Pharmaceuticals

Immunosuppressive drugs, which are required to maintain graft function in transplant recipients, are associated with many unavoidable side effects including posttransplant diabetes mellitus (PTDM) that involves both peripheral insulin resistance and impairment of insulin secretion. To characterize in detail the concentration-dependency of the effect of well-known immunosuppressive drugs on glucose-stimulated insulin secretion (GSIS), we performed dynamic perifusion studies with human pancreatic islets. The effect on the time-profile of GSIS has been assessed over a wide concentration range for several clinically relevant immunomodulatory therapies, including small-molecule drugs (cyclosporine, sirolimus, tacrolimus, prednisolone acetate, and loteprednol etabonate) and biologics (abatacept and anti-CD40L), plus a prospective {beta}-cell proliferation-inducing agent (harmine). While biologics showed no significant detrimental effects after one-day treatment even at relatively high concentrations (5 {micro}M), all small-molecule drugs inhibited insulin secretion in a concentration-dependent manner, although glucocorticoids showed a distinct response pattern. Calcineurin and mTOR inhibitors preserved GSIS within their therapeutic ranges but progressively distorted its time-profile at higher concentrations and completely suppressed secretion at the highest levels. Cyclosporine exhibited the least, only about 35-fold, separation between its therapeutic target (Ctarg) and half-maximal GSIS inhibitory (IC50) concentrations. Glucocorticoids did not alter the shape of the time-profile but inhibited overall insulin secretion even at therapeutic levels. Their inhibitory effect only increased slowly with concentration and did not follow a classic sigmoid pattern that has unity Hill slope. These findings establish quantitative benchmarks for immunosuppressant-induced {beta}-cell toxicity and provide a framework for optimizing immunosuppressive regimens to reduce the risk of PTDM.

Lung cancer continues to be the leading cause of cancer-related mortality globally, with non-small cell lung cancer (NSCLC) representing the most prevalent subtype. Tumor hypoxia is a characteristic feature of the neoplastic microenvironment in NSCLC, facilitating tumor progression and conferring resistance to oxidative stress through the stabilization of hypoxia-inducible factor-1 alpha (HIF-1). In this study, we investigated the combined anticancer effects of baicalein (Bai), a natural flavonoid, and thermal-cycling stimulation (TCS), a physical treatment that minimizes damage to normal cells, under cobalt (II) chloride (CoCl2)-induced hypoxic conditions in NSCLC. In A549 NSCLC cells, the combination of Bai and TCS significantly decreased cell viability and induced apoptosis, while exhibiting minimal cytotoxicity on IMR-90 normal human lung fibroblast cells. On a mechanistic level, this combined treatment suppressed the expression of HIF-1 and superoxide dismutase 2 (SOD2) proteins, elevated intracellular reactive oxygen species (ROS) levels, and impaired DNA repair capability by downregulating MutT homolog 1 (MTH1) protein expression. Additionally, disruption of mitochondrial membrane potential and increased poly (ADP-ribose) polymerase (PARP) cleavage further confirmed the induction of apoptosis. These findings indicate that combining Bai with TCS offers a promising synergistic approach to treating NSCLC under hypoxic conditions.

