Abstract Physical exercise recruits endogenous opioid mechanisms in the central nervous system, but whether this physiological opioid tone potentiates the effects of exogenous μ-agonists, and whether such interactions differ between sexes, remains unknown. Here, we used the Straub tail response as a behavioral index of central μ2-opioid receptor activity to investigate whether acute submaximal running modulates the pharmacodynamic effects of meperidine in male and female Swiss mice. In resting animals, meperidine produced a dose- and time-dependent Straub tail response across a 12-fold dose range, and the submaximal doses of 8 and 12 mg/kg were selected for treadmill experiments. Running at approximately 70% of V̇O2 max potentiated the Straub tail response at 12 mg/kg in both sexes, with the effect substantially larger in females, as indicated by a significant Sex × Exercise interaction. Thermal antinociception was equivalent between sexes at 12 mg/kg, indicating that the dimorphism was specific to the exercise-opioid interaction rather than to baseline drug sensitivity. A separate experiment testing whether running alone elicits a naloxone-sensitive response was inconclusive in both sexes; under opioid receptor blockade, however, females maintained higher running-induced scores than males, indicating that the sex difference persists during naloxone treatment. These findings indicate that acute submaximal running potentiates the Straub tail response to meperidine in a dose- and sex-dependent manner. The sexually dimorphic nature of this interaction suggests that biological sex and exercise intensity may jointly modulate opioid pharmacodynamics during physical activity.
Abstract Members of the DYRK kinase family have been implicated in the regulation of key metabolic processes. In particular, DYRK1B plays a central role in hepatic lipid metabolism and fatty liver disease. Notably, these functions depend not only on the enzyme’s catalytic activity but also on scaffolding properties. Consequently, therapeutic ambitions aiming to target the entire spectrum of DYRK1B functions must inactivate or eliminate the protein itself rather than merely inhibiting its enzymatic activity, as is achieved by current kinase inhibitors. In this study, we describe a small molecule from the leucettinib class of DYRK inhibitors (LCTB-92) that not only suppresses DYRK kinase activity but also promotes selective ablation of the DYRK1B protein while sparing the closely related paralog DYRK1A. The identification of this compound establishes a foundation for the development of selective agents capable of targeting the entire DYRK1B protein scaffold, thereby addressing catalytic-dependent as well as catalytic-independent kinase functions.
Abstract The degradation efficiency of a PROTAC─bivalent compounds that link together a ligand for a protein of interest with a ligand for an E3 ligase─is governed, in part, by the length and flexibility of its linker (“linkerology”), as well as the accessibility of ubiquitinatable surface lysines. Based on the prior serendipitous discovery of the cyclin-dependent kinase 9 (CDK9)-selective PROTAC degrader “THAL-SNS-032”, developed from the multi-CDK2/CDK7/CDK9 inhibitor SNS-032, we speculated that other multi-CDK inhibitors might likewise be transformed into CDK9-selective PROTACs, establishing a “linkerology” platform approach to enhancing CDK family member specificity (and beyond). We tested this hypothesis using AT7519, a multi-CDK2/CDK5/CDK9 inhibitor, and employing cereblon as the targeted E3 ligase. Accordingly, we built candidate PROTACs by linking AT7519 to one of two thalidomide derivatives; those molecules in which AT7519 and the thalidomide derivative were coupled via rigid linkers caused greater degradation of the CDK9 protein than those with more flexible linkers. Western blotting of human acute myeloid leukemia (AML) cells treated with AMC-3-221, our most CDK9-selective PROTAC, resulted in almost-complete reduction of the CDK9 protein, with no detectable degradation of CDK1, CDK2, or CDK5, and minimal degradation of CDK4 and CDK6, corroborated by global proteomics analysis. Subtle shortening or lengthening of AMC-3-221’s rigid linker altered the CDK degradation selectivity profile, suggesting that continued linker variation may further enhance selectivity. Molecular modeling provided insight into the observed selectivity through the analyses of ternary complex stabilities. AMC-3-221 treatment rapidly decreased RNA and protein levels of oncogenic downstream targets of CDK9─MCL-1 and c-/N-MYC─and potently inhibited human AML and other types of human cancer cell lines, with less inhibition of a non-transformed cell line, via an on-target mechanism of apoptosis. AMC-3-221’s CDK9 degradation potency (DC50 < 10 nM) approximated its antileukemic inhibitory potency (IC50 < 50 nM). The maximum tolerated dose in mice of AMC-3-221 was higher than that of another PROTAC, AMC-3-225, consistent with AMC-3-221’s more selective CDK degradation profile. In summary, we demonstrate that converting a multi-CDK inhibitor into a PROTAC with selectivity for CDK9 over other family members is not an isolated phenomenon. Our findings further support the concept that PROTAC linkerology may serve as a platform for enhancing kinase selectivity.
