ACS Pharmacology & Translational Science

Gabapentin (GBP) and pregabalin (PGB) are widely used gabapentinoids. Previously, we have demonstrated, for the first time, that GBP and PGB modulate the nociceptive response to noxious heat in C. elegans at an optimal concentration. In the current study, we use C. elegans and paired thermal nociception assays with direct internal drug concentration measurements to characterize the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of both compounds. Neither drug altered baseline mobility or quadrant preference, confirming that behavioral effects reflected genuine antinociceptive action. Both GBP and PGB produced dose- and time-dependent reductions in thermal avoidance, with 500 uM exposures generating a biphasic, V-shaped time course in which suppression of thermal sensitivity deepened before partially reversing. This partial reversal occurred later with PGB than with GBP. Internal concentrations confirmed dose-dependent absorption and retention for both drugs, yet at 500 uM, internal drug levels remained elevated through 360 min even as behavioral avoidance recovered, indicating that the recovery limb reflects active counter-regulation rather than passive clearance, consistent with previously reported transcriptional and proteomic signatures. Exposure-response profiles were notably flat, suggesting a saturable pharmacodynamic ceiling. Molecular modeling revealed conserved electronic pharmacophores supporting shared alpha-2-delta engagement, alongside shape-descriptor differences that may contribute to divergent absorption kinetics. These findings position C. elegans as a valuable model for dissecting gabapentinoid PK/PD relationships. Beyond mechanistic insight, these findings support the continued investigation of C. elegans as a screening platform whose validation could help address the 3R (Replacement, Reduction, Refinement) principles guiding animal research.

We published recently that one of the main constituents of cannabis products, cannabidiol (CBD), is an efficacious negative allosteric modulator (NAM) of the mu opioid receptor (MOR1) (Bosquez-Berger et al., 2023). Here, we investigated how the presence of cannabidiol (CBD) is associated with fentanyl (FEN) binding across MOR1 conformations. We performed molecular dynamics simulations of systems containing FEN alone or FEN+CBD in three mouse MOR1 conformational backgrounds: active-like 5C1M, inactive-like 4DKL, and a modeled Morph50 intermediate between the 5C1M and 4DKL conformations. Three independently seeded 200 ns trajectories were analyzed per model and condition (18 trajectories total), with the trajectory treated as the independent unit. Across the matched 0-200 ns window, consensus CBD contacts and CBD-associated changes in FEN contacts were strongly state dependent. Corrected intracellular TM3 to TM6 analyses separated the expected active-like, intermediate, and inactive-like backgrounds but did not identify a CBD-associated shift that was consistent across both geometric definitions and all three replicates. Equal-weight replicate-composite density maps preserved both the shared ligand distributions and this between-trajectory variability. These descriptive results support receptor-state-dependent CBD, FEN, MOR1 interactions while emphasizing the limited inferential power of three trajectories per condition.

Background: Subtype-selectivity predictions are scored against measured selectivity and judged against an assumed noise ceiling. We asked what an 2-adrenergic benchmark rewards and which controls change its interpretation. Research design and methods: On a frozen benchmark of 586 paired 2A/2C compounds we evaluated Glide SP docking, CNN rescoring, ligand-only fingerprint models, receptor descriptors and pose contacts, with dopamine D3/D2 as comparator, applying five controls: a measured ceiling, a cluster-identity null, a nonselective reference, a same-receptor floor and a trivial-descriptor baseline. Results: Five descriptors from SMILES reached Spearman 0.645, 72% of the measured ceiling, against 0.071 for Glide SP and 0.188 for CNN rescoring; receptor properties and pose contacts reduced to size under control, while a non-size signal of 0.258 survived. Measured rather than propagated noise raised that ceiling from 0.704 to 0.897; cluster identity alone reached R2 0.499 on D3/D2 and none on 2; a nonselective reference received +1.43 to +4.79 kcal/mol where zero is expected; and a same-receptor floor reached 1.77-fold against 1.88-fold across subtypes. Conclusions: Such benchmarks reward molecular size first; a method must exceed 0.645 before its score indicates structural reasoning. The controls are inexpensive; conclusions rest on two receptor pairs, a three-pair floor and static structures.