Epigenetics regulate cell-cycle kinetics, differentiation, apoptosis, and migration. Nuclear receptor-binding SET Domain (NSD) histone methyltransferases represent a family of oncoproteins with aberrant expression in cancer. Emerging reports suggest that NSD1 could be an attractive target as its expression is correlated with poor prognosis and tumorigenesis. Previously, we reported the target validation and structure-based virtual screening against NSD1, leading to the selection of several hit molecules with relatively high docking and MMGBSA delta G Bind scores. One of the best-fit molecules identified was 5-O-sulfamoyl adenosine (5-SA) and was compared with the S-Adenosyl-l-Cysteine (SAC), a structural analog of S-Adenosyl-l-Methionine (SAM) for its inhibitory activity against NSD1. IC50 values for 5-SA and SAC against NSD1 were 53.819 {micro}M and 115.003 {micro}M respectively. 5-SA significantly reduced the viability of DU145 and HepG2 cells with IC50 values calculated as 198{micro}M and 168.3 {micro}M respectively. It also reduced the RNA and protein expression levels of NSD1 and subsequently prevented dimethylation of lysine 36 on histone H3 (H3K36me2). Furthermore, 5-SA impeded proliferation, and migration, altered the cell cycle phase, and induced cell apoptosis. Interestingly, 5-SA potentiated the anticancer activity of 5-Fluorouracil (5-FU) against cancer cells. The xenograft model of prostate cancer also showed that 5-SA significantly reduced the tumor growth kinetics. However, the combination of 5-SA and 5-FU synergistically reduced tumor growth and improved survival of animals. To the best of our knowledge, we report for the first time that 5-SA mediated inhibition of NSD1 enhanced the tumor sensitivity to 5-FU and thereby, improved the tumor growth and progression. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/731397v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@e2f0bdorg.highwire.dtl.DTLVardef@12b2ca6org.highwire.dtl.DTLVardef@1807613org.highwire.dtl.DTLVardef@c7f8f0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Hepatitis C virus (HCV) infection remains a major global health challenge despite the success of direct-acting antivirals (DAAs), which are limited by high cost, restricted accessibility, and the emergence of resistant strains. Natural products, particularly phytochemicals, represent a promising reservoir of antiviral agents with diverse mechanisms of action and favorable safety profiles. In this study, we combined wet-lab experimentation with computational approaches to identify plant-derived molecules capable of inhibiting HCV entry. Guided by ethnobotanical evidence, methanolic leaf extracts of Psidium guajava L., Plumeria alba L., Syzygium cumini L., and Tamarindus indica L. were prepared and evaluated for cytotoxicity in Huh7 hepatoma cells. Entry inhibition was assessed using EGFP-labelled HCV pseudoparticles (HCVpp) by qRT-PCR and confocal microscopy. Among the tested plants, Tamarindus indica extract significantly reduced KGFP expression (p < 0.05), confirmed by {Delta}{Delta}Cq analysis and impaired membrane fusion events, while Psidium guajava and Plumeria alba impaired intracellular trafficking without blocking initial attachment. Syzygium cumini showed no inhibitory effect under the tested conditions. Complementary in silico analyses included homology modelling, molecular docking, ADME/toxicity profiling, and molecular dynamics simulations of HCV E2-ligand complexes. Literature mining identified 39 candidate compounds, among which lupeol exhibited stable binding interactions with HCV E2 and favorable pharmacokinetic properties. Critically, in vitro binding assays confirmed that lupeol disrupted the E2-CD81 interaction, reducing bound E2-EGFP to 6% compared to controls. This was supported by HCV-pseudoparticle entry assays confirming inhibition of entry. Together, these findings establish Tamarindus indica and lupeol as potent HCV entry inhibitors.

Therapy of ischemic stroke is currently limited to pharmacological and/or mechanical recanalization. There are no neuroprotective therapies approved for use during the rehabilitative phase of ischemic stroke, which is characterized by neurodegenerative changes. Thus, the search for neuroprotective compounds capable of preventing neuronal death caused by pathogenetic cascades triggered during hypoxia is an urgent task. In this study, we demonstrate increased culture viability following pre- and post-incubation with salicyl-carnosine (SC) in a model of oxygen glucose deprivation on a primary culture of rat cortical neurons. Its neuroprotective properties were greater than that of acetylsalicylic acid and carnosine, and it was effective in lower concentrations. In addition, SC protected the culture from NMDA-induced excitotoxicity. We also showed the passage of SC into neurons, and the presence of its direct antioxidant activity in a model of paraquat-induced oxidative stress. The neuroprotective effects of SC are associated with a decrease in the level of pro-apoptotic protein Bak and a decrease in the activation of kinase p38, as well as an increase in the activation of kinase ERK1/2. The acquired data suggests that SC is a promising neuroprotective compound, and warrants further investigation in vivo.