Abstract Calcium oxalate monohydrate (COM) crystals are major contributors to nephrolithiasis-associated renal tubular injury, primarily through mechanisms involving oxidative stress, inflammation, apoptosis, and epithelial–mesenchymal transition (EMT). While nesfatin-1 is known for its antioxidant and cytoprotective actions, the specific role of endogenous nesfatin-1 in regulating COM-induced epithelial injury has not been fully defined. In this study, nesfatin-1 was silenced using antisense oligonucleotides in NRK-52E and primary renal tubular epithelial cells to examine how loss of endogenous nesfatin-1 peptide influences cellular susceptibility to COM. COM exposure reduced nesfatin-1 levels, and this decline was markedly greater in the nesfatin-1 silenced + COM group. Silencing nesfatin-1 intensified COM-driven oxidative stress, as reflected by reduced catalase activity and increased levels of malondialdehyde, nitric oxide, and ROS. Pro-inflammatory markers including NF-κB, TNF-α, IL-6, and IL-1β were upregulated, whereas IL-10 was decreased. Apoptotic indicators such as elevated intracellular Ca2+, diminished Bcl-2, increased caspase-3 activation, and higher proportions of apoptotic and necrotic cells were also evident. EMT and fibrotic markers (TGF-β1, α-SMA, Smad2/3, Smad4, vimentin) were increased, alongside reduced E-cadherin expression. Exogenous nesfatin-1 mitigated oxidative, inflammatory, apoptotic, and fibrotic responses in COM-treated cells; however, these protective effects were minimal in nesfatin-1 silenced + COM cells. Overall, the findings demonstrate that endogenous nesfatin-1 is indispensable for maintaining epithelial stability under COM-induced stress, and its absence amplifies cellular injury while diminishes responsiveness to exogenous peptide. Nesfatin-1 thus emerges as a potential therapeutic target for limiting crystal-induced renal cells damage.
Abstract Topiramate is a widely prescribed antiepileptic drug with a multimodal mechanism of action. However, its effects on male reproductive function remain poorly characterized. This study was conducted to investigate the impact of this drug on testicular and epididymal function in adult male Wistar rats. Animals were treated orally for 21 days with topiramate at 40 or 160 mg/kg. Serum testosterone levels, testicular histomorphology, spermatogenic indices, sperm parameters, and epididymal duct contractility were subsequently evaluated. The treatment did not alter serum testosterone levels at either dose. However, both doses reduced Sertoli cell and round spermatid numbers, increased primary spermatocyte counts, and decreased meiotic and overall spermatogenic yields, whereas only the higher dose (160 mg/kg) reduced Sertoli cell efficiency and daily sperm production. The lower dose (40 mg/kg) increased sperm morphological abnormalities, whereas the higher dose reduced total and progressive sperm motility and induced both head and tail defects. Additionally, treatment with topiramate at 160 mg/kg prolonged sperm transit time in the cauda epididymis and reduced contractile responses of the distal cauda epididymal duct to KCl, noradrenaline, and carbachol, suggesting impaired epididymal transport and potential ejaculatory dysfunction. Altogether, these findings demonstrate that topiramate adversely affects male reproductive function. Given its widespread use in individuals of reproductive age, these results underscore the need for translational studies in humans to assess potential reproductive risks associated with this therapy.