BackgroundCannabis derivatives have been reported to possess antinociceptive properties. However, oral delivery is limited by poor bioavailability, stability, and reliability of effects. Previously, we reported an analgesic effect of the aqueous complex {Delta}9-tetrahydrocannabinol/2-hydroxypropyl-{beta}-cyclodextrin (THC/HP{beta}CD) after intracerebroventricular administration in male rats. MethodsHere, we investigated the analgesic effects of the THC/HP{beta}CD complex after oral administration (0.3 and 3 mg/kg) by the tail flick test after both acute and chronic administration (15 days) in female rats. Locomotor activity and anxiety-like behavior were also evaluated at the same experimental conditions. Moreover, dopamine and glutamate content in the periaqueductal gray (PAG), a key area for the antinociceptive action of THC, were also measured by HPLC. ResultsAfter acute administration, the antinociceptive effect of the complex was seen at 3 but not 0.3 mg/kg THC, with a maximum effect observed at 30 min (MPE 60%). Similar results were obtained after 15 days of treatment, although partially reduced (max MPE 20%). Reductions in locomotor activity with the dose of 3 mg/kg and a slight biphasic effect of the two doses on anxiety-like behavior were also observed. Finally, neurochemical analyses revealed that the dose of 3 mg/kg significantly increased dopamine and glutamate content in the PAG, an effect no longer present after 15 days of treatment. ConclusionsOur results highlight the antinociceptive efficacy of the THC/HP{beta}CD complex also after oral administration, notably higher than that previously seen with other carriers, although with some degree of tolerance after chronic administration. From a translational point of view, these results are relevant for the development of THC-based oral formulations with analgesic properties for the treatment of pain in humans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/742765v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@fff791org.highwire.dtl.DTLVardef@d672f4org.highwire.dtl.DTLVardef@1150b3forg.highwire.dtl.DTLVardef@956403_HPS_FORMAT_FIGEXP M_FIG C_FIG

Edible plant derived extracellular vesicles (PDEVs) are emerging as biocompatible, orally deliverable nanocarriers with therapeutic potential; however, their phytometabolite cargo, gastrointestinal stability, and source specific biological functions remain poorly characterized. Here, PDEVs were isolated from four phytochemically distinct plant based foods (black carrot, ginger, garlic, and turmeric), selected for their diverse bioactivity, and characterized by transmission electron microscopy, nanoparticle tracking analysis, and zeta potential. Gastrointestinal stability was evaluated in simulated digestion model. Source specific phytometabolites were profiled by untargeted LC MS MS metabolomics. Functionally validated in ammonia stressed epithelial cells and steatotic hepatocytes. PDEVs exhibited characteristic cup shaped morphology with particle sizes ranging from 60 to 214 nm and zeta potentials of -6.0 to -49.0 mV. PDEVs retained colloidal stability, supporting their suitability for oral delivery. We identified 572 phytometabolites with distinct source specific signatures, including lignin and quercetin in carrot EVs, [6] gingerol and silymarin in ginger EVs, diosgenin in garlic EVs, and curcumin in turmeric EVs. These metabolites found associated to antioxidant, anti inflammatory, epithelial barrier, lipid metabolic, and apoptotic pathways. Functional validation demonstrated carrot EVs significantly enhanced epithelial barrier integrity by increasing claudin (>8-fold, p<0.05), occludin (>2-fold, p<0.05). Ginger EVs restored ZO 1 while suppressing cyclin D1 and MMP9(p<0.05). Garlic and turmeric EVs attenuated inflammatory signaling by reducing STAT3, AKT1, and TNF , whereas turmeric EVs additionally decreased caspase 3 and PTGS2(p<0.01). In steatotic hepatocytes, garlic EVs significantly reduced PNPLA3 (p<0.001) and SREBP 1c while increasing PPAR- (p=0.002). Hence, our results indicate that edible PDEVs are gastrointestinally stable, phytometabolite enriched nanocarriers with distinct source specific functional properties, supporting their potential as orally deliverable nutraceuticals for improving gut liver functions.