It has been previously shown that chronic social defeat stress caused by paired agonistic interactions between male mice is accompanied by the development of depression-like state and immune deficiency. The aim of this study was to investigate changes in the expression of C1qtnf superfamily genes (encoding the complement component related with tumor necrosis factor) in the hypothalamus, thymus and lungs against the background of the Lewis lung adenocarcinoma growth. In the experiments, on the 5th day of social stress, male mice were injected with tumor cells into the tail vein. Chronic social stress continued for the next two weeks. The transcriptomes of the hypothalamus, thymus and lungs of mice were sequenced at the Genoanalytica Collective Center (http://genoanalytica.ru/, Moscow). Changes in the expression of the C1qtnf genes in the tissues of stressed mice were studied compared with the control and mice that were additionally injected with tumor cells. Overall, significant correlations were found between expression of most genes in each tissue of the experimental groups. In the hypothalamus of stressed animals, when tumor cells were introduced, an increase in the expression of the genes C1qtnf1, C1qtnf2, C1qtnf3, C1qtnf6 and C1qtnf7 was observed compared to controls. In the thymus of these animals, tumor cell injection increased expression of the C1qtnf1, C1qtnf5, and C1qtnf6 genes. In the lung of tumor-injected stressed mice, expression of the C1qtnf1, C1qtnf2, C1qtnf7, and C1qtnf9 genes was decreased relative to controls and non-tumor-injected depressed mice, reaching near-zero levels in some mice. Analysis of C1qtnf superfamily gene expression in the all tissues revealed negative correlations between the expression of the C1qtnf1, C1qtnf2, and C1qtnf7 genes in the hypothalamus and lungs indicating synchronization of processes against the background of social stress and Levis lung adenocarcinoma.

Dengue Virus Serotype 2 is a human pathogenic flavivirus that encodes a non-structural protein 3 (DEN2-NS3) containing a helicase domain essential for viral replication. DEN2-NS3 utilizes energy derived from NTP hydrolysis to unwind dsRNA and dsDNA. A galloylated catechin, (-)-epigallocatechin gallate (EGCG), was previously reported to be highly potent against the Zika Virus NS3 helicase, with an IC50 value observed at 295.7 nM. This prompted an investigation to determine if three catechins, namely, (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and EGCG, would act as potent inhibitors of DEN2-NS3. Enzyme-inhibition assays revealed that the helicase catalytic domain, DEN2-NS3(S171-K618), is strongly inhibited by these galloylated catechins. We observed Ki values of 400 {+/-} 86.6 nM for EGCG (mixed-mode inhibition with respect to ATP) and 550 {+/-} 250 nM for ECG (uncompetitive inhibition with respect to ATP). Furthermore, using a computational workflow starting with SiteMap, we provide evidence that a highly druggable pocket exists within the RNA-binding cavity, involving residues ASP290, ARG387, ASP409, MET429, HIS487, ASP541, ARG599, and ASP603. These catechins were each analyzed through 200-ns molecular dynamics (MD) simulations to evaluate the binding stability within the target DEN2-NS3 binding pocket. Computational results revealed that EGCG and ECG maintained high stability, forming shared, highly persistent amino acid contacts (>45% occupancy) with ASP603, ARG599, ASP541, and ARG387. In conclusion, we have demonstrated that EGCG and ECG achieve strong binding and allosteric disruption of the critical RNA-binding channel. We suggest that future structural optimization of these compounds into stable prodrug derivatives could yield promising antiviral therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/733882v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@2db363org.highwire.dtl.DTLVardef@5c2fdaorg.highwire.dtl.DTLVardef@49bf8eorg.highwire.dtl.DTLVardef@1bf31f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Non-small cell lung cancer (NSCLC), as the predominant subtype of lung cancer, presents a considerable clinical challenge due to its high rates of recurrence and the significant adverse effects associated with conventional therapeutic modalities. In response to these challenges, this study explored the new combined anticancer effects of epigallocatechin gallate (EGCG) together with thermal cycling-stimulation (TCS). The findings demonstrated that the combination of EGCG and TCS synergistically decreased the viability of A549 and NCI-H460 NSCLC cells, while exhibiting minimal cytotoxic effects on IMR-90 normal lung fibroblasts. Further investigation revealed that EGCG mitigated the TCS-induced upregulation of heat shock proteins HSP70 and HSP105 and concurrently diminished the expression levels of proteasome subunits. This combined effect disrupted proteostasis, resulting in pronounced endoplasmic reticulum (ER) stress. Subsequently, a positive feedback mechanism was established between inositol 1,4,5-trisphosphate receptor (IP3R)-mediated ER Ca2+ release and excessive reactive oxygen species (ROS) production, ultimately leading the cells to undergo mitochondrial apoptosis. This combined treatment reduces the necessary dosage of EGCG, thereby overcoming limitations related to its poor bioavailability and systemic toxicity, while also preventing the development of thermotolerance induced by TCS. Consequently, this method offers a new and potentially practical therapeutic strategy for treating NSCLC.