Abstract Uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes play a critical role in hepatic and other metabolism of endogenous and exogenous small molecules. The addition of glucuronic acid via UGT increases the polarity and subsequent solubility of small molecules, helping direct them for biliary excretion and renal excretion by organic anion transporter OAT3 (SLC22A8). Mutations in UGTs are associated with disorders in biliary metabolism (e.g., Gilbert’s syndrome, Crigler-Najjar syndrome). Here, we used comprehensive serum metabolomic profiling of pan Ugt1-knockout (Ugt1–/–) mice to define the metabolic consequences of UGT deficiency. All 7 knockout mice exhibited markedly elevated bilirubin (mean = 5.96 mg/dL). Partial Least Squares Discriminant Analysis (PLS-DA) showed clear separation between Ugt1–/– and wild type (WT), and volcano plots revealed significant endogenous metabolite alterations. These included upregulation of numerous bilirubin degradation products as well as major disturbances in lipid and amino acid metabolism. In particular, pathway enrichment analyses identified disruptions in fatty acid biosynthesis, glycine-serine metabolism, heme breakdown, porphyrin metabolism, and β-oxidation of fatty acids. Thus, the UGT1 proteins regulate different aspects of endogenous metabolism and signaling. Consistent with this notion, there were increases in liver expression of several nuclear receptors (e.g., HNF4a, PPARa, PXR) involved in metabolic signaling. These results support the remote sensing and signaling theory (RSST), which proposes that UGT1 proteins play a key role in organ crosstalk via routing small molecules between the liver and kidney, with glucuronidated molecules being mainly directed to the multispecific SLC kidney transporter OAT3. We propose that this glucuronidation-regulated RSST loop is essential to signaling along the gut-liver-kidney axis.
Chemotherapy-induced hepatorenal toxicity is one of the most predominant dose-limiting obstacles in oncological pharmacotherapy, and the molecular mechanisms surrounding its pathogenesis are still poorly defined. Lipid peroxide toxicity-induced, iron-dependent, nonapoptotic types of regulated cell death called ferroptosis have emerged as a mechanically important process in the organ injury caused by chemotherapy. In this Keynote Review, we will summarize recent evidence showing the implication of ferroptosis in liver and kidney toxicity of platinum-based drugs, anthracycline drugs, tyrosine kinase inhibitors, and some targeted drugs. We identify key mechanisms underlying ferroptosis, including abnormalities of the axis of system Xc-/glutathione peroxidase4 (GPX4) axis, changes in iron homeostasis, and activation of ferritinophagy and the maladaptive effects of polyunsaturated fatty acid oxidation. The role of the hepatorenal microenvironment is discussed in detail, with a particular focus on mitochondrion-related abnormalities, interaction between endocrine and immune cells, and mitochondrion-endoplasmic reticulum-stress coupling, as well as interactions with ferroptosis suppressor protein 1 (FSP1)/CoQ10 and dihydroorotate dehydrogenase (DHODH) pathways. Data from the preclinical models are combined with new treatment data to develop a linear toxicological story. The utility of the use of biomarker strategies such as plasma malondialdehyde, 4-hydroxynonenal adducts, circulating GPX4, and urinary prostaglandin-like isoprostanes in diagnosis and monitoring is examined. The role of therapeutic interventions such as Nrf2 activators, liproxstatin1, ferrostatin1, and iron chelators for reducing organ-specific ferroptotic injury without reducing effectiveness as antineoplastic is briefly explored. This Review highlights ferroptosis as a therapeutic target that is amenable to therapy and suggests a systematic approach for its integration into toxicological risk classifications during drug development for cancer treatment.
MYCN is a key oncogenic driver in hepatocellular carcinoma (HCC) and a therapeutic challenge due to the historical undruggability of MYC transcription factors (TFs). Using a high-throughput MYCN promoter-luciferase reporter, we identified PhiKan 083 (PK83), a small molecule previously recognized as a mutant p53 activator, that dose-dependently suppresses MYCN expression in HCC cells. PK83 impaired the proliferation and survival of MYCN-high HCC cells, inducing DNA damage, apoptosis, and loss of clonogenic and spheroid growth potential, while sparing MYCN-low HCC cells and normal hepatocytes. Structure-activity analysis revealed that polar, hydrogen-bond-capable substituents on PK83's tricyclic scaffold are critical for its activity. Although PK83 broadly activates p53 signaling, its cytotoxicity in MYCN-high cells is not strictly dependent on intact p53, as confirmed in p53-knockout systems. Transcriptome profiling and pathway analysis demonstrated robust suppression of MYC/MYCN targets along with modulation of pathways linked to stress, differentiation, and metabolism. In primary HCC tumors, PK83-downregulated TFs, including oncogenic TFs ZMIZ1 and TARBP1, positively correlated with MYCN, whereas upregulated stress-responsive TFs ATF3 and FOSL2 showed a negative correlation. These findings suggest that PK83 suppresses MYCN expression and preferentially affects MYCN-high HCC cells in a p53-independent manner, warranting further preclinical investigation of PK83 and related compounds in MYCN-associated cancers.
