quantification further revealed that these combinations produced net cytotoxicity (GR < 0) at sub-IC concentrations of each component. Single-cell fate mapping by holotomographic microscopy identified a temporally ordered death programme: an initial phase of extensive cytoplasmic vacuolisation associated with focal perinuclear space swelling and progressive nuclear compression, morphological hallmarks of autosis, which is followed by a transition to apoptotic execution. The autotic nature of the primary death phase was confirmed by pharmacological rescue with digoxin, a selective inhibitor of the Na,K-ATPase. To the best of our knowledge, this sequential engagement of autosis followed by apoptotic execution represents the first documented instance of such a two-stage death programme in any cellular model. ConclusionThese findings provide robust evidence that specific phytocannabinoid-terpene ratios engage a Na,K-ATPase-regulated autotic programme as an upstream commitment step, followed by apoptotic execution, effectively circumventing the caspase-independent resistance mechanisms characteristic of TNBC. This study establishes a rational, quantitatively validated framework for transitioning from empirical botanical use to evidence-based, multi-target cannabinoid polypharmacology in aggressive breast cancer.

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels (pLGICs) essential for inhibitory synaptic transmission throughout the central nervous system. Despite progress in understanding their three-dimensional structure, the molecular basis for how neurotransmitter binding is transduced to ion channel gating remains poorly understood. Furthermore, relatively little is known about the contributions of distinct subunits to this coupling within typical heteromeric receptors. A highly conserved proline (site 1) in the M2-M3 linker of pLGIC subunits is involved in channel gating - e.g., P273 in the GABAAR {beta}2 subunit. In GABAARs, only the {beta} subunits have an additional proline in the M2-M3 linker (site 2) - e.g., {beta}2(P276) - whereas all other subunits have a non-proline at the homologous site 2 position. Here, we investigate the functional contribution of proline at site 2 in distinct subunits of 1{beta}2{gamma}2 GABAARs. We expressed wild type or mutant 1{beta}2{gamma}2 GABAARs in Xenopus laevis oocytes and used two-electrode voltage clamp electrophysiology to record channel currents in response to GABA and/or other ligands. First, we introduced a proline at site 2 in 1 or {gamma}2 subunits: 1(A280P) and {gamma}2(S291P). Second, we replaced the site 2 proline in the {beta}2 subunit with its homologous non-proline residue from 1 or {gamma}2 subunits: {beta}2(P276A) or {beta}2(P276S). We show that 1(A280P) confers enhanced GABA-sensitivity and spontaneous unliganded channel activity, whereas {gamma}2(S291P) has minor effects on channel activation. In contrast, {beta}2(P276A) or {beta}2(P276S) either had no effect or enhanced GABA-activation, respectively, indicating complex functional dependence on the side chain at site 2 in the {beta}2 subunit. When in combination with other substitutions, the presence or absence of 1(A280P) was consistently correlated with enhanced GABA-sensitivity and spontaneous activity. Thus, introduction of a proline at site 2 in the 1 M2-M3 linker biases the channel towards an activated state and prevents it from remaining closed at rest.

In the management of atrial fibrillation, the most frequently prescribed oral anticoagulant is apixaban, given at a fixed dose of 5mg BID. Apixaban is predominantly metabolized by cytochrome P4503A4 (CYP3A4) and is also a substrate for the drug efflux transporter P-glycoprotein (P-gp). In nearly 300,000 Medicare patients with AF receiving apixaban, we previously showed that concomitant therapy with drugs that inhibit both CYP3A4 and P-gp, specifically amiodarone or diltiazem, significantly increased serious bleeding that caused hospitalization and/or death. We hypothesized that this adverse effect was mediated by an increase in apixaban plasma concentrations caused by concomitant therapy that reduced drug elimination. Utilizing left-over samples obtained from clinically indicated blood draws that would typically be discarded, the Vanderbilt University Medical Center biobank BioVU contains >353,000 samples linked to de-identified electronic medical records (EMRs), with both DNA and plasma harvested. Of 35 samples drawn from patients taking apixaban 5mg BID, 5 were identified to be drawn from patients concomitantly taking drugs inhibiting both CYP3A4 and P-gp. Using a chromogenic anti-Xa assay, we found that plasma concentrations of apixaban were significantly higher (347{+/-}64 ng/mL; mean{+/-}SEM) for patients receiving concomitant CYP3A4/P-gp-inhibiting drugs compared to those not treated with these drugs (166{+/-}67 ng/mL; P=0.025, Mann Whitney). There were no differences between the 2 patient groups with respect to age, weight, or serum creatinine. The results of this pilot study provide preliminary data to support our hypothesis, and they demonstrate the practicality of obtaining pharmacokinetic data from a large cohort of plasma samples linked to deidentified EMRs. This approach could be used to define the role of apixaban levels in high-risk clinical scenarios and to better understand the relationship between drug levels and bleeding risk.