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.

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.

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 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: 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.

Mocravimod (KRP203) is a selective sphingosine 1-phosphate (S1P) receptor modulator currently in development for patients with haematological malignancies undergoing allogenic haematopoietic cell transplantation (HCT). This first-in-human, randomised, double-blind, placebo-controlled, single ascending oral dose study evaluated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of mocravimod in 136 healthy adult participants (EudraCT No. 2006-006814-13). Participants received single doses ranging from 0.01 to 40 mg or placebo, with a cohort dedicated to studying food-effect at 3 mg. Mocravimod demonstrated slow absorption (mean Tmax 6-11 hrs), extensive distribution, and a long terminal half-life (91-132 hrs). Exposure increased dose-proportionally for doses [&ge;]2 mg. The most common adverse events were headache, dizziness, and fatigue, all graded as mild or moderate; no serious adverse events or deaths occurred. Mocravimod-phosphate induced robust, dose-dependent reductions in lymphocyte counts, with significant decreases at doses [&ge;]2 mg and recovery to baseline observed in all but the highest dose groups. Cardiac effects included transient bradycardia and benign second-degree atrioventricular (AV) block at higher doses, without clinically significant arrhythmias. Food intake had minimal impact on PK. No clinically meaningful changes in pulmonary function or laboratory safety signals were detected. These results indicate that single oral doses of mocravimod up to 40 mg are safe and well tolerated in healthy adults, with predictable PK and expected PD effects. The findings support further clinical development of mocravimod as a targeted immunomodulator in settings such as allogeneic HCT for haematological malignancies.

In some countries, melatonin is sold without a physician prescription and dosage is unregulated. Transdermal products have become popular including those marketed for children. We measured consumer assumptions about these products among adult residents of the United States, analyzed lot-to-lot variability, and compared the pharmacokinetics of melatonin administered in oral, lotion, and bath product forms. Survey respondents (n=199) believed oral melatonin was more effective than transdermal products and that all melatonin products were relatively safe. Melatonin lotion products analyzed by HPLC displayed lot-to-lot variability as well as changes in formulation and product claims. To determine pharmacokinetics, three different treatments (oral tablets, lotion, and bath immersion) were administered to twelve undergraduate participants in a randomized, crossover design. Five additional participants completed bath product treatment only. Participants collected saliva samples up to 48 hours after administration, which were analyzed for melatonin by enzyme-linked immunosorbent assay. Oral (n=11) and lotion formulations (n=12) caused maximum salivary melatonin levels within 30 minutes after administration, but bath immersion did not cause increases in saliva melatonin (n=17). The half-life of oral melatonin was 1.17 [0.69 -- 1.65] hours versus 5.72 [3.75 -- 7.68] hours for lotion treatment (p = 0.011, effect size r = 0.770). Melatonin lotion may pose a risk to consumers who assume it is safe and less effective than oral tablets, when in fact it may be very potent and remain at high physiological levels into the following day. This study is registered on clinicaltrials.gov (NCT06382610) and was funded by the Sleep Research Society.

ABSTRACT Background: Self medication with analgesics and non steroidal anti inflammatory drugs (NSAIDs) is common in low- and middle income countries and may expose users to preventable adverse outcomes. Evidence from Guinea remains scarce. This study aimed to estimate the prevalence of self medication with analgesics and NSAIDs among pharmacy clients in urban Conakry, identify associated factors, and describe clinical risk situations. Methods: We conducted a pharmacy based analytical cross sectional study in 30 private pharmacies across Conakry, Guinea. A total of 1,032 participants seeking analgesics or NSAIDs were enrolled between November 3, 2012, and April 5, 2013. Self-medication was defined as acquisition or use without a valid medical prescription. Factors associated with self-medication were analysed using multivariable logistic regression. Results: Among 1,032 participants, 603 reported self medication (prevalence 58.4%). Previous unsupervised use was reported by 78.7%. The most frequently used medicines were paracetamol (56.9%, n=587), diclofenac (21.3%, n=220), ibuprofen (17.9%, n=185), and aspirin (3.9%, n=40). Overall, 68.0% (n=702) reported no knowledge of potential adverse effects. Clinical risk situations were frequent: gastrointestinal disorders (41.3%, n=426), hypertension (9.2%, n=95), and pregnancy exposure among reproductive age women (26.0%). In multivariable analysis, self medication was independently associated with previous analgesic/NSAID use (aOR = 2.8, 95% CI: 2.1 to 3.6), lack of knowledge of adverse effects (aOR = 1.9, 95% CI: 1.4 to 2.5), informal occupation (aOR = 1.6, 95% CI: 1.2 to 2.2), and age 18 to 59 years (aOR = 1.5, 95% CI: 1.1 to 2.1). Conclusions: In this pharmacy based study conducted in urban Conakry, self medication with analgesics and NSAIDs was common and frequently associated with limited awareness of potential adverse effects. These findings support the need for strengthened pharmaceutical regulation, pharmacist-led counselling, health literacy interventions, and improved access to primary care. Keywords: self medication; analgesics; NSAIDs; paracetamol; diclofenac; ibuprofen; pharmacy; Guinea; Conakry; drug safety; public health.

