Molecular Pharmacology

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.

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.

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.

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.

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.

Cannabinoid receptors are therapeutically promising GPCRs that are also interesting test systems for structure-based methods, which have targeted them previously. Here we used the CB2 receptor as a template to explore several topical questions in library docking. Whereas an earlier campaign against the CB1 receptor led to potent but relatively non-selective ligands, here we found that targeting interactions with polar, orthosteric site residues led to subtype-selective ligands. Docking hit rate and especially hit affinity improved in moving from a 7 million to a 2.6 billion molecule library. Similar to earlier studies, docking against active and inactive states of the receptor did not reliably bias toward the discovery of agonists or inverse agonists. Cryo-EM structures of two of the new agonists, each in a different chemotype, superposed well on the docking predictions. Correspondingly, structure-based optimization led to 10- to 140-fold improvements within three different series, also consistent with well-behaved ligand families. Hit rates with a fully enumerated 2.6 billion molecule library resembled those of an implied 11 billion molecule library from a building-block method, consistent with the latters ability to explore this space, though higher affinities were discovered from the fully enumerated set. Overall, eight diverse families of ligands, with potencies <100 nM and mostly unrelated to previously known ligands were found. Implications for future studies are considered.

Measuring the activity of the tumor suppressor p53 in living systems is essential for understanding its dysregulation in cancer and other conditions, such as aging and diabetes. Zebrafish (Danio rerio) are a powerful vertebrate model that enable such studies, due to the evolutionary conservation of p53 structure and function. However, p53 activity in zebrafish has mainly been assessed using pharmacological methods that induce DNA damage or have off-target effects, making it difficult to isolate p53-specific responses from broader stress responses. Here, by using biophysical assays, molecular dynamics, and molecular assays, we show that sulanemadlin, a stapled peptide inhibitor of MDM2, binds to zebrafish Mdm2 and transcriptionally activates downstream targets of p53, including cdkn1a, isoform{Delta} 113p53, and Mdm2. No effect on gene expression was observed in embryos treated with a point-modified control peptide or in embryos carrying a mutation that renders p53 transcriptionally inactive. RNA sequencing further confirmed upregulation of p53 signaling and downregulation of DNA replication pathways, while an acridine orange assay showed no detectable increases in apoptosis. In contrast, the tested small molecule Mdm2 inhibitors exhibit reduced binding affinity to zebrafish Mdm2 due to an amino acid variation in the zebrafish Mdm2 binding pocket. By overcoming a species-specific barrier in p53-MDM2 binding, the stapled peptide sulanemadlin is the first pharmacological tool to specifically activate p53 in zebrafish without inducing measurable apoptosis, enabling direct in vivo studies of p53 regulation in cancer and other disease contexts.

Neuroactive steroids modulate GABAA and NMDA receptors allosterically, typically requiring specific structural features for their activity. In this study, we characterize YX84, a novel neuroactive steroid bearing a 3{beta} sulfate and p-trifluoroacetylbenzyl alcohol attached in an ether linkage to a hydroxyl group at steroid carbon 17. This compound and similar analogues exhibit an atypical pharmacological profile, with three distinct actions at GABAA receptors. First, YX84 is a full agonist, with EC50 near 1 {micro}M and comparable efficacy to GABA at GABAA receptors in native hippocampal neurons. It presents as a full agonist relative to GABA at 4/{delta} subunit-containing receptors. Second, YX84 acts as a slow-onset, potent positive allosteric modulator (PAM) of GABAA receptors at concentrations below those that gate a response. Finally, YX84 exhibits rapid desensitizing and/or blocking kinetics; voltage dependence is consistent with a contribution of channel block. Structure- activity relationship analyses reveal that both functional groups are essential for gating activity, while classical requirements such as carbon 3 hydroxyl stereoselectivity and carbon 5 reduction are dispensable. YX84 also modestly inhibits NMDA receptor currents, suggesting weak negative allosteric modulation. Behavioral assays show that intraperitoneal administration of YX84 (30 mg/kg) does not impair sensorimotor function, unlike allopregnanolone. These findings identify YX84 as a structurally distinct neuroactive steroid with dual receptor activity and favorable behavioral tolerability, offering a promising scaffold for therapeutic development targeting excitatory/inhibitory imbalance in neuropsychiatric disorders if pharmacokinetic considerations can be overcome.