Glioblastoma remains a highly aggressive and therapy-resistant brain tumor, with limited benefit from the current standard-of-care regimen combining surgery, radiotherapy, and temozolomide. Overcoming chemoresistance therefore represents a critical unmet clinical need. Here, we investigate the anticancer potential of Succisa pratensis and its ability to enhance TMZ efficacy in GBM models. Treatment with S. pratensis markedly reduced cell proliferation and migration while significantly increasing sensitivity to TMZ. Integrated multi-omics analyses revealed extensive metabolic rewiring, characterized by suppression of central carbon metabolism and activation of stress-adaptive pathways. Mechanistically, we identify the Pregnane X Receptor, a key regulator of drug metabolism and chemoresistance, as a central node affected by treatment. Although S. pratensis increased PXR expression, this was not accompanied by induction of canonical downstream targets, including MDR1 and ALDH1A1, indicating a functional impairment of PXR transcriptional activity. Consistently, pharmacological inhibition of PXR using the antagonist SPA70 further potentiated the cytotoxic effects of S. pratensis and TMZ. Docking analyses suggest that specific secondary metabolites, including apigenin-derived compounds, may interact with the PXR ligand-binding domain, providing a potential molecular basis for this effect. Collectively, our findings indicate that S. pratensis enhances TMZ efficacy by inducing metabolic vulnerability and functionally impairing PXR signaling. These results highlight the therapeutic potential of plant-derived metabolites as adjuvant strategies to overcome chemoresistance in glioblastoma. Article HighlightsO_LISuccisa pratensis enhances temozolomide efficacy in glioblastoma by reducing proliferation, migration, and clonogenic growth. C_LIO_LIIntegrated proteomic and metabolomic analyses reveal extensive metabolic rewiring, with suppression of central carbon metabolism and induction of stress-adaptive pathways. C_LIO_LIPregnane X Receptor (PXR), a key regulator of chemoresistance, is functionally impaired despite increased expression, resulting in reduced activation of drug-resistance genes. C_LIO_LIPharmacological inhibition of PXR further potentiates the antitumor effects of Succisa pratensis and temozolomide, promoting apoptotic cell death. C_LIO_LIApigenin-derived metabolites show high affinity for the PXR ligand-binding domain and emerge as promising candidates to overcome temozolomide resistance in glioblastoma. C_LI

Glucocorticoids (GCs) remain the fastest-acting anti-inflammatory agents but are constrained by systemic exposure that suppresses the hypothalamic pituitary adrenal (HPA) axis, silences adaptive immunity, and drives chronic toxicities. Chronic inflammatory diseases are sustained by long-lived CD206+ macrophages containing immune-resistant pathogenic material not cleared physiologically. We developed 101-PGC-005 ('005), a macrophage-targeted type 1a dexamethasone prodrug engineered for low-affinity, recycling-compatible uptake via CD206, with intracellular release triggered by acidic endosomes. We evaluated '005 in mechanistic assays, pathogen-diverse preclinical models, three human pharmacokinetic (PK) studies, and an adaptive-design randomized Phase II/III trial in 309 hospitalized patients with moderate COVID-19. In two completed Phase I human studies, a first-in-human dose-escalation and repeated-dose study and a dedicated single/multiple-dose PK and safety study; '005 circulated as intact prodrug with rapid systemic clearance (Tmax ~0.5 h; terminal half-life ~1.9 h), with no measurable free dexamethasone after single dosing and only low, clinically non-significant free dexamethasone after repeated dosing, and intact prodrug recovered unchanged in urine. Morning cortisol and ACTH were preserved after 30 mg once daily for three consecutive days (1.5 times the intended therapeutic dose). A cerebrospinal fluid PK study is evaluating central-compartment penetration. In the Phase II/III trial, powered for non-inferiority, conducted across six sites in India under GCP with Ministry of Health approval and independent DSMB oversight; '005 (20 mg IV daily for 3 days) was superior to dexamethasone (6 mg IV daily for 3 -10 days) on the primary endpoint of time to > a 2-point improvement on the WHO ordinal scale (HR 2.31; 95% CI 1.83-2.93; p < 0.0001; median 3 vs. 4 days). '005 was also superior on viral clearance (HR 1.47; 95% CI 1.17-1.84; p = 0.0001), hospital discharge rate, SpO2; recovery, and fever resolution. Zero patients in the '005 arm received investigator-initiated corticosteroid supplementation despite protocol allowance. All 309 randomized patients completed the study (ITT = per-protocol). Safety profiles were equivalent (TEAEs 54.8% vs 54.5%; p = 0.958), with no Grade 3+ events, SAEs, deaths, or discontinuations in either arm. Mechanistically, '005 delivered dual benefit: acute debulking of inflammatory macrophages and selective depletion of chronically activated pathology-sustaining macrophages, while preserving CXCL10 antiviral signaling and physiologic HPA control. Critically, HPA preservation is not merely a safety feature, it is a core efficacy mechanism: by clearing the pathogenic macrophage burden that was overriding HPA regulation, '005 restores the conditions for endogenous cortisol to resume its pulsatile, demand-responsive anti-inflammatory role across all GR-expressing cells, lymphocytes, endothelial cells, neurons, and newly differentiated macrophages, that '005 itself cannot reach. These findings support regulatory-grade evidence for macrophage-targeted corticosteroid therapy and provide the foundation for further development across acute inflammatory indications (sepsis, viral pneumonia, cytokine-release syndromes) and chronic macrophage-driven diseases (atherosclerosis, metabolic steatohepatitis, neurodegeneration, tumor-associated macrophages).