Background: In the United States, several states have restricted sales of flavoured tobacco products, including popular menthol- and mint-flavoured Oral Nicotine Pouches (ONP). In response, tobacco companies introduced "unflavoured" ONP containing odorless synthetic cooling agents. Since these, in turn, have become targets of legislative bans, the tobacco industry may seek out flavourants with other sensory effects to increase the appeal of "unflavoured" ONP. Methods: Online merchants were searched for "unflavored" ONP marketed to consumers in jurisdictions with flavour bans. Sensory effects of aqueous extracts from identified "heat", "spicy" and "unflavoured" ONP were analyzed by Ca2+ microfluorimetry in HEK293 cells expressing the human heat/chili pepper flavourant (capsaicinoid) receptor, hTRPV1. ONP were analyzed for capsaicinoids and sweeteners by Liquid Chromatography/Mass Spectrometry (LC/MS). Results: A new category of "heat" or "spicy" ONP was identified, including products marketed as "unflavoured". Extracts from all these ONP robustly activated TRPV1, with "unflavoured" Lucy Heat the most potent. Chemical analysis demonstrated that Lucy Heat contained the synthetic capsaicinoid nonivamide at high levels (~675 microgram/pouch), while others contained mixtures of capsaicinoids (5-25 microgram/pouch) combined with other characterizing flavours (tropical, fruit). All tested ONP contained sweeteners. Conclusions: The tobacco industry continues to probe regulatory loopholes by claiming that newly introduced capsaicinoid flavourants and sweeteners in ONP do not represent characterizing flavours. This is contradicted by industry and regulatory determinations assigning characterizing flavour properties to these additives. The toxicological health risks of repeated capsaicinoid exposures due to ONP use, in combination with nicotine and other constituents, need to be assessed.

Several potentially potent anticancer drugs have been identified by in vitro evaluation, such as Andrographolide. These compounds show strong anticancer activity in vitro, but struggle to reach effective concentrations in the bloodstream when taken orally because they dissolve poorly in water or break down rapidly in the body. Bioenhancers, which are compounds that have potential to improve drug stability in the body, offer an alternative solution to overcome this limitation. Naringin and Quercetin have been identified as candidate bioenhancers, and have been hypothesized to potentially slow rapid first pass metabolism of poorly bioavailable drugs. Our work focuses on testing Naringin and Quercetin because they are flavonoids with therapeutic potential, due to their anti-inflammatory and antioxidant properties. Data from the hepatic microsomal assays performed on Naringin and Quercetin suggest moderate to proficient periods of stability in the body, with Naringin having 91.86% remaining, while Quercetin had 74.84% remaining. When administered alongside Andrographolide, a drug known to rapidly degrade in the body, Naringin raised its metabolic stability from 38.93% to 80.77% and on the other hand, Quercetin raised Andrographolide metabolic stability from 38.93% to 86.70%. In addition, plasma protein binding assays show the percentage of compounds available at the target site where Naringin was observed to be 49.32% bound and Quercetin found to be 50.14% bound, implying 50.68% of Naringin, and 49.86% of Quercetin available at the target site, respectively. This preliminary study explores whether Quercetin and Naringin could act as bioenhancers by remaining stable and available in plasma and by slowing the metabolism of poorly bioavailable drugs such as Andrographolide.