Background Plasma gelsolin (pGSN) is a non-immunosuppressive anti-inflammatory immunomodulator with demonstrated efficacy in animal models of acute lung injury. Its potential role in moderate-to-severe acute respiratory distress syndrome (ARDS) is currently under investigation. Methods We conducted a phase 1, randomized, double-blind, placebo-controlled study to evaluate the safety, tolerability, and pharmacokinetics of recombinant human pGSN (rhu-pGSN) following intravenous (IV) administration to healthy volunteers. Thirty-two participants were assigned to 4 sequentially ascending dose cohorts (6, 12, 18, 24 mg/kg of body weight) to receive five IV infusions of rhu-pGSN or saline placebo. Each cohort includes 8 subjects randomized 3:1 with rhu-pGSN or placebo. Doses were administered at 0 hours, 12 hours, 36 hours, 60 hours, and 84 hours. The primary outcome is the incidence and severity of clinical and laboratory AEs regardless of causality. Secondary outcomes include the pharmacokinetics of IV rhu-pGSN and the presence of anti-rhu-pGSN antibodies at Day 28. Results Overall, 10 subjects (41.7%) who received rhu-pGSN reported a total of 13 adverse events (AEs), and 1 subject (12.5%) who received placebo reported an AE. All AEs were mild or moderate. AEs in system organ classes that were reported by 2 or more subjects in either arm were skin and subcutaneous tissue disorders (12.5% rhu-pGSN; 0% placebo), gastrointestinal disorders (8.3% rhu-pGSN; 0% placebo), and nervous system disorders (12.5% rhu-pGSN; 12.5% placebo). No AEs by preferred term were reported by more than 1 subject in either arm. Three subjects (12.5%) experienced an AE assessed as related to study drug. No serious AEs occurred, and no AEs led to study discontinuation, dose interruption/reduction, or death. There were no apparent between-treatment differences in laboratory abnormalities, vital signs, or electrocardiogram findings. Conclusions Overall, in this study, IV rhu-pGSN (up to 24 mg/kg daily) appeared safe and well tolerated compared to placebo. The median half-life of rhu-pGSN exceeded 14 h across all dosing regimens, supporting once daily IV dosing in healthy subjects. Trial registration This study was registered with ClinicalTrials.gov on 2023-03-29 under the registration identifier NCT05789745.

B7-H3 (CD276) is an immune checkpoint molecule frequently overexpressed in hepatocellular carcinoma (HCC) and represents a promising therapeutic target. However, its roles in tumor cell adhesion, metastatic behavior and immune evasion--particularly in interactions with natural killer (NK) cells--remain incompletely understood. In the present study, B7-H3 was depleted using small interfering RNA and CRISPR/Cas9 in epithelial (Huh7 and HepG2) and mesenchymal (SNU449) HCC cell lines. Tumor cell survival, apoptosis, adhesion, migration and invasion were evaluated using functional assays. Expression of adhesion molecules and immune checkpoint proteins was assessed by flow cytometry and western blotting. Oncogenic signaling pathways were analyzed by examining phosphorylation of Akt, ERK, FAK and STAT3. NK cell-mediated cytotoxicity was assessed using primary human NK cells. B7-H3 depletion reduced clonogenic survival and increased apoptosis in mesenchymal HCC cells under anchorage-independent conditions. Loss of B7-H3 impaired cell adhesion, migration and invasion, accompanied by downregulation of PTGFRN, E-cadherin, integrin 3 and integrin V, and reduced cell-to-cell aggregation under anchorage-independent conditions. B7-H3 depletion also decreased surface expression of PD-L1, PD-L2 and CD47. Notably, B7-H3-deficient cells exhibited enhanced susceptibility to primary NK cell-mediated cytotoxicity. Mechanistically, B7-H3 promoted tumorigenic signaling through Akt/S6, MVP/ERK and FAK/Src pathways in epithelial cells, and through FAK/Src and JAK2/STAT3 pathways in mesenchymal cells. Together, these findings reveal previously unrecognized roles for B7-H3 in coordinating adhesion and NK immune evasion in HCC, and support its therapeutic targeting for next-generation immunotherapies.