Functional excipients are increasingly recognized as active components in drug formulations because they can influence drug stability, solubility, release behavior, delivery efficiency, biological barrier penetration, transporter activity, and metabolic clearance. Machine learning is becoming useful in formulation research because it can connect scattered formulation data to excipient selection, property prediction, and experimental decision-making. In particular, machine learning models can help extract useful patterns from fragmented formulation data, prioritize candidate excipients, and guide formulation decisions before extensive experimental screening. This review summarizes recent advances in machine-learning-driven optimization of functional excipients and their biointeractions in drug formulations. We first discuss the methodological foundations of this field, including data acquisition, feature engineering, model architecture selection, optimization, and evaluation strategies. Representative application scenarios are then reviewed, including the identification of functional excipients related to drug efflux inhibition, formulation stability enhancement, biological barrier penetration, and metabolic clearance reduction. This review also discusses practical barriers that still limit this field related to data quality, representation design, experimental feedback, computational cost, and deployability. These issues need to be addressed before artificial intelligence (AI)-assisted formulation systems can become reliable tools for routine pharmaceutical development.
The wound healing process typically consists of four stages; however, it can be delayed by several external factors, including bacterial contamination, irritation from dressings, and internal conditions such as diabetes, etc. These challenges have driven a growing interest in advanced dressings incorporating bioactive materials, which can protect the wound while simultaneously promoting healing. Collagen peptides derived from collagen hydrolysis have gained increasing attention in this context due to their bioactive properties. Notably, fish collagen has gained prominence in recent years due to its favorable profile compared to traditional sources. These attributes make fish collagen peptides highly advantageous for use in wound care. In this context, literature and patent searches were conducted to assess the application of these materials in the context of biomaterial production and wound healing. An initial screening resulted in 289 patents from the European Patent Office and Cortellis Drug Discovery Intelligence, of which 10 were deemed eligible. The largest concentration of patents originated in China (n = 6), Japan and Taiwan (n = 2 for both). These patents focused on topical cosmetics and oral formats, which are cosmetic products without a primary focus on therapeutic use. The effects cited in the patents include cell and collagen proliferation, skin hydration, scar repair, antiaging, antioxidant activity, etc. Therefore, it was concluded that there has been an increase in the application of collagen peptides in recent years and that there are several benefits regarding the appearance and repair of the skin, making it interesting to conduct in-depth future research, including clinical trials.
Breast cancer is the most prevalent cancer among women worldwide, with a complex pathogenesis and diverse treatment options. However, conventional treatments are often accompanied by large side effects and limited efficacy. In recent years, mitochondria, as the core organelles of cellular energy metabolism, oxidative stress regulation, and apoptosis execution, have played a key role in breast cancer development, progression, metastasis, and treatment resistance. Therefore, many researchers have turned their attention to mitochondria to open up new pathways for the treatment of breast cancer. This review systematically outlines the relationship between mitochondrial energy metabolism, antiapoptotic proteins, mitochondrial fusion/fission, mitochondrial autophagy, and ferroptosis and breast cancer cell proliferation, metastasis, and drug resistance, summarizes and analyzes the inhibitory effects of drugs or therapeutics targeting mitochondria, and explores the strategies of targeting mitochondria for the treatment of breast cancer to provide new ideas and methods for the treatment of breast cancer.
G protein-coupled receptors (GPCRs) can trigger distinct signaling pathways depending on their localization. Because their appearance is not restricted, GPCRs can signal not only from the plasma membrane but also from internalized compartments. The pH of endosomes is significantly lower compared to the extracellular environment, and thus, location-biased ligands can be developed by carefully adjusting their physicochemical properties. This work describes the development of pH-sensitive β-adrenergic receptor antagonists with weak basicity, conferring enrichment in acidic compartments. Starting from FDA-approved β-blockers including bisoprolol, metoprolol, propranolol, and nebivolol, chemical synthesis, physicochemical studies, in vitro pharmacological investigations, and structure-activity relationship studies led to ligands with considerably higher potency at representative acidic pH values of 6 and 6.5 compared to a pH of 7.4, which is typical for ligand binding at the plasma membrane. Bis-fluorination in the beta position of nitrogen substituents was the most effective and led to pH-sensitive analogs of bisoprolol, metoprolol, and propranolol with 10- to 12-fold higher potencies at endosome-specific pH values. Because endosomal β-adrenergic receptors are suggested to be associated with heart hypertrophy and cardiac dysfunction, the newly developed ligands may serve as lead compounds for the development of powerful and safe cardioprotective drugs.