Background: The nicotine analog 6-methyl nicotine (6-MN) has appeared in commercial e-cigarette liquids, and other products, spurring interest in determining the extent to which it conveys similar effects to those of nicotine. Objective: To determine if 6-MN acts like nicotine to decrease body temperature, decrease nociception, suppress wheel activity and reinforce operant behavior when delivered by vapor inhalation using an Electronic Nicotine Delivery System (ENDS; "e-cigarette") approach in a rat model. Methods: Male and female (N=8 per sex) young adult Sprague-Dawley rats were evaluated for rectal temperature and nociceptive responses (warm water tail-withdrawal) to the inhalation of vapor from (-)-6-MN or (-)-nicotine in concentrations ranging from 5-30 mg/mL in the propylene glycol vehicle. Rats were then assessed for the reinforcing effects of nicotine and 6-MN using a vapor self-administration procedure and the rate suppressing effects of nicotine and 6-MN on wheel activity following injection. Results: Inhalation of nicotine or 6-MN for 30 minutes decreased the rectal temperature and increased tail-withdrawal latency of female and male rats in a concentration-dependent manner. The magnitude of the effects of 6-MN and nicotine were similar at similar vapor concentrations. Operant responding for 6-MN vapor was increased by pre-treatment with the antagonist mecamylamine. 6-MN was more potent than nicotine at suppressing wheel activity after injection. Conclusions: 6-MN induces effects very similar to those of nicotine, at a similar potency when inhaled and at a slightly increased potency when injected.

Introduction: Osteoarthritis (OA) is a degenerative joint condition characterized by chronic pain and the need for pain management. Locally targeting the endocytotic AP2 complex in nociceptors presents a potential strategy for providing sustained pain relief in individuals with OA. Objective: We investigated whether pain behavior associated with OA can be mitigated by genetically silencing the AP2alpha2 subunit of the AP2 complex in nociceptors and by pharmacologically inhibiting the AP2 complex through the intraarticular administration of a small lipidated decoy peptide. Method: Monoiodoacetate (MIA) was employed to induce knee joint OA in mice and rats. Pain behavior was assessed using dynamic weight-bearing and von Frey filaments. Upon confirmation of established OA pain behavior, in vivo AP2alpha2 genetic knockdown in mice was achieved through sciatic nerve transfection of a targeting AP2alpha2 short hairpin RNA (shRNA). To pharmacologically target endocytosis, a single intraarticular injection of peptide was administered into the arthritic knee of rats. The injection contained either the AP2 inhibitor peptide or a scrambled peptide control. Results: Pain behavior was significantly reduced after both genetic and pharmacological disruption of AP2-driven endocytosis. Animals treated with the Ap2 inhibitor peptide exhibited reduced pain behavior throughout the 28-day assay period. Following the completion of behavioral testing, arthritic knee joints and contralateral healthy knee joints were subsequently collected to assess the impact of the treatment on disease progression. Micro-computed tomography analysis revealed a preservation of bone volume in the arthritic joints that received the AP2 inhibitor peptide treatment, in contrast to the scrambled peptide group. Conclusion: These findings demonstrate that the inhibition of nociceptor endocytosis by a small lipidated peptide presents a promising approach to provide sustained relief from joint pain in individuals with arthritis.

G protein-coupled receptor (GPCR) allosteric modulators (AMs) offer significant therapeutic advantages over orthosteric drugs, yet structure-based virtual screening lacks validated protocols accounting for the conformational complexity of GPCR allosteric sites. We benchmark docking protocols using PDB experimental structures and structural ensembles derived from Gaussian accelerated Molecular Dynamics (GaMD) simulations across four Class A GPCRs (including the muscarinic M2 and M4 receptors, the {beta}2-adrenergic receptor, and the C-C chemokine receptor type 2) with four programs (Glide HTVS, AutoDock Vina, DOCK3.8, and Boltz-2) against experimentally validated modulator libraries and property-matched decoys. GaMD ensemble docking improved early AM enrichment across all four targets under at least one program. Glide ensemble docking was the only protocol to consistently improve early AM recovery across all four targets, ranking known actives almost exclusively within the top 0.5% of compounds at CCR2 and improving M2R active recovery nearly 9-fold relative to the PDB structure. GaMD free-energy landscape topology governed ensemble re-ranking strategy selection: population-skewed landscapes favored top binding energy ranking (BEmin) while flat, multi-populated landscapes favored average binding energy ranking (BEavg), and at targets with dominant low-energy states, a single GaMD cluster matched or exceeded full ensemble or PDB performance. Taking the union of top percentile hits identified by both ensemble re-ranking methods, BEmin / BEavg, maximizes chemical diversity at the earliest percentiles. Program-specific scaffold recovery biases further motivated a consensus BEmin / BEavg approach to maximize hit diversity. The Boltz-2 deep-learning program showed minimal sensitivity to GaMD templates and underperformed conventional docking, suggesting its affinity predictions complement rather than replace physics- and empirical-based docking approaches for GPCR AM screening.