While Trastuzumab emtansine (T-DM1) and other HER2-targeting antibody-drug conjugates (ADCs) are used to treat cancer patients with HER2-amplified tumors, there is a need to improve the efficacy through the understanding of their mechanism of uptake into cells. Flotillin-2 (FLOT2) regulates the internalization of epidermal growth factor receptor (EGFR), leading us to investigate FLOT2 effects on HER2 internalization. Higher FLOT2 expression in nine HER2 amplified cell lines correlated with a higher T-DM1 IC50 in vitro, and breast cancer patients with high FLOT2 expression had worse survival when receiving either T-DXd (16.2 months (m) vs 18.3 m, p=0.04) or T-DM1 (38.0 m vs 41.3 m, p=0.1) in real-world Caris Life Sciences data. FLOT2 interacts with HER2 and positively regulates HER2 activation and downstream signaling, while FLOT2 knockdown reduces the viability of HER2 amplified cancer cells. FLOT2 knockdown results in increased HER2 internalization upon binding of T-DM1, mediated by ubiquitination by the Cbl ubiquitin ligases. We investigated the effects of various small molecules and discovered that zoledronic acid binds to FLOT2 and disrupts the HER2/FLOT2 interaction, which enhances T-DM1 internalization and cytotoxicity. In conclusion, FLOT2 regulates the internalization and cytotoxicity of T-DM1 mediated by Cbl-dependent ubiquitination of HER2. Zoledronic acid disrupts the HER2/FLOT2 interaction, therefore increasing the internalization and cytotoxicity of T-DM1, providing proof of principle that a small molecule inhibitor of the HER2/FLOT2 interaction can enhance the activity of the HER2-targeting ADC.

BackgroundWomen experience drug-induced Torsades de Pointes (TdP) at approximately twice the rate of men across more than 50 QT-prolonging drug classes, yet the quantitative ionic basis of this sex disparity remains incompletely characterised. The slow delayed rectifier current (IKs) is reduced by [~]45% in female compared with male human ventricular cardiomyocytes, reducing the repolarization reserve available to compensate pharmacological IKr block. MethodsWe implemented the OHara-Rudy (ORd) 2011 undiseased human ventricular epicardial action potential model in Python and parameterised sex variants using the most robustly established human ionic difference: GKs reduced by 45% in females [Kurokawa et al., 2016]. We simulated graded IKr blockade (0-95% in steps of 5%) at three physiologically relevant pacing rates (2 Hz, 1 Hz, 0.5 Hz) after 60 beats of warm-up to approach electrophysiological steady state. Action potential duration at 90% repolarization (APD90), triangulation (APD90-APD30), and repolarization failure (defined as APD90 > 500 ms, a conservative cellular risk marker informed by clinical QTc safety thresholds, or failure to repolarize within the cycle length) were quantified. All simulations used SciPys Radau solver (rtol = 10-, atol = 10-8) with a Numba-JIT-compiled right-hand side for computational efficiency. ResultsAt baseline (0% block), the female model exhibited longer APD90 than the male at all pacing rates (+2.8 ms at 2 Hz; +4.6 ms at 1 Hz; +4.6 ms at 0.5 Hz). Under progressive IKr blockade, the absolute sex difference in APD90 amplified non-linearly: at 85% block and 1 Hz pacing the female APD90 exceeded the male by 60.4 ms (versus 4.6 ms at baseline; 13-fold amplification). At slow pacing (0.5 Hz), the sex gap was most pronounced: at 85% block, female APD90 was 1127 ms versus 939 ms for the male (+188 ms; 20% more prolonged). The critical APD threshold (>500 ms) was reached by female cells at 5 percentage points lower IKr block than male cells at 1 Hz pacing (55% vs. 60% block), both reported at the first simulated 5%-grid block level exceeding the criterion. Repolarization failure occurred 5 percentage points earlier in females at 1 Hz (90% vs. 95% block). Action potential triangulation was consistently greater in the female model at all block levels and pacing rates. ConclusionA 45% reduction in IKs conductance is sufficient in this model to produce measurably greater APD90 prolongation under IKr blockade across all tested pacing rates. The non-linear amplification of the sex gap is consistent with the hypothesis that reduced IKs repolarization reserve contributes to greater female susceptibility to drug-induced QT prolongation, and supports testing sex-specific parameterizations in CiPA-style in silico cardiac safety workflows.