Chronic wounds, such as diabetic and ischemic ulcers, involve impaired perfusion and delayed healing. TOP-N53 is a novel bifunctional molecule combining nitric oxide (NO) release with phosphodiesterase-5 (PDE5) inhibition to enhance local NO-cGMP signalling, resulting in vasodilation and angiogenesis. This first-in-human, randomized, double-blind, vehicle-controlled Phase I trial assessed the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of single subcutaneous TOP-N53 doses in 29 healthy male volunteers. Each participant received injections of TOP-N53 and vehicle in the same forearm, but either at the proximal or at the distal site in an intra-individually blinded manner. Safety assessments included local and systemic parameters. PK and PD responses were evaluated by analysis of TOPN53 and its bioactivation metabolite TOP-52 in plasma, and by Laser Speckle Contrast Imaging (LSCI), a non-invasive method to measure skin perfusion, respectively. TOP-N53 was safe and well tolerated, with no serious adverse events or local or systemic adverse reactions. Plasma concentrations remained below the quantification limit and LSCI showed sustained dose-dependent increases in local skin perfusion at doses of 4.84 ug and 9.075 ug TOP-N53 SC for up to 24 h post injection when compared to vehicle. These findings support the favourable safety and tolerability profile of TOP-N53 associated with locally improved skin perfusion, encouraging its further clinical development as a topical treatment for chronic wounds with microvascular dysfunction.

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder marked by numerous renal cysts that impair kidney function, with about half of affected individuals progressing to kidney failure by midlife. Patients exhibit reduced circulating apelin, a ligand of the apelin receptor, known to regulate cardiovascular function including hypertension. We tested whether diminished apelin signaling contributes to cystogenesis and if exogenous apelin receptor activation can improve disease outcomes. MethodsPlasma samples from age- and sex-matched healthy controls and ADPKD participants were analyzed for circulating apelin peptides. To assess direct cystic effects, primary ADPKD renal epithelial cells were grown as 3D collagen-embedded cysts and treated with apelin agonists. Male and female Pkd1RC/RC; Pkd2+/- (PKD) mice were treated for 27 days with apelin agonists, vehicle, or the standard of care drug, Mozavaptan. Kidney and heart weight ratios, BUN, renal cAMP, and kidney transcriptional profiles were evaluated. ResultsCirculating apelin peptides were significantly reduced in ADPKD patients despite normal kidney function (eGFR, BUN, and creatinine). In vitro, both apelin and the small molecule apelin receptor agonist Azelaprag inhibited cyst growth. Apelin and Mozavaptan reduced kidney weight, cystic index, blood urea nitrogen and renal cAMP in PKD mice, whereas Azelaprag did not. Apelin downregulated expression of genes associated with cyst progression, including Lcn2 (Ngal), Postn, and Havcr1 (Kim-1). Mozavaptan, but not apelin, induced diuresis and reduced urinary concentration. ConclusionApelin receptor activation by exogenous apelin inhibited cAMP synthesis and cyst growth and improved kidney function in an orthologous mouse model of ADPKD. We propose that the apelin receptor may be a potential therapeutic target in ADPKD.

