Competitive Inhibitor Research Articles

Substrate recognition principles for the PP2A-B55 protein phosphatase.

The PP2A-B55 phosphatase regulates a plethora of signaling pathways throughout eukaryotes. How PP2A-B55 selects its substrates presents a severe knowledge gap. By integrating AlphaFold modeling with comprehensive high-resolution mutational scanning, we show that α helices in substrates bind B55 through an evolutionary conserved mechanism. Despite a large diversity in sequence and composition, these α helices share key amino acid determinants that engage discrete hydrophobic and electrostatic patches. Using deep learning protein design, we generate a specific and potent competitive peptide inhibitor of PP2A-B55 substrate interactions. With this inhibitor, we uncover that PP2A-B55 regulates the nuclear exosome targeting (NEXT) complex by binding to an α-helical recruitment module in the RNA binding protein 7 (RBM7), a component of the NEXT complex. Collectively, our findings provide a framework for the understanding and interrogation of PP2A-B55 function in health and disease.

Comprehensive stable-isotope tracing of glucose and amino acids identifies metabolic by-products and their sources in CHO cell culture

Mammalian cell culture processes are widely utilized for biotherapeutics production, disease diagnostics, and biosensors, and hence, should be optimized to support robust cell growth and viability. However, toxic by-products accumulate in cultures due to inefficiencies in metabolic activities and nutrient utilization. In this study, we applied comprehensive 13C stable-isotope tracing of amino acids and glucose to two Immunoglobulin G (IgG) producing Chinese Hamster Ovary (CHO) cell lines to identify secreted by-products and trace their origins. CHO cells were cultured in media formulations missing a single amino acid or glucose supplemented with a 13C-tracer of the missing substrate, followed by gas chromatography-mass spectrometry (GC-MS) analysis to track labeled carbon flows and identify by-products. We tracked the sources of all secreted by-products and verified the identity of 45 by-products, majority of which were derived from glucose, leucine, isoleucine, valine, tyrosine, tryptophan, methionine, and phenylalanine. In addition to by-products identified previously, we identified several metabolites including 2-hydroxyisovaleric acid, 2-aminobutyric acid, L-alloisoleucine, ketoisoleucine, 2-hydroxy-3-methylvaleric acid, desmeninol, and 2-aminobutyric acid. When added to CHO cell cultures at different concentrations, certain metabolites inhibited cell growth while others including 2-hydroxy acids, surprisingly, reduced lactate accumulation. In vitro enzymatic analysis indicated that 2-hydroxy acids were metabolized by lactate dehydrogenase suggesting a possible mechanism for lowered lactate accumulation, e.g., competitive substrate inhibition. The 13C-labeling assisted metabolomics pipeline developed and the metabolites identified will serve as a springboard to reduce undesirable by-products accumulation and alleviate inefficient substrate utilization in mammalian cultures used for biomanufacturing and other applications through altered media formulations and pathway engineering strategies.

Xanthine oxidase inhibitors: Virtual screening and mechanism of inhibition studies

Xanthine oxidase (XO), which plays a key role in purine metabolism, is an important target enzyme for the prevention and treatment of hyperuricemia. Inhibitory activity against XO is a common criterion for the screening of compounds with potential anti-hyperuricemic activity. In this study, 22 XO inhibitors were used to construct a 3D-QSAR pharmacophore model. Subsequently, molecular docking and in vitro activity evaluations were used to identify strong XO inhibitors from a list of 2000 natural compounds. The interaction mechanisms of these compounds with XO were analyzed based on inhibition kinetics and multi-spectral analyses. The pharmacophore model was composed of three hydrogen bond receptors and a hydrophobic center. The screened compounds — Diosmetin, Fisetin, and Genistein — all showed good XO inhibitory activity, with IC50 values of 1.86 ± 0.11 μM, 5.83 ± 0.08 μM, and 7.56 ± 0.10 μM, respectively. Kinetic analysis, fluorescence quenching assays, and molecular docking experiments showed that Diosmetin, Fisetin, and Genistein docked near the same active site of XO, mainly affecting the microenvironment of tryptophan residues. These molecules showed static binding to XO via hydrogen bonds, hydrophobic interactions, and van der Waals forces. Diosmetin and Genistein were competitive inhibitors, whereas Fisetin was a mixed inhibitor. Infrared spectroscopy showed that Diosmetin, Fisetin, and Genistein increased the α-helix content of XO from 7.4 % to 16.6 %, 21.4 %, and 11.2 %, respectively, thereby enhancing its stability. In summary, the pharmacophore model constructed in this study was accurate. The flavonoids Diosmetin, Fisetin, and Genistein effectively inhibited the activity of XO, and the amino acid residues LEU257, ILE353, and VAL259 played a key role in the interaction between the flavonoids and XO. These findings are of great significance for the screening and development of new XO inhibitors.