Histone deacetylases (HDACs) are pivotal epigenetic regulators that modulate diverse cellular pathways by removing acetyl groups from lysine residues on both histone and non-histone proteins. Histone deacetylase 11 (HDAC11), the sole member of class IV HDACs, exhibits both deacetylation and fatty acid deacylation activities. Accumulating evidence implicates HDAC11 as a key epigenetic regulator of fundamental cellular processes, including metabolism, immune responses, and tissue development. Dysregulation of HDAC11 activity has been associated with inflammatory diseases, metabolic disorders, neurodegenerative conditions, and cancer, highlighting its potential as a therapeutic target. Although several HDAC11-specific inhibitors have been identified, none have progressed to clinical development. In this study, we aimed to discover HDAC11-selective inhibitors by integrating in silico and in vitro validation approaches. Homology modelling of the HDAC11 structure was conducted, followed by model validation, structure-based virtual screening, molecular dynamics (MD) simulations, and binding free energy calculations. We identified and validated three lead compounds and their intermediates using biochemical and cell-based assays. Fluorescence-based and HPLC-based enzymatic assays demonstrated potent inhibition of both the deacetylase and deacylase activities of HDAC11, with Inhibitor 6 and Inhibitor 3 exhibiting the strongest effects among the six compounds tested. Further, a decrease in lipid accumulation, reduced stability of the HDAC11 substrate SHMT2, as determined by immunoblot analysis and decreased cell viability, as assessed by MTT assay, confirmed HDAC11 inhibition in cellular models. The study shows that new HDAC11 inhibitors significantly reduce the viability of breast cancer cells and induce apoptosis; inhibitor 6, in particular, showed high potency, similar to the reference compound SIS-17. Flow cytometry showed that treated MDA-MB-231 cells exhibited cell-cycle arrest and increased apoptosis, a finding further confirmed by Annexin V/PI staining. Molecular analysis showed that BAX increased while BCL2 decreased, indicating that apoptotic pathways were activated in novel compound-treated MDA-MB-231 cells. The results suggest that inhibiting HDAC11 is an effective way to induce cancer cell death and provide a basis for further assessment of these compounds as potential treatments for breast cancer. Collectively, this study identifies novel zinc-chelating HDAC11 inhibitors containing a nitro-sp2 group, providing promising candidates for further therapeutic development.

Breast cancer (BC) is the second most common noncutaneous cancer and the second leading cause of cancer-related death in women. BC is classified into three primary subtypes, with triple-negative breast cancer (TNBC) having the poorest prognosis because it lacks specific targetable markers. Preclinical studies on TNBC indicated a common occurrence of diminished tumor-suppressor activity of PTEN, activating the PI3K/AKT/mTOR signaling pathway. Notably, published studies reveal that the WWP1 enzyme plays a pivotal role in driving PTEN degradation via ubiquitination, unveiling a promising therapeutic target for treating TNBC. In the search of new WWP1 inhibitors, we used artificial intelligence (AI)-driven computational strategies for de novo design of peptide-based WWP1 inhibitors and identified a hexapeptide, termed WI23-B, which demonstrated high nanomolar binding affinity to WWP1. In TR-FRET enzymatic assays, WI23-B inhibited WWP1 activity with an IC of approximately 11 {micro}M. In MCF7 and MDA-MB-231 breast cancer cell lines, WI23-B showed promising cytotoxic efficacy, particularly in combination with the PI3K inhibitor BYL719, also when it was loaded into nanocapsules. Collectively, these findings highlight WI23-B as a promising lead peptide with potent WWP1 inhibitory activity and synergistic antiproliferative effects when combined with PI3K inhibitors. While further structural optimization is required to enhance its potency and pharmacological properties, our results provide a strong foundation for the development of next-generation WWP1 inhibitors. Such agents have the potential to reshape therapeutic strategies for BC and TNBC by enabling more effective and less toxic treatment regimens, ultimately reducing the reliance on high-dose chemotherapy and minimizing adverse effects.