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterised by a highly immunosuppressive tumour microenvironment and limited therapeutic responses. Tumour-derived extracellular vesicles (EVs) contribute to PDAC progression by transferring immunomodulatory molecules and tumour-associated signals, suggesting EV-associated processes as potential intervention opportunities. However, the heterogeneity of EV biology and the complexity of tumour-immune interactions make single-target intervention strategies challenging. Here, we developed a computation-driven dual-layer candidate-prioritisation framework to identify potential modulators associated with PDAC EV-mediated immune escape through complementary production-side and action-side strategies. For the production-side layer, we focused on upstream processes related to EV biogenesis, cargo regulation, inflammatory signalling, and tumour-associated pathways. An 88-gene PDAC EV-associated target framework was integrated with cell-type-resolved prognosis annotations from ctPANDA and predicted targets of 18 natural products derived from Scutellaria baicalensis, Epimedium spp., and Cornus officinalis to prioritise natural-product candidates with disease relevance and potential chemical tractability. In parallel, key targets with experimentally resolved ligand-binding structures were subjected to pocket-guided de novo small-molecule design based on co-crystal ligand-defined binding sites, followed by structural, docking-based, and physicochemical screening of generated compounds. For the action-side layer, VHH and scFv binders were computationally designed and screened against extracellular regions of MET and CD81 to prioritise candidates potentially suitable for EV recognition and capture. This study provides a computational strategy for narrowing candidate spaces across both EV-associated production pathways and released vesicle recognition. The resulting small molecules, antibody-like binder models, and screening workflows provide a resource for future experimental validation of strategies targeting PDAC EV-associated immune regulation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/740294v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@11829b0org.highwire.dtl.DTLVardef@158efb5org.highwire.dtl.DTLVardef@1e17cc5org.highwire.dtl.DTLVardef@c69696_HPS_FORMAT_FIGEXP M_FIG C_FIG

Malignant brain tumors are among the most aggressive and difficult to treat human cancers. Glioblastomas (World Health Organization grade IV gliomas) are particularly lethal and refractory to treatment. Few drugs exist that are even somewhat effective. Our investigation of the physiologic role of fatty acid (FA) activating enzymes (acyl-CoA synthetase; ACS) identified an ACS that was widely expressed in gliomas but not in normal glial cells. Depletion of this enzyme, ACSVL3 (very long-chain ACS3), by knockdown or knockout decreased the malignant behavior of several glioma cell models including U87MG and Mayo-22 cells both in culture and when grown as xenografts. Hypothesizing that ACSVL3 is a potential therapeutic target in glioma, we conducted a search for inhibitors of this enzyme and found that CB5 (grassofermata) was a promising candidate. Treating U87MG glioma cells with CB5 slowed growth in monolayer culture; the growth rate was similar to that seen in cells in which ACSVL3 was either knocked down or knocked out. CB5 inhibited growth in a dose-dependent manner over a narrow range, and concentrations above 10 M were toxic. Treatment at the lower dose of 3 M inhibited growth of U87MG cells but was reversible, suggesting that this dose was not toxic. CB5- treated U87MG cells exhibited an altered morphology with a larger size and longer projections. In contrast, normal human fibroblasts treated with 10 M CB5, a concentration that was toxic to U87MG cells, showed no effect on either growth rate or morphology. Treating U87MG cells with 3 M CB5 induced differentiation as shown by increased expression of the astrocyte-specific marker glial fibrillary acidic protein (GFAP). In contrast, GFAP levels remained low in ACSVL3 knockdown cells. CB5- treated U87MG cells were less invasive, and thus less malignant, than either untreated cells or ACSVL3 knockout cells when assessed by a scratch wound healing assay. Acute treatment of U87MG cells with 3 M CB5 decreased the ability of these cells to degrade FA of differing chain lengths from 16-24 carbons by {beta}-oxidation, suggesting that decreased ACS enzyme activity contributes at least in part to the drugs mechanism of action. NOD/SCID mice receiving up to 32 mg/kg/day CB5 by intraperitoneal injection showed no obvious side effects, suggesting that the drug was well-tolerated. Xenografts induced by subcutaneous injection of U87MG cells in the flanks of NOD/SCID mice were allowed to grow for 8 days after which half of the mice were treated with 2 mg/kg/day CB5. After 7 days of treatment, xenograft growth slowed in the treated mice and by 12 days tumor size had begun to decrease, suggesting therapeutic efficacy. When a similar study was done using xenografts induced by subcutaneous injection of Mayo-22 cells, which are maintained as subcutaneous tumors in mice rather than in cell culture, the effect of CB5 on tumor growth or weight at sacrifice was not statistically significant. The results of these studies suggest that CB5 may have therapeutic value in malignant glioma. Additional studies using other glioma models and other drugs chemically related to CB5 seem warranted.