Characterising and preventing the gut microbiota's inactivation of trifluridine, a colorectal cancer drug

The gut microbiome can metabolise hundreds of drugs, potentially affecting their bioavailability and pharmacological effect. As most gut bacteria reside in the colon, drugs that reach the colon in significant proportions may be most impacted by microbiome metabolism. In this study the anti-colorectal cancer drug trifluridine was used as a model drug for characterising metabolism by the colonic microbiota, identifying correlations between bacterial species and individuals’ rates of microbiome drug inactivation, and developing strategies to prevent drug inactivation following targeted colonic delivery. High performance liquid chromatography and ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometry demonstrated trifluridine's variable and multi-route metabolism by the faecal microbiota sourced from six healthy humans. Here, four drug metabolites were linked to the microbiome for the first time. Metagenomic sequencing of the human microbiota samples revealed their composition, which facilitated prediction of individual donors’ microbial trifluridine inactivation. Notably, the abundance of Clostridium perfringens strongly correlated with the extent of trifluridine inactivation by microbiota samples after 2 hours (R2 = 0.8966). Finally, several strategies were trialled for the prevention of microbial trifluridine metabolism. It was shown that uridine, a safe and well-tolerated molecule, significantly reduced the microbiota's metabolism of trifluridine by acting as a competitive enzyme inhibitor. Further, uridine was found to provide prebiotic effects. The findings in this study greatly expand knowledge on trifluridine's interactions with the gut microbiome and provide valuable insights for investigating the microbiome metabolism of other drugs. The results demonstrate how protection strategies could enhance the colonic stability of microbiome-sensitive drugs.

Influence of micro- and nano-plastics on phenanthrene adsorption at the root-water interface

The adsorption of organic pollutants onto plant root surfaces is an initial crucial stage in the transfer of these pollutants from aqueous or soil solutions to plant systems. This study explored the potential influence of micro- and nano-plastics (MNPs) (0.1–100 μm) on the process of phenanthrene (Phe) adsorption at the root–water interface. The findings indicate that MNPs can exhibit either promotional or inhibitory effects on the root adsorption of organic pollutants, depending on both their particle size and content. Specifically, both the sorption capability of MNPs for Phe and their tendency to accumulate on or within root tissues increase with decreasing particle size. These dual effects facilitate the accumulation of nanoscale MNPs (0.1–6 μm in this work) within roots, which function as efficient carriers for Phe and increase Phe accumulation in root tissues. In contrast, MNPs with larger particle sizes (1–100 μm in this work) exhibit reduced affinity for roots and are more prone to remain in solution, thereby exerting more pronounced competitive inhibition against the adsorption of Phe by roots. However, when the concentration of nano-plastics in the rootwater system exceeded the retention capacity of the roots, the excess nano-plastics could compete with the roots for Phe, potentially reducing overall Phe sorption by the roots. These conditional effects of MNPs as either competitors or facilitators (vectors) in the root adsorption processes of organic pollutants may account for the different roles observed in previous studies where MNPs exhibited varying effects on the plant accumulation of organic pollutants.

Ergosterol Biosynthesis and Regulation Impact the Antifungal Resistance and Virulence of Candida spp.