People are widely exposed to the antimicrobial cetylpyridinium chloride (CPC) via consumer products, but CPC is a mitochondrial toxicant with potency comparable to that of canonical mitotoxicants. CPC is largely unregulated despite growing usage, bioavailability, and ability to cross the blood-brain barrier. Previously, we showed, in several cell types at non-cytotoxic and exposure-relevant doses, CPC inhibits ATP and OCR, endpoints of the electron transport chain (ETC). Mitochondrial toxicity is linked to multiple diseases (e.g., diabetes, Parkinsons, myalgic encephalomyelitis), but CPC has not been studied epidemiologically, and little mechanistic information is available. To determine why OCR and ATP are hampered by CPC, we hypothesized that CPC inhibits individual ETC components, cardiolipin, or TCA enzymes. Here, we show that, in primary human skin cells, an immune mast cell model, and isolated mitochondria, CPC apparently inhibits multiple ETC Complexes. Detailed investigation pinpointed the mechanism to the distal end of ETC: Complex III-cytochrome C-Complex IV. Using multiple approaches, we show that CPC does not directly inhibit any of the Complexes (not even Complex I as earlier reported), nor TCA enzymes, nor coenzyme Q. Yet, we found that CPC exhibits mitotoxicity as potent as cyanide. Anionic lipid cardiolipin attracts cytochrome C to the inner mitochondrial membrane so that it may shuttle electrons from Complex III to IV. Despite not altering levels of cardiolipin, CPC hinders cytochrome C by electrostatically interfering with cardiolipin. To aid epidemiology, risk analysis, and predictive toxicology, we have determined the precise biochemical mechanism of action of this ubiquitous compound.

Alzheimer's disease is characterized by progressive cognitive decline and early cerebrovascular dysfunction, including impaired neurovascular coupling (NVC) and reduced cerebral blood flow (CBF). Tau pathology is a major driver of these deficits, yet therapeutic strategies targeting tau-induced neurovascular dysfunction remain limited. The M1 muscarinic acetylcholine receptor (M1 mAChR) is a promising therapeutic target because of its critical role in cognition. We previously demonstrated that pharmacological activation of M1 mAChR improves cognitive function and neuronal survival in amyloid-based Alzheimer's disease mouse models through sex-specific mechanisms. However, whether M1 mAChR activation restores tau-mediated NVC deficits remains unknown. P301S mice were used as a model of tauopathy. Cognitive function was evaluated using the novel object recognition and Morris water maze tests, and NVC was assessed by measuring whisker stimulation-induced changes in CBF using laser speckle contrast imaging. Following baseline measurements, mice received an acute intraperitoneal injection of VU0486846, a selective M1 mAChR positive allosteric modulator (3 mg/kg), and CBF responses were reassessed over time. P301S tau mice exhibited impaired recognition and spatial memory functions, associated with reduced whisker stimulation-induced increase in CBF, indicative of impaired NVC response, while acute treatment with VU0486846 reversed these changes in NVC. This rescuing effect of VU0486846 was observed earlier in female tau mice compared to males, suggesting a sex-biased effect of M1 mAChR modulation. These findings demonstrate that M1 mAChR positive allosteric modulation reverses tau-induced neurovascular dysfunction, supporting M1 mAChR activation as a promising disease-modifying approach for Alzheimer's disease. The earlier improvement observed in females further suggests that therapeutic efficacy is influenced by biological sex.