A cassane diterpene, 6{beta}-cinnamoyl-7-hydroxyvouacapen-5-ol (6{beta}CHV), isolated from Caesalpinia pulcherrima, has emerged as a promising anticancer drug lead with reported Wnt/{beta}-catenin pathway inhibitory activity and in vivo safety. The present study reports the in vivo pharmacokinetics and tissue distribution of 6{beta}CHV in Wistar rats following a single oral dose of 200 mg/kg. A reproducible RP-HPLC-UV method was developed and validated for quantifying 6{beta}CHV in rat plasma and tissues. Chromatographic separation was achieved using a gradient elution of methanol and water. The method was subsequently applied to investigate the pharmacokinetics and tissue distribution of 6{beta}CHV. Plasma pharmacokinetic analysis revealed delayed and moderate absorption, with a Tmax of 4 h and a Cmax of 1314.12 ng/mL. Following absorption, 6{beta}CHV is distributed widely across peripheral tissues, including the liver, heart, lungs, spleen, and kidneys, as well as pharmacological sanctuary sites such as the brain and testes. The highest concentrations were observed in the stomach, small intestine, and liver, with detectable levels persisting up to 24 h, reflecting extensive tissue partitioning and retention. Overall, these findings demonstrate that oral administration of 6{beta}CHV is feasible. However, the delayed absorption suggests that further optimization of formulation or alternative administration routes may enhance systemic exposure. This study provides the first comprehensive pharmacokinetic and tissue distribution profile of 6{beta}CHV, supporting its continued preclinical development as a potential anticancer therapeutic. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/715187v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@4ae86forg.highwire.dtl.DTLVardef@1e1e51aorg.highwire.dtl.DTLVardef@1881c43org.highwire.dtl.DTLVardef@f7789f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Crosstalk across two major receptor families involved in signal transduction, namely receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs), have been observed at different levels of their signaling cascades. Using newly developed enhanced bystander bioluminescence resonance energy transfer (ebBRET)-based biosensors that monitor the recruitment of SH2 domains to activated RTKs, we assessed the ability of GPCRs to modulate cellular localization of SH2 domains. Receptor-mediated activation of either Gq/11 or G12/13 but not Gs or Gi/o (e.g., thromboxane A2 receptor, TP, and type-2 protease activated receptor, PAR2) resulted in the plasma membrane (PM) dissociation of SH2 domains derived from RTKs effectors such as GRB2, STAT5 and PLC{gamma}1. The role of Gq/11, G12/13, Rho and downstream kinases in the subcellular SH2 domain redistribution was further confirmed using both pharmacological and genetic approaches. BRET imaging and spectrometric analyses showed that the dissociation of SH2 domains from the PM was accompanied by their accumulation in the nucleus and a reduction in RTK signaling activity, as determined using a STAT5 transcriptional assay. The effect of Gq/11 and G12/13 activation on STAT5 transcriptional activity was observed both in engineered systems and in HeLa cells endogenously expressing all the components of the regulatory mechanism. The Gq/11 / G12/13-mediated redistribution of SH2 domain-containing proteins represents an undescribed mechanism through which GPCRs regulate RTKs activity. Significance StatementThis study reveals a novel crosstalk mechanism between G protein coupled receptors and receptor tyrosine kinases showing that Gq/11 and G12/13 activation triggers Rho-dependent translocation of SH2-containing effector proteins, such as GRB2, PLC{gamma}1 and STAT5. This process causes compartmentalization inside the nucleus and thus reduces their availability at the plasma membrane, leading to attenuated RTK responses.

Despite decades of research, current understanding of the spectrum of targets bound by kinase inhibitors remains incomplete. This complicates mechanism of action studies, drug repurposing, and understanding of adverse responses. Here, we describe kinome-wide profiling of an optimal kinase library (OKL) comprising 192 small molecules selected based on stage of clinical development, chemical diversity, and target coverage. Our results show that polypharmacology is widespread among kinase inhibitors independent of regulatory approval. The generally understood ("assigned") targets of approved molecules are not necessarily the most potently inhibited and off targets include multiple understudied kinases. Moreover, median selectivity has not increased over time. We illustrate the use of synoptic OKL-kinome profiles in identifying potential toxicity targets, repurposing anti-inflammatory drugs for neurodegenerative and infectious diseases, and performing chemical genetic studies. Our studies illustrate how much remains to be discovered about the chemistry and biology of one of the largest classes of human therapeutics.

Acute kidney injury (AKI) is a serious and common clinical syndrome that currently has no effective treatment. Emerging evidence links coagulation pathways to kidney injury, particularly through coagulation proteases. Protease-activated receptors (PARs) are a family of G-protein coupled receptors (GPCRs) that are activated by proteolytic cleavage of their N termini, exposing a tethered ligand that initiates receptor signaling. PARs have been shown to play a major role in inflammation, vascular regulation, and tissue injury. PARs play key roles in inflammation, vascular regulation, and tissue injury. Previous work from the Hamm laboratory demonstrated that PAR4 contributes to AKI progression, as PAR4 knockout mice were protected in both unilateral ureteral obstruction and ischemia-reperfusion-based models of kidney disease. In this study, we investigated the potential of a PAR4 antagonist, VU6073819, at mitigating AKI progression in an ischemia-reperfusion injury (IRI) mouse model. PAR4 antagonism not only alleviated kidney injury and inflammatory response, but it significantly improved the survival. These findings identify PAR4 as a promising therapeutic target for AKI.