Prader-Willi syndrome (PWS) is a rare multisystemic disorder characterized by obesity, endocrine dysfunctions, and psychiatric comorbidities, which imply frequent use of psychotropic medications. They account for atypical responses to standard dosages of psychiatric drugs. Pharmacogenetics could be part of the reason for this situation, potentially offering a valuable tool for individualized treatment. This study analyzed allelic and phenotypic frequency distributions of five of the main cytochrome P450 enzymes (CYP2D6, CYP2B6, CYP2C19, CYP2C9, CYP3A4) involved in psychiatric drug metabolism in 47 patients with genetically confirmed diagnosis of PWS and compared them to reference frequencies in the general European population. Allelic frequency comparisons between the European reference population and the overall PWS cohort revealed a significant global difference for CYP2B6, with CYP2C19 and CYP2D6 showing trends toward significance. Although no global allelic differences remained significant after false discovery rate correction, post-hoc analyses consistently identified an enrichment of reduced- or non-functional alleles CYP2B619 and CYP2D610 in patients with PWS. Predicted metabolizer phenotype analyses showed a significant shift toward intermediate metabolizers of CYP3A4 in the PWS cohort, with corresponding depletion of normal metabolizers. Subgroup analyses indicated that allelic differences were more pronounced in maternal uniparental disomy and non-deletion subtypes, particularly for CYP2B6, although no significant differences were observed between PWS genetic subtypes. Overall, results imply potential differences in metabolizing activity in PWS patients, and subsequent implications in drug efficacy and tolerability. These results support the idea that pharmacogenetic testing may improve therapeutic decision-making in PWS for psychiatric treatment. Larger studies are needed to confirm these preliminary results.

BackgroundNeuropathic pain is a major source of disability and distress with few pharmacological options for treatment. Opioid drugs can be effective, but high doses are needed, leading to unwanted effects. BMS-986122 is a positive allosteric modulator of the mu opioid receptor that potentiates acute opioid antinociception without increasing opioid-induced constipation, reward, or respiratory depression. Therefore, we asked if BMS-986122 could increase the effects of low-dose opioid analgesics in chronic neuropathic pain. MethodsWe employed the spared nerve injury and tibial neuroma models in rats and assessed the tactile hypersensitivity of the hind paw and site of neuroma, respectively. ResultsAdministration of low doses of (R)-methadone, morphine, or buprenorphine slightly reduced the tactile hypersensitivity of the hind paw the in spared nerve injury model. Pretreatment with BMS-986122 significantly enhanced the reversal of hypersensitivity, reaching the effect of high-dose gabapentin, a standard of care in neuropathic pain. Pretreatment with BMS-986122 similarly increased the anti-allodynic effects of low dose (R)-methadone on neuroma pain. A similar effect of (R)-methadone in the absence of BMS-986122 was only observed at a dose where respiratory distress was seen. ConclusionsThese findings show that allosteric modulators of the mu opioid receptor such as BMS-986122 can enhance opioid activity that could translate to a safe and effective treatment for chronic neuropathic pain.