Ergosterol is a key fungal sterol that is mainly found in the plasma membrane and is responsible for the proper membrane structure, rigidity, permeability and activity of membrane proteins. Ergosterol plays a crucial role in the ability of fungi to adapt to environmental stresses. The biosynthesis of ergosterol is also intimately connected with the antifungal resistance and virulence of pathogenic fungi. The most common etiological agents of life-threatening fungal infections are yeasts belonging to the genus Candida. The antifungal agents mostly used to treat Candida spp. infections are azoles, which act as competitive inhibitors of sterol demethylase, a key enzyme in the fungal ergosterol biosynthetic pathway. Although most studies on ergosterol biosynthesis, its regulation and the uptake of sterols are from the baker’s yeast Saccharomyces cerevisiae, the study of ergosterol biosynthesis and its relationship to antifungal drug resistance and virulence in pathogenic fungi is of utmost importance. The increasing antifungal drug resistance of Candida spp. and the limited armamentarium of antimycotics pose a challenge in the development of new therapeutic approaches. This review summarizes the available data on ergosterol biosynthesis and related phenomena in Candida albicans and non-albicans Candida species (Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida auris) with special emphasis on C. albicans and C. glabrata as the most common etiological agents of systemic candidiasis.

Overload of dissolved organic matter (DOM) in riparian infiltration zone increasing the pollution risk of naphthalene, insight from the competitive inhibition of naphthalene biodegradation by DOM.

Riparian infiltration zones are crucial for maintaining water quality by reducing the aqueous concentrations of polycyclic aromatic hydrocarbons (PAHs) through adsorption and biodegradation within the aquatic ecosystem. Dissolved organic matter (DOM) are ubiquitous in riparian infiltration zones where they extensively engage in the adsorption and biodegradation of PAHs, thereby influencing PAHs natural attenuation potential within riparian infiltration zones. Few studies have explored the natural attenuation mechanisms of PAHs influenced by DOM in riparian infiltration zones. In this study, the natural attenuation mechanisms of naphthalene (a typical PAHs component), under the influence of DOM, were explored, based on a case riverside source area. Analysis of microbial community structures, and the electron acceptor (e.g., Fe(III), DO/NO3-, SO42-)/electron donor (naphthalene and DOM) concentration changes within the riparian infiltration zone revealed a competitive inhibition relationship between DOM and naphthalene during microbial metabolism. Biodegradation experiments showed that when the concentration of DOM is higher than 4.0 mg·L-1, it inhibits the biodegradation of naphthalene. DOM competitively inhibits the biodegradation of naphthalene through the following mechanisms: (i) triggering microbial antioxidative defense mechanisms, diminishing the available resources for microbial participation in naphthalene degradation; (ii) altering microbial community structure; (iii) modulating microbial EPS composition, reducing the efficiency of microorganisms in utilizing carbon sources; and (iv) inhibiting the expression levels of downstream genes involved in naphthalene degradation. The competitive inhibition constants of DOM with concentrations of 1.0, 2.0, 4.0, 8.0, and 16.0 mg·L-1 on naphthalene biodegradation are -2.0 × 10-3, -5.0 × 10-3,1.0 × 10-3, 4.0 × 10-4, and 1.0 × 10-4, respectively. These findings enhance understanding of PAHs attenuation in riparian infiltration zone, providing a basis for assessing and managing PAHs pollution risks during riparian extraction.

RRFERV stabilizes TEAD1 expression to mediate nasopharyngeal cancer radiation resistance rendering tumor cells vulnerable to ferroptosis.

Long noncoding RNAs (lncRNAs) regulate various essential biological processes, including cell proliferation, differentiation, apoptosis, migration, and invasion. However, in nasopharyngeal carcinoma (NPC), the clinical significance and mechanisms of lncRNAs in malignant progression are unknown. RNA sequencing and bioinformatic analysis were used to determine the potential function of RRFERV (radiation-resistant but ferroptosis vulnerable), and its biological effects were investigated using in vitro and in vivo experiments. Western blotting, quantitative real-time reverse transcription PCR, RNA immunoprecipitation (RIP) assays, and flow cytometry detected RRFERV expression. Ferroptosis and lipid peroxidation were added to evaluate the relationship between it and radiotherapy resistance. LncRNA-RRFERV was both highly expressed in NPC tissues and radiation-resistant cells. RRFERV is associated with poor clinical outcomes of NPC patients and stabilizes TEAD1 by competitive binding with microRNA-615-5p and microRNA-1293. RRFERV-TEAD1 signaling axis leads to malignant progression and radiotherapy resistance of NPC. Furthermore, we observed that NPC radiotherapy resistance cells exist in a fragile oxidative stress equilibrium, which makes them more sensitive to ferroptosis inducers. Surprisingly, we found that RRFERV-TEAD1 signaling axis also plays a key role in mediating the lipid peroxidation levels of NPC radiotherapy resistance cells through transcriptional activation of ACSL4/TFRC. RRFERV serves as an independent prognostic factor in NPC. During the malignant progression of NPC caused by high expression of RRFERV, ferroptosis can be induced to effectively kill cancer cells and reverse the radiotherapy resistance of NPC cells, suggesting a potential treatment approach for recurrent and refractory NPC.