Recent models describe drug release from polymeric nanoparticles through an analogy with the quantum tunneling effect, treating the delivery system as a static rectangular potential barrier. In this work, we argue that this analogy is structurally identical to the standard solution of the Schrodinger equation for a rectangular barrier, and that the introduction of a multifractal formalism to describe time evolution -- obtained via a formal Wick rotation (x [-&gt;] t) -- lacks direct physical justification. We propose, instead, to treat the barrier height as an effective function of time, Ueff(t) = U0 f(t), with f(t) varying slowly within the barrier region, reflecting the progressive degradation/swelling of the polymeric matrix, under two hypotheses for f(t)-- exponential decay and rational decay (Hill-type). Rather than the thick-barrier WKB approximation, the exact transmission formula is used throughout, which is real-analytic in f(t) and continues smoothly into the resonance (over-barrier) regime once the barrier collapses, avoiding the artificial step-like transitions produced by the WKB approximation used in earlier drafts of this work. Both hypotheses for f(t) predict a finite barrier collapse time, t*, whose dependence on the energy ratio m = U0/E differs qualitatively between them (t* {propto}ln m vs. t* {propto}(m-1)1/n), offering a distinguishable criterion from experimental release data. The model was tested against ex-vivo chicken-skin permeation kinetics of 5-FU digitized from Rata et al. [1] (three systems: NCA-1-5-FU, G-NCA-1-5-FU, G-5-FU). The exact formula substantially improved fit quality relative to the WKB approximation for all three systems. Fits were obtained by global optimization (differential evolution, polished with scipy.optimize.curve_fit for covariance estimates) rather than a single local search, which proved necessary: for G-5-FU and NCA-1-5-FU, the exponential family is well-identified (all parameter uncertainties below 11% and 6% of the estimates, respectively; R2 > 0.999), while the rational (Hill) family remained poorly identified for all three systems despite the improved formula - favoring, by parsimony, the simpler exponential model throughout. Only G-NCA-1-5-FU remained non-identified with m free. A sensitivity check fixing m at the G-5-FU-derived value (m = 1.377) resolves this non-identifiability for G-NCA-1-5-FU at negligible cost in fit quality, consistent with a shared energy ratio for that system; the same constraint applied to NCA-1-5-FU, however, degrades its (already well-identified) fit by a factor of [~]4 in maximum residual; its own energy ratio (m = 1.188 {+/-} 0.007) differs from the shared value by [~]4{sigma}, a formally significant difference, so a single universal m is rejected for the complete set of systems studied. Reference values from the original multifractal study [2-4] and candidate extensions to further aptamer-functionalized nanocarrier systems [5-7] are also discussed. We discuss the implications of this treatment and its limits of validity, and point out paths for further empirical validation.