The epidermal growth factor receptor (EGFR) family network comprises 4 receptors (EGFR, ERBB2, ERBB3, ERBB4) and numerous ligands, and is dysregulated in many cancers. Since anti-cancer drugs that target these receptors are cardiotoxic for some patients, it is important to understand the network in cardiac cells. Data from the Human Protein Atlas established that EGFR family members and their ligands are differentially expressed in cardiac cell types. Ligand expression was altered in human failing hearts and may contribute to disease. These ligands stimulated extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt in rat cardiomyocytes but to different degrees. Afatinib (at a concentration to inhibit all EGF family receptors) was used to assess the role of the network in a mouse model of cardiac hypertrophy induced by angiotensin II (AngII). Echocardiography and segmental strain analysis demonstrated that afatinib reduced AngII-induced cardiac hypertrophy and caused cardiac dysfunction. This was associated with loss of cardiomyocyte hypertrophy, enhanced cardiac fibrosis, and reduced expression of Nrg1. NRG1 binds to ERBB4 in cardiomyocytes which homodimerizes or heterodimerises with ERBB2. The role of ERBB2 in the cardiomyocyte response to NRG1 compared with EGF was dissected using tucatinib (a selective ERBB2 inhibitor) and mRNA expression profiling. Most, but not necessarily all, of the response to NRG1 required ERBB2 signalling; most, but not all, of the response to EGF did not. Thus, the EGFR family network plays an important role in the heart. Understanding this network may identify therapeutic approaches to avoid cardiotoxicity associated with EGFR family anti-cancer drugs. Clinical perspectivesO_LIAnti-cancer drugs that target the epidermal growth factor receptor (EGFR) family are cardiotoxic for some patients; it is therefore important to understand the network in cardiac cells. C_LIO_LIThe EGFR family and their ligands are differentially expressed in cardiac cells with changes in ligand expression in heart failure; inhibition of all receptors in a mouse model of hypertrophy reduces cardiac hypertrophy and causes cardiac dysfunction with attenuation of cardiomyocyte hypertrophy and enhanced cardiac fibrosis and loss of neuregulin 1 (NRG1); in rat cardiomyocytes, NRG1 signalling to gene expression is largely mediated via ERBB2. C_LIO_LIThe EGFR family network plays an important role in the heart; understanding this network may identify therapeutic approaches to avoid cardiotoxicity associated with anti-cancer drugs targeted against it. C_LI

BackgroundIn RAS-mutant tumors, ERK phosphorylates the mitochondrial fission GTPase DRP1 to promote mitochondrial fission. DRP1 activity is tumor-promoting in pancreatic and other RAS-driven cancers, but its role in therapeutic resistance is unknown. MethodsWe developed a panel of patient-derived pancreatic cancer cell lines resistant to the MEK inhibitor trametinib. We used immunofluorescence imaging, in vitro growth assays and orthotopic xenografts to determine the role of DRP1 in trametinib resistance. ResultsWe find that trametinib-resistant cells exhibit increased expression and phosphorylation of DRP1 compared to sensitive counterparts. Quantitative analysis of mitochondrial structure reveals that mitochondria in resistant cells are morphologically distinct and relatively smaller than sensitive cells treated with trametinib. Genetic and pharmacological inhibition of both c-Myc and CDK6 are sufficient to block DRP1 phosphorylation in resistant cells, suggesting that activation of a c-Myc-CDK6 signaling axis drives reactivation of mitochondrial fission in the absence of MAPK signaling. Importantly, deletion of DRP1 leads to either growth inhibition or re-sensitization to trametinib in resistant lines. ConclusionThese findings suggest DRP1 contributes to drug resistance, and that inhibition of mitochondrial fission might be a promising therapeutic strategy to combat resistance to MAPK and RAS inhibitors.

Syntaxin-binding protein 1 (STXBP1) mutations lead to severe epilepsy, intellectual disability, developmental delay, and movement disorder. Effective treatments for these conditions do not exist. Recent studies in Munc18-1 (STXBP1) C. elegans models demonstrate that 4-phenylbutyrate (4-PBA) or related pharmacological chaperones stabilize Munc18-1 protein levels and rescue locomotion deficits. These studies suggest a novel treatment strategy for these patients. Here, we used a stxbp1a zebrafish model with a profound movement disorder to screen 4-PBA and alternative structural analogs identified using artificial intelligence (AI)-based screening. Automated locomotion assays conducted on larval stxbp1a mutant zebrafish at 5 days post-fertilization (dpf) confirm and extend the movement disorder endophenotype. Drug treatment (4-PBA or 16 identified candidates) failed to rescue the stxbp1a mutant zebrafish locomotion deficit. Electrophysiology studies in a stxbp1b zebrafish model characterized by spontaneous seizure activity (i.e., epilepsy) failed to detect a reduction in ictal-like events with 4-PBA treatment. Taken together, our results suggest caution in repurposing 4-PBA or related compounds for treatment of STXBP1 disorders.