The cluster of differentiation 36 (CD36) is a membrane protein with broad physiological roles in health and disease, and its function is regulated in part by phosphorylation. Experimental evidence shows that phosphorylation of Thr92 reduces CD36 affinity for thrombospondin-1 (TSP-1), binding of which initiates antiangiogenic signaling, whereas phosphorylation of Ser237 decreases CD36-mediated fatty acid uptake, with implications for energy metabolism. However, the only available crystal structure of CD36 lacks phosphorylation, and the molecular mechanisms by which phosphorylation regulates CD36 function remain largely unknown. This study provides an atomically detailed computational characterization of CD36 in unphosphorylated and dual phosphorylated states, using molecular dynamics simulations with a total sampling time of 30 microseconds in combination with Markov state models. We present, to our knowledge, the first evidence of a cryptic pocket on CD36 surface that is formed by phosphorylation. This cryptic surface pocket and a loop spanning residues 121-131 form a high affinity binding site for TSP-1 derived ligands, shifting their binding away from the canonical site. We propose that this altered binding provides a molecular basis for the disruption of antiangiogenic signaling upon CD36 phosphorylation. Additionally, our data indicate that, phosphorylation increases helicity and compaction within the helix-loop region spanning residues 296-331, narrowing one of the entrances to the internal cavity and reducing its overall volume. These conformational changes provide a potential mechanistic explanation for the decrease in fatty acid uptake upon CD36 phosphorylation. Our findings provide structural insights that may inform the future design of CD36 modulators and emphasize the importance of targeting phosphorylation induced CD36 conformations in angiogenic and metabolic diseases.

Solid tumors are often surrounded by abnormal vasculature and a dense collagen-rich extracellular matrix that severely restrict the infiltration of T cells, including tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR)-T cells. These physical barriers represent a major obstacle to the efficacy of adoptive T cell therapies in solid tumors. We previously identified Formin-like 1 (FMNL1) as a cytoskeletal regulator critical for T cell extravasation and migration through restrictive environments, making it a promising target to improve T cell infiltration into tumors. Here, we developed a bioengineering platform to enhance T cell cytoskeletal dynamics by overexpressing FMNL1 in TILs and CAR-T cells. FMNL1 overexpression significantly increased T cell migration through restrictive pores in transwell assays, supporting enhanced migratory capacity of T cells under mechanically constraining conditions. Importantly, FMNL1 overexpression did not impair T cell reactivation or cytotoxic function in vitro. In murine models of melanoma and lung carcinoma characterized by limited effector T cell infiltration, FMNL1-overexpressing TILs and CAR-T cells had significantly increased accumulation at tumor sites compared to controls. Importantly, enhanced tumor accumulation resulted in improved therapeutic activity, as adoptive transfer of FMNL1-overexpressing CAR-T cells limited tumor growth and prolonged the survival of tumor-bearing mice in multiple melanoma models. Together, our findings identify FMNL1 as a broadly applicable cytoskeletal engineering target to enhance T cell accumulation and persistence in restrictive tumor microenvironments, thereby overcoming a fundamental limitation of adoptive cellular immunotherapy in solid tumors.

"Revealing the structure of pharmacobehavioral space through motion sequencing" by Wiltschko et al. (2020) has been highly influential in behavioral phenotyping research. In a cohort of nearly 700 mice, the authors demonstrated that Motion Sequencing (MoSeq) could distinguish behavioral effects across a large and diverse set of neuroactive and psychoactive compounds. A central conclusion of the study is that MoSeq syllable features substantially outperform more traditional scalar behavioral features in treatment classification tasks. Although this comparison is not emphasized outside the Results section, the reported advantage corresponds to an increase in classification performance exceeding 50% relative to scalar feature representations. While reproducing parts of the analysis using the publicly available dataset, we found that much of this apparent performance difference can be attributed to differences in preprocessing, classifier selection, and hyperparameter optimization. Under alternative, but comparably standard, analytical choices, the performance gap between scalar features and MoSeq syllables was reduced to approximately 11%. Furthermore, in our reanalysis, the performance advantage of MoSeq syllables became statistically significant primarily in highly dense pharmacobehavioral spaces. These findings do not contradict the utility of MoSeq syllables. Rather, they suggest that the magnitude and generality of their advantage over simpler scalar features may depend strongly on analytical methodology and dataset structure. This distinction is practically relevant, as scalar feature approaches are substantially less computationally demanding and often easier to interpret biologically. Consequently, for laboratories with limited computational resources or for studies focused on specific treatment effects, conventional scalar representations may provide a competitive and more accessible alternative. Our findings highlight the importance of analytical standardization and reproducibility in comparative behavioral representation studies.