The combined therapy of immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKIs) is now an approved first-line approach for unresectable hepatocellular carcinoma (HCC). Hepatic arterial infusion chemotherapy (HAIC) with the FOLFOX regimen (oxaliplatin, fluorouracil, and leucovorin) has also shown promising outcomes for advanced HCC. However, the efficacy of these two therapeutic strategies remains to be further improved. This retrospective study evaluated the safety and the efficacy of combining adebrelimab (a PD-L1 inhibitor), apatinib (a TKI), and FOLFOX-HAIC for unresectable HCC. The medical records of unresectable HCC patients who received adebrelimab, apatinib, and FOLFOX-HAIC from March 2023 to November 2023 at the First Affiliated Hospital of Sun Yat-sen University (FAH-SYSU) were retrospectively reviewed. Safety was assessed by the frequency and severity of adverse events (AEs), and efficacy was evaluated by tumor response criteria including objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS). A total of 19 patients were enrolled. There was no treatment-related death. The most frequent grade ≥ 3 treatment-related AEs were increased ALT/AST (47.4%) and thrombocytopenia (15.8%). The ORR was 26.3% and 57.9% as per RECIST 1.1 and mRECIST, respectively. The DCR was 84.2% and 89.5% as per RECIST 1.1 and mRECIST, respectively. The median PFS was 9.0 months by both RECIST 1.1 and mRECIST, and median OS was 22.0 months. The combination of adebrelimab, apatinib, and FOLFOX-HAIC showed manageable safety and encouraging efficacy for unresectable HCC. This regimen may represent a novel therapeutic option for advanced HCC and warrants further investigation.
Abstract Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by aberrant fibroblast activation and excessive extracellular matrix accumulation, predominantly collagen, leading to irreversible lung remodeling. Sinigrin (SNG), a naturally occurring glucosinolate with reported anti-inflammatory and antioxidant activities, has not been investigated in the context of pulmonary fibrosis. In the present study, we evaluated the therapeutic activity of SNG using complementary in vitro and in vivo models of IPF. In the current investigation, inflammatory responses were induced by lipopolysaccharide (LPS) in RAW 264.7 macrophages and precision-cut lung slices (PCLS), whereas fibrotic activation was elicited by TGF-β in LL29, DHLF cells, and PCLS. Treatment with sinigrin and dexamethasone significantly suppressed LPS-induced inflammatory mediators (IL-6, TNF-α, IL-1β, CCL2, MMP-9, and COX-2). In TGF-β-stimulated fibroblasts/PCLS, SNG and pirfenidone markedly attenuated collagen accumulation and downregulated the expression of key fibrotic markers (FN-1, α-SMA, TIMP-1, TIMP-3, COL1α1, and COL3α1). SNG administration in a bleomycin-induced pulmonary fibrosis mouse model significantly improved body weight, survival rate, and pulmonary index. Histopathological analyses revealed reduced collagen deposition and lowered Ashcroft scores, accompanied by decreased oxidative stress and improved lung function in SNG treatment compared to BLM and PFD groups. Consistently, gene and protein expression analyses demonstrated that SNG dose-dependently attenuated the bleomycin-induced inflammatory and fibrotic marker expression. Further mechanistic investigations, including an AMPK siRNA-based approach, demonstrated that SNG exerts its anti-inflammatory and antifibrotic effects partly through activation of AMPK signaling. Collectively, these findings suggest that SNG may represent a therapeutic candidate for the treatment of IPF.