Background and aimsTherapeutic outcomes for advanced hepatocellular carcinoma remain inadequate, despite recent advances using immunotherapy. Long-term effectiveness of systemic therapies, including second-line multi-tyrosine kinase inhibitor sorafenib, is limited by resistance mechanisms and adverse effects. Upregulated deubiquitinase UCH-L1 is frequently correlated with poor prognosis in cancers. Here, we investigated the therapeutic potential of combining pharmacological UCH-L1-inhibition with sorafenib in HCC. MethodsUCH-L1 expression was analysed in TCGA-LIHC data and patient-derived HCC tissues. Sorafenib and LDN57444 effects were evaluated in vitro in cytotoxicity and invasion assays. Gene and protein expression were examined by RT-qPCR, Western blotting and immunohistochemistry. In vivo efficacy of drug synergy was assessed in an orthotopic xenograft mouse HCC model. ResultsIn silico data-analysis revealed significantly higher UCH-L1 levels in patient HCC tumours versus non-tumour, associated with reduced overall survival. Low-dose sorafenib upregulated UCH-L1 in HCC cell line Hep3B. Paradoxically, this also promoted invasiveness and sustained MEK1/2-ERK1/2-pathway activation. Combining low-dose sorafenib with LDN57444 produced strong synergistic cytotoxicity in vitro, reverted MAPK-activation and suppressed invasion. Consistently, at low sorafenib dose co-treatment with LDN57444 completely inhibited tumour growth of Hep3B xenografts and enhanced sorafenib efficacy. ConclusionLDN57444 sensitises HCC cells to low-dose sorafenib by reverting drug-induced pro-oncogenic signalling and thereby strongly synergises with sorafenib to enhance anti-tumour efficacy in a HCC mouse model. This presents UCH-L1 as a player in treatment-induced adaptive response and supports further exploring UCH-L1-targeting in combination with sorafenib as therapeutic avenue for advanced HCC. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/725527v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@176dc91org.highwire.dtl.DTLVardef@8acae8org.highwire.dtl.DTLVardef@f71bborg.highwire.dtl.DTLVardef@1f3c5aa_HPS_FORMAT_FIGEXP M_FIG C_FIG Lay summaryThis study explores a new treatment approach for hepatocellular carcinoma (HCC) by combining two drugs: LDN57444, which blocks the enzyme UCH-L1, and sorafenib, a FDA-approved multi-tyrosine kinase inhibitor. We evaluated the effect of this drug combination in vitro using a HCC cell line and in an mouse HCC-model. The drug combination displayed strong, synergy in lowering HCC cell viability, and greatly reduced invasiveness and in vivo tumour growth. LDN57444 sensitised HCC cells to low doses of sorafenib by preventing UCH-L1-mediated activation of pro-oncogenic signalling. These findings highlight the potential of this new drug combination for treating advanced HCC thereby potentially reducing side-effects and countering drug resistance. Impact and implicationsOur preclinical research introduces a novel combination strategy against advanced HCC that holds potential to improve existing therapies, particularly the second-line multi-tyrosine kinase inhibitor sorafenib. The proposed combination of sorafenib with an inhibitor of the deubiquitinase UCH-L1 not only enhances sorafenib efficacy but present promise to also counter resistance mechanisms. Moreover, because effective responses are achieved at lower drug doses, this may in addition reduce therapy-associated adverse effects further increasing potential impact. While sorafenib is FDA-approved, the UCH-L1 inhibitor LDN57444 needs further (clinical) development to bring our promising findings to full translational potential for HCC patients and physicians.

The p21-activated kinases (PAKs) are a group of serine-threonine kinases central to multiple signaling pathways that govern cell survival and proliferation. Aberrant activity of PAK1, the most well characterized member of the PAK family, drives progression of several malignancies and brain disorders, including Alzheimers disease and neurodevelopmental disorders. Despite growing interest in PAK1 as a drug target for these diseases, there is no assay to evaluate the intracellular target engagement of PAK1 inhibitors. To address this need, we developed first-in-class NanoBRET assays for wild-type PAK1 and a neurodevelopmental disorder-causing gain-of-function PAK1 mutant. Furthermore, we executed our novel PAK1 NanoBRET assay to evaluate target engagement of PAK1 inhibitors in primary hippocampal neurons. To the best of our knowledge, this is the first demonstration of a NanoBRET cellular target engagement assay in primary neurons, thereby increasing the relevance of our work by confirming PAK1 inhibitor binding to the aberrant form of the protein in primary neurons.

UT-018, a stem cell chemoattractant formulation, demonstrated significant regenerative activity across independent murine wound-healing and hair-regeneration studies. Topical treatment accelerated wound closure, enhanced granulation tissue formation, improved collagen organization, increased fibroblast proliferation, and enhanced dermal vascularization. Separate hair-growth studies demonstrated increased follicular density, deeper follicular penetration, enhanced dermal vascularization, and induction of anagen-phase transition by UT-018. Mechanistic studies demonstrated strong intracellular cAMP generation and activation-associated {beta}-catenin phosphorylation consistent with GPCR-mediated regenerative signaling.