N, N-dimethyltryptamine (DMT) and 5-methoxy-N, N-dimethyltryptamine (5-MeO-DMT) are structurally related tryptamine psychedelics with emerging therapeutic potential, yet their comparative acute pharmacology and longer-term neuroplastic effects remain incompletely defined. Here we show that DMT produces a bell-shaped dose-response curve in the mouse head-twitch response (HTR) assay, whereas 5-MeO-DMT elicits a monotonic increase. Selective antagonism at 5-HT2A or 5-HT1D receptors, or agonism at 5-HT1A, robustly attenuates HTR for both compounds without abolishing their ability to reduce marble burying, a screening assay for OCD-like behavior. Acutely, both agents elevate TrkB phosphorylation in a region-specific manner, with broader engagement by DMT across default-mode-network and hippocampal territories. Twelve days after a single dose, both compounds increase synaptic proteins (PSD-95, synaptophysin; SV2A for DMT), while DMT uniquely lowers hippocampal BDNF and reprograms frontal-cortex glutathione and energy metabolism. These findings demonstrate that acute hallucinogenic-like activity and selected therapeutic-like behavioral and plasticity outcomes can be pharmacologically dissociated, informing the rational design of more tolerable, scalable psychedelic-based treatments.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by progressive motor neuron degeneration. Mutations in the SOD1 gene represent the second most common genetic cause of ALS (ALS), and distinct SOD1 missense variants present with markedly different clinical profiles. A4V leads to an aggressive form of the disease (median survival [~]1y), H46R confers a mild, slowly progressive course and I113T exhibits an intermediate phenotype. The molecular basis by which these mutations produce divergent clinical outcomes remains poorly understood. We performed extensive classical molecular dynamics simulations of wild-type SOD1 and the three ALS-associated variants in the apo monomeric state to attempt to investigate the mechanisms behind such phenotypic differences. Structural stability, global compactness, and conformational flexibility, as well as analysis of collective motions between residues and estimation of free energy, were assessed. The H46R, A4V, and I113T variants exhibited distinct dynamic behaviours, highlighting differences in structural stability, local flexibility, and intramolecular interactions. These findings suggest that specific structural regions may contribute differently to protein dysfunction and could represent key elements for understanding the relationship between molecular dynamic properties and the differing clinical severity associated with these variants. Most strikingly, H46R exhibited exceptional structural stability across every analytical level, the lowest global deviation, most attenuated local flexibility, strongest internal dynamic coordination, and the deepest, most confined free energy basins of any system examined. This convergent multi-layered evidence of structural restraint provides a compelling mechanistic basis for the mild and slowly progressive clinical course of H46R ALS, suggesting that enhanced conformational rigidity, rather than bulk destabilisation, is the defining biophysical feature of this variant, and that its pathogenic mechanism operates through a route fundamentally decoupled from the aggregation-driven toxicity that characterises the more aggressive SOD1-ALS mutations.

The de novo design of proteins that bind chemically complex small molecules has broad chemical and biological implications, but strategies typically rely on a small set of protein scaffolds and require extensive experimental screening. Here, we computationally designed proteins around a minimal aromatic {pi}-stacking motif to bind the anthracycline anticancer drug doxorubicin. Experimental characterization of twelve proteins revealed a {micro}M doxorubicin binder; two additional design cycles improved scaffold stability and binding affinity to yield an 85-residue protein that binds doxorubicin with a dissociation constant of 85 nM. An X-ray crystal structure of the protein-drug complex confirmed the accuracy of the designed {pi}-{pi} stacking interactions. The designed protein could act to protect cultured cells from doxorubicin-induced cytotoxicity. Unlike previous ligand-binding protein designs based on repeat proteins or naturally occurring folds, the designed protein adopts a previously unobserved 5-helix globular fold, indicating that a broader space of folded, functional proteins exists even for compact tertiary structures smaller than 100 residues. These results demonstrate that motif-guided generative protein design can discover compact de novo protein folds capable of high-affinity recognition of chemically complex small molecules.