Introduction: Nicotine Pouches are increasingly used as a smokeless alternative to cigarettes and other nicotine products, yet their acute cardiovascular effects remain poorly documented. While nicotine's impact on heart rate and electrocardiogram (ECG) parameters is well-documented in smoking, no trials have evaluated these effects specifically for nicotine pouches. Methods: This study is a single-center, double-blind, placebo-controlled, crossover trial which will include 20 healthy adult nicotine users. Participants will undergo three sessions, receiving either a placebo, 6 mg, or 14 mg nicotine pouch in random order. Heart rate obtained by an ECG and various other ECG parameters, vital signs, and subjective symptoms will be measured at baseline, and multiple time points over 30 minutes. Conclusions: This study aims to determine whether nicotine pouches cause acute changes in heart rate, ECG parameters, vital signs, and self-reported symptoms. We hypothesize that higher nicotine pouch does will lead to measurable increases in heart rate and other autonomic effects compared to placebo.

Cell-penetrating peptides (CPPs) are widely used to deliver therapeutic cargoes into cells. Although numerous computational methods have been developed for identifying CPPs and several predictors are available for protein subcellular localization, no method has been developed to predict the subcellular localization of CPPs. Here, we present CPPLocPred, a hierarchical machine-learning (ML) framework that predicts CPPs and their subcellular localization. In the first stage, we developed ML models to identify CPPs, achieving an AUC of 0.953 with an MCC of 0.7842 on an independent set, exhibiting performance equivalent to or better than existing state-of-the-art methods. In the second stage, we developed a method for predicting the subcellular localization of CPPs. Subcellular localization methods were trained (80% data using five-fold cross-validation) and validated (20% data) on experimentally validated CPPs for 663 Cytoplasm, 287 Nucleus, 57 Mitochondria, 186 Endo_lysosome, and 328 Others. Our primary analysis revealed that Mitochondrial and Nuclear associated CPPs are abundant in positively charged arginine- and lysine-rich patterns, whereas Endo_lysosomal CPPs preferentially comprise glycine-, proline-, and cysteine-rich motifs. We used a wide range of traditional peptide features, along with the embedding of protein language models, to develop ML models. Among all evaluated models, the CatBoost-based subcellular localization models with Distance Distribution of Residues (DDR) achieved AUCs of 0.814, 0.775, 0.970, 0.782, and 0.798 for Cytoplasm, Nucleus, Mitochondria, Endo_lysosome, and Others, respectively, on validation dataset. We developed CPPLocPred, which offers a practical platform for functional annotation and rational design of localization-specific CPPs for therapeutic applications (https://webs.iiitd.edu.in/raghava/cpplocpred/). HighlightsO_LIPrediction and subcellular localization of cell-penetrating peptides. C_LIO_LILocalization of CPPs depends on their amino acid and dipeptide composition. C_LIO_LIBest feature for subcellular localization was Distance Distribution of Residues. C_LIO_LICatBoost model achieved the highest performance for subcellular localization. C_LIO_LIA web server and standalone software to facilitate its use by the scientific community. C_LI

Polo-like kinase 1 (PLK1), a key regulatory protein controlling entry into and passage through mitosis, has been targeted through its kinase domain (KD) with mixed clinical success. Inhibition through the Polo-box domain (PBD) is a viable alternative through targeting the sub-cellular localization and kinase activity of PLK1. Novel non-peptidic compounds, termed abbapolins, were discovered through the REPLACE strategy and have been lead optimized through structure-based strategies and screening analogs in the NCI-60 tumor cell panel. Proteomic analysis revealed a correlation between abbapolin activity and PLK1 protein levels in the cell lines part of the NCI-60. Prostate cell lines were identified as among the most sensitive and led to further detailed studies of their activity in prostate cancer models. Compounds were evaluated for their pharmacokinetic properties, and in vivo efficacy, and results showed significant antitumor xenograft activity with no observable gross toxicity. Treated tumors were analyzed for loss of PLK1, which was previously shown to be induced by abbapolin binding. Results obtained showed a significant degradation of PLK1 in abbapolin-treated vs untreated tumors, thereby confirming on-target action in vivo and revealing PLK1 levels as a potential pharmacodynamic marker. Lead compounds were shown to sensitize PC tumors resistant to androgen deprivation therapy paving the way for future combination studies in vivo. These data provide an alternative pathway for effective PLK1 therapeutics that avoid the reported problems of molecules targeting the KD, in vivo proof-of-concept for the REPLACE strategy and validation for targeting the PBD as an anti-tumor drug development strategy.