AimThe ERG b-wave is primarily attributed to ON bipolar cell activity, while the contribution of the OFF pathway and the differential role of voltage-gated sodium (NaV) channels in these pathways remain unclear. This study investigated whether pharmacological modulation of NaV channels differentially alters ON and OFF cone bipolar cell responses and ERG b-wave amplitudes. MethodsDark- and light-adapted ERGs were recorded from rats across stimulus intensities spanning rod, mixed rod-cone, and cone pathways (1-1000 lux). ON and OFF cone bipolar cell pathways were pharmacologically isolated using intravitreal cis-PDA. NaV channel activity was modulated via intravitreal administration of lidocaine and lamotrigine (blockers) and veratridine (agonist). Changes in b-wave amplitudes were analysed to assess pathway-specific effects. ResultsBoth lidocaine and lamotrigine significantly globally reduced ERG b-wave across all stimulus intensities, confirming a role for NaV channels in bipolar cell signalling. Pathway isolation revealed differential effects: lidocaine predominantly suppressed ON pathway, whereas lamotrigine preferentially reduced OFF pathway responses. In contrast, veratridine enhanced both ON and OFF pathway activity. These findings indicate that NaV channel activity in ON and OFF cone bipolar cells can be independently and differentially modulated. ConclusionThe ERG b-wave reflects integrated contributions from both ON and OFF cone bipolar cells. Differential NaV channel modulation alters these pathways distinctly, highlighting sodium channel-mediated mechanisms as potential targets for physiologically relevant retinal stimulation strategies in degenerative retinal conditions.

Primary mitochondrial diseases are clinically and genetically heterogeneous disorders, commonly caused by defects in the oxidative phosphorylation system. This heterogeneity presents major challenges for therapeutic development; however, a shared hallmark across these diseases is the accumulation of dysfunctional mitochondria. Enhancing mitochondrial turnover, by activating the selective degradation of dysfunctional mitochondria via mitophagy, concurrently with the activation of mitochondrial biogenesis, could represent a shared therapeutic strategy for mitochondrial diseases. Here, we describe a novel mitophagy inducer, CAP-1902. CAP-1902 is a new agonist of the MAS G-Protein Coupled Receptor (MasR). In fibroblasts from patients carrying a BCS1L mutation that impairs complex III (CIII) assembly, CAP-1902 increased mitochondrial turnover by promoting both mitophagy and biogenesis. Specifically, MasR activation triggered the AMPK/ULK1/FUNDC1 mitophagy pathway. Knockdown of FUNDC1 blocked mitophagy but not AMPK activation, confirming pathway specificity. Additionally, a decrease in the occurrence of depolarized mitochondria with treatment indicated the selective targeting of accumulated damaged mitochondria in the disease context. MasR activation by CAP-1902 also stimulated the nuclear translocation of PGC-1, promoting increased expression of transcripts associated with mitochondrial biogenesis, respiratory chain components, and mitochondrial translation. Remarkably, CAP-1902 was ultimately able to restore key defects in CIII-deficient fibroblasts by rescuing bioenergetics and correcting both the aberrant lysosomal distribution and the elevated integrated stress response markers, which is consistent with a shift toward a healthier mitochondrial population. In summary, we describe the first potential GPCR-mediated treatment of mitochondrial diseases and demonstrate that MasR activation by CAP-1902 induces mitochondrial turnover and improves mitochondrial function.

Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in elderly individuals for which no effective treatments are currently available. The photoreceptor loss in dry AMD is secondary to the demise of the retinal pigment epithelium (RPE) cells. The accumulation of extracellular deposits, known as drusen, resulting in part from deficient lysosomal and autophagosomal degradation, is a key feature of dry AMD pathogenesis. Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway that maintains proteostasis by targeting specific cytosolic proteins for lysosomal translocation and degradation. LAMP2A (lysosome-associated membrane protein 2A) functions as the key lysosomal receptor required for CMA. Using Lamp2a knockout mouse, we show that selective CMA dysfunction recapitulates AMD-like pathologies, including sub-RPE lipid and protein deposits, RPE atrophy, Bruchs membrane thickening, and impaired autophagic activity. Furthermore, we identify large-conductance Ca{superscript 2}-activated K (BK) channels as a therapeutic target for restoring autophagic activity. Mechanistically, pharmacological activation of BK channels with the small-molecule agonist GLA-1-1 enhances macroautophagy and stimulates autophagic flux by promoting autophagosome-lysosome fusion. Importantly, oral administration of GLA-1-1 in markedly attenuates structural, functional, and molecular retinal abnormalities in Lamp2a-deficient mice, suggesting that pharmacological activation of macroautophagy through facilitating autophagosome-lysosome fusion can partially compensate for CMA deficiency. Together, these findings demonstrate that pharmacological activation of macroautophagy can ameliorate the retinal phenotype resulting from CMA dysfunction and support BK channel activation by GLA-1-1 as a promising therapeutic strategy for dry AMD.