Precision oncology has evolved from single-gene biomarker testing toward multimodal molecular and clinical profiling; however, most therapeutic decisions remain based on static baseline assessments that inadequately capture tumor evolution, treatment response, and emerging resistance. In this perspective, we propose a forward-looking framework that integrates federated learning, pharmacogenomic digital twins, and hybrid quantum-classical optimization to support the development of adaptive, privacy-preserving precision oncology systems. The framework enables collaborative model training across institutions without sharing raw patient data, thereby addressing major barriers associated with data fragmentation and privacy regulations. Patient-specific digital twins serve as continuously evolving computational representations that integrate longitudinal multiomics, imaging, pathology, and clinical information to simulate disease trajectories, estimate therapeutic response, and anticipate resistance patterns. Federated learning allows these models to benefit from geographically distributed patient cohorts while maintaining data sovereignty and institutional privacy. In contrast to near-term deployable components such as federated learning and digital twin modeling, quantum computing is presented as a future-oriented computational strategy that may assist selected combinatorial optimization tasks, including treatment selection, dose optimization, and scheduling, through hybrid quantum-classical workflows. Rather than assuming immediate clinical utility or quantum advantage, the framework emphasizes realistic translational pathways that acknowledge current limitations in quantum hardware, scalability, validation, and regulatory readiness. We further discuss technical feasibility, challenges associated with heterogeneous clinical data, privacy considerations, validation requirements, and regulatory pathways for adaptive AI-enabled clinical decision-support systems. By providing a formal conceptual architecture and translational roadmap, this perspective outlines how federated artificial intelligence, continuously learning digital twins, and future quantum-assisted optimization may collectively contribute to the next generation of adaptive precision oncology.
Cardiovascular events such as myocardial infarction (MI) show an early morning peak, suggesting circadian regulation of cardiac vulnerability. However, the mechanisms through which sleep loss may contribute to time-of-day myocardial risk remain unclear. We examined whether insomnia-associated circadian and thrombotic alterations are associated with morning cardiac vulnerability. Rats were subjected to pharmacologically induced insomnia using para-chlorophenylalanine (PCPA), isoproterenol-induced myocardial stress, and their combination. Temporal profiling across zeitgeber times (ZTs) assessed locomotor activity, biochemical markers (melatonin, serotonin, and cardiac troponin I), cardiac electrophysiology, and coagulation parameters. Integrative transcriptomic analysis was performed using human insomnia and MI data sets. Experimentally, insomnia and myocardial stress altered rest-activity, temporal electrophysiology, biochemical, and coagulation circadian profiles, and the combined condition showed the most pronounced alterations. These included changes in cardiac conduction and repolarization indices, altered melatonin rhythmicity, altered serotonin levels, elevated troponin I, reduced clotting time, and increased platelet aggregation, predominantly during ZT6-ZT12, corresponding to the light-dark transition, analogous to the human early morning phase. PCPA effects were restricted to the light phase, whereas ISO-containing groups showed sustained alterations. Integrative transcriptomic analysis identified a candidate circadian-thrombotic molecular signature involving CRY1, F2R, LEP, SSTR2, and IL7R, with mRNA profiling in rats showing altered Cry1 expression in heart and Lep expression in both SCN and the heart, suggesting altered SCN-heart coordination under combined insomnia and MI. These findings suggest a shift toward a pro-thrombotic cardiac profile during the early day window with insomnia-associated circadian alterations, supporting future investigation of circadian-informed chronotherapeutic approaches for morning cardiac vulnerability.
Zuotin-related factor 1 (ZRF1) is an essential chromatin-associated regulator with established roles in transcription, differentiation, and cancer; however, its contribution to paracrine signaling within the tumor microenvironment (TME) remains unexplored. Here, we identify ZRF1 as a regulator of nutrient-sensitive tumor-derived paracrine signaling in triple-negative breast cancer (TNBC). Across multiple breast cancer subtypes, ZRF1 depletion altered the expression, secretion, and stress-associated protein patterns of the inflammatory mediator S100A9. This regulation was influenced by metabolic status, where nutrient deprivation selectively destabilized specific S100A9 proteoforms through proteasome-sensitive mechanisms. Bioinformatic analyses revealed a strong association between S100A9 expression and neutrophil infiltration in breast tumors, prompting functional investigation of ZRF1-mediated tumor-neutrophil communication using a multilayered experimental framework. Conditioned medium from ZRF1-deficient tumor cells induced a nonbinary neutrophil activation spectrum rather than a fixed polarization state. This phenotypic remodeling was characterized by altered inflammatory signaling and cytokine (IL-8) dynamics in both differentiated HL-60 neutrophil-like cells and primary human neutrophils. Functionally, ZRF1-dependent paracrine cues regulated neutrophil transendothelial migration over time. Reciprocal coculture platforms and real-time xCELLigence assays further demonstrated that ZRF1 loss sensitizes tumor cells to neutrophil-derived signals, enhancing invasion and driving state-dependent architectural remodeling in 3D tumor spheroids. Collectively, these findings support a model in which a nutrient-sensitive ZRF1-dependent secretory network involving S100A9 contributes to human neutrophil plasticity under metabolic stress. Consequently, this study highlights S100A9 as one component of a broader ZRF1-regulated secretory network and identifies nutrient-sensitive tumor-immune communication as a potential therapeutic vulnerability in aggressive breast cancer.