Favipiravir (FPV) is an RdRp inhibitor developed and licensed in Japan for influenza but which has shown promising in-vitro activity against a range of RNA viruses. A physiologically-based pharmacokinetic model was developed for oral FPV and its metabolite M1 in order to optimise the dose regimen against plasma concentration targets for a number of viral pathogens. The model was validated using clinical data and was able to capture the variability in plasma concentrations for a population of individuals. FPV doses predicted to cause in-vivo exposures exceeding in-vitro IC90 targets against influenza, Ebola, Lassa fever, CCHF, SFTS, Andes virus and SARS-CoV-2, lie within the window of observed safe dosing, with SARS-CoV-2 requiring predicted doses of 2400 mg twice daily due to lower in-vitro potency. Simulations showed that a loading dose on day one of treatment should allow plasma targets to be exceeded on day one. Simulations of chronic kidney disease (CKD) showed no change to FPV plasma concentration in individuals with CKD3 and CKD5 compared to healthy individuals. Clinical data suggested active renal efflux of M1 which led to a predicted 2.2 and 11.5 fold increase in the maximum plasma concentrations of M1 in individuals with CKD3 and CKD5 respectively in comparison with healthy individuals.

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.

BackgroundReactive Oxygen Species (ROS) are the small, unstable and highly reactive species, having DNA oxidizing ability. Oxidation of the DNAs purine and pyrimidine bases can lead to single or double strands in this macromolecule. In this situation, the ATM molecule, a serine-threonine kinase, targets several proteins for phosphorylation, which causes the cell cycle to stop and the DNA damage repair begins. It has previously been proven that natural polyphenols have the cancer inhibiting properties due to their high efficacy and low side effects. Silibinin is the main herbal and medical ingredient in Milk Thistle (Silybum marianum) is a polyphenol flavonolignan, which has been widely considered as an antioxidant and anticancer agent. The purpose of the present study was to investigate the ATM gene expression and measurement of reactive oxygen species (ROS) in SKBR3 cell line, treated with Silibinin. Materials and MethodsAt first, the SKBR3 cell line was cultured in RPMI1640 culture medium and MTT assay was carried out to evaluate the Silibinin cytotoxicity. Flow Cytometry was carried out for cell cycle analysis, apoptotic induction, and ROS detection. While, Real Time PCR was used to evaluate the ATM gene expression in the Silibinin-treated and un-treated SKBR3 cells. ResultsPresent results have shown that 150 {micro}M Silibinin had the most significant cytotoxicity and apoptotic induction influence after the treatment period of 48 h. Flow cytometry data have shown that Silibinin induced considerable amount of apoptosis and caused cell cycle arrest at G1/S phase and induced production of ROS. Real-time PCR results have revealed that Silibinin increased the ATM expression in SKBR3 cell line. ConclusionSilibinin causes increased ATM gene expression by inducing ROS production, which initiates cell cycle arrest and apoptotic induction in SKBR3 cells line.

ObjectiveIn recent years, the number of cats kept as companion animals has increased, leading to a growing demand for veterinary care. Although some histone deacetylase (HDAC) inhibitors are promising for the treatment of human cancers and neurological diseases, comprehensive systematic research on HDAC inhibitors in domestic cats remains insufficient. Therefore, this study aimed to investigate the effects of HDAC inhibitors on the transcriptome of feline cells. MethodsTwo types of cells derived from domestic cats, Crandell-Rees Feline Kidney (CRFK; kidney-derived) cells and PG-4 cells (astrocyte-derived), were treated with four HDAC inhibitors (panobinostat, trichostatin A, valproic acid, and vorinostat) for 24 h. Transcriptomic changes after treatment were examined using RNA sequencing. ResultsHDAC inhibitor treatment upregulated the expression of intercellular chemical interactions and signal transduction-related genes, similar to observations in human cells. Although HDAC inhibitors did not suppress the expression of cell cycle-related genes in CRFK cells, as observed in human cells, the inhibitors downregulated the expression of organogenesis-related genes. Consistent with observations in human cells, HDAC inhibitors suppressed the expression of cell cycle- and cancer-related genes in PG-4 cells. Importantly, valproic acid, which is thought to be more effective for neurological diseases than for cancer, suppressed the expression of more cancer-related genes in PG-4 cells than the other three HDAC inhibitors. Conclusion and relevanceOur findings revealed that the responses of cells derived from feline organs to various HDAC inhibitors varied considerably depending on the organ of origin and species. Since few studies, including human studies, have comprehensively compared transcriptomic responses to multiple HDAC inhibitor classes across multiple cell types, the results of this study provide a foundation for future research on the treatment and prevention of cancer and neurological diseases in domestic cats and other mammals.