This paper discusses how targeted pharmacologic experiments can elucidate the mechanism of allosteric receptor inhibitors (NAMs) to identify NAMs that may have exceptionally useful properties and target residence times in vivo. Specifically, experiments can be done to identify positive allosteric modulator (PAM) antagonists, NAMs that increase the affinity of the receptor for the agonist but decrease agonist efficacy. These molecules would be predicted to have long receptor offset times in vivo (in the presence of ambient concentrations of agonist) and favorable receptor residence times for therapy and also selectively target agonist prebound receptors. Flux analysis is used to demonstrate the salient properties of PAM antagonists and their favorable pharmacodynamic effects for blocking physiological signals.
The effect of flexible versus conformationally constrained phosphonate internucleotide linkages on CpG-mediated TLR9 activation was investigated. Sixteen analogues of the CpG oligonucleotide ODN 2006, a well-known TLR9 agonist, uniformly modified at all CpG motifs, were synthesized and evaluated in a human TLR9 (hTLR9) reporter cell line and peripheral blood mononuclear cells. Analogues bearing conformationally constrained 2',3'-cytidine phosphonate modifications (ODN1-ODN10) generally exhibited enhanced activity relative to ODN 2006, with ODN1 (incorporating modification I) displaying ∼30-fold increased potency. Analogues with flexible phosphonate linkages (ODN11, ODN13, ODN15, ODN16) showed comparable activity to that of the parent ODN, whereas additional sugar modification abolished activity (ODN12, ODN14). A hexamer (ODN-B) incorporating the most potent modification I, when combined with CpG DNA, enhanced hTLR9 activation, consistent with interaction at the auxiliary 5'-xCx site. This study evaluates phosphonate internucleotide linkages in CpG oligonucleotides and demonstrates that a conformationally constrained phosphonate backbone in ODN 2006 analogues enhances hTLR9 activation, offering a potential strategy for the development of potent immunostimulatory oligonucleotides.
Cancer cachexia is frequently associated with altered pharmacokinetics and increased chemotherapy toxicity due to the downregulation of cytochrome P450 (CYP) enzymes. However, the molecular mechanisms driving this broad metabolic suppression remain poorly understood. This study investigated whether tumor-derived parathyroid hormone-related protein (PTHrP) is associated with, and may contribute to, suppression of multiple CYP families. In a rat cachexia model, protein expression of CYP3A, CYP1A, CYP2C, CYP2D, and CYP2E1 was significantly downregulated in both the liver and small intestine. Consistent with these changes, pharmacokinetic analyses using a CYP substrate cocktail demonstrated markedly increased AUC and reduced clearance for probe drugs. In vitro experiments showed that PTHrP treatment reduced these CYP isoforms in primary rat hepatocytes. In human data sets, analysis of The Cancer Genome Atlas (TCGA) hepatocellular carcinoma (HCC) data set revealed a significant negative correlation between PTHrP and CYP gene expression, together with enrichment of NF-κB-related transcriptional programs. Furthermore, multimodal analysis using single-cell RNA sequencing and spatial transcriptomics demonstrated that PTHrP-high tumor regions exhibit suppressed xenobiotic metabolism. Additionally, in breast cancer liver metastases, high tumor PTHrP expression correlated with reduced CYP expression in surrounding nontumor hepatocytes, consistent with a possible paracrine relationship. Collectively, these results support an association between tumor-derived PTHrP and suppression of drug-metabolizing programs. They further suggest that PTHrP may be one contributing factor, but not definitive proof of a principal suppressor, and should therefore be considered a candidate biomarker requiring further mechanistic and clinical validation.