Investigation of the anti-inflammatory, anti-pruritic, and analgesic effects of sophocarpine inhibiting TRP channels in a mouse model of inflammatory itch and pain

Ethnopharmacological relevanceSophocarpine is a bioactive compound extracted from the dried root of Sophorae Flavesentis Aiton, a plant that has been used for thousands of years for various conditions including skin itch and pain. Its antipruritic and analgesic effects are suggested in publications, while the molecular mechanisms underneath interacting with TRP channels are not understood. Aim of the studyWe investigated the anti-inflammatory, antipruritic, and analgesic effects of sophocarpine in a murine inflammatory itch and pain model to elucidate the underlying mechanisms. Materials and methodsWe evaluated sophocarpine's anti-pruritic and analgesic effects by monitoring mice's scratching and wiping behaviors, and the anti-inflammatory effect by measuring psoriasis area and severity index (PASI) score. The mRNA and protein expression of TRPA1/TRPV1 was analyzed by real-time quantitative polymerase chain reaction and western blotting. We further investigated the relationship between sophocarpine and TRPA1/TRPV1 in mice administered allyl-isothiocyanate (AITC) or capsaicin and by molecular docking. ResultsWe found that sophocarpine decreased scratching bouts, wipes, and the PASI score, reduced the TNF-α and IL-1β in the skin and TRPA1 and TRPV1 in the trigeminal ganglion. Pretreatment of sophocarpine decreased AITC-induced scratching bouts and wipes and capsaicin-induced wipes. We also found potential competitive bindings between sophocarpine and AITC/capsaicin to TRPA1/TRPV1. ConclusionsSophocarpine is a potential competitive inhibitor of TRPA1 and TRPV1 channels eliciting strong anti-inflammatory, anti-pruritic, and analgesic effects, suggesting its significant therapeutic potential in treating diseases with inflammatory itch and pain.

Real-time live imaging of cytosolic hydrogen peroxide and Ca2+ of Marchantia polymorpha gemmalings reveal immediate initial responses of plant cells triggered by nonthermal plasma irradiation.

Cold atmospheric pressure plasma generators capable of generating plasma under normal pressure and temperature conditions have recently been developed, and their biological applications have been extensively studied. Plasma irradiation has been reported to affect plant germination and growth; however, the molecular mechanism underlying these effects and initial cellular responses to plasma irradiation remains poorly understood. To unravel the molecular and cellular mechanisms underlying the effects of plasma irradiation on plants, we have been establishing novel experimental systems using a model liverwort Marchantia polymorpha. We here focused on the initial responses of plant cells to plasma irradiation. To investigate immediate cellular responses following plasma irradiation, we developed a new plasma device that allows irradiation under a microscope. Through integration with live fluorescence imaging, we established an experimental setup to track, the dynamics of intracellular concentration of H2O2 and Ca2+ as representative initial cellular responses. We revealed that plasma irradiation induced a rapid and transient increase in intracellular concentration of H2O2 and Ca2+ in Marchantia gemmalings. Pharmacological analyses suggested that the long-lived reactive species, H2O2, generated by the plasma generator was directly delivered into the plant cells. Competitive inhibitors of Ca2+ channels abolished the Ca2+ rise, suggesting that plasma irradiation immediately activate plasma membrane Ca2+ channel(s) to induce Ca2+ influx. Importantly, this study marks the inaugural demonstration of real-time monitoring of cytosolic H2O2 and Ca2+ dynamics in plants, triggered by plasma irradiation.

Probe substrates assay estimates the effect of polyphyllin H on the activity of cytochrome P450 enzymes in human liver microsomes.

Cytochrome P450 enzymes (CYPs) play a crucial role in phase I metabolic reactions. The activity of CYPs would affect therapeutic efficacy and may even induce toxicity. Given the complex components of traditional Chinese medicine, it is important to understand the effect of active ingredients on CYPs activity to guide their prescription. This study aimed to evaluate the effect of polyphyllin H on the activity of CYPs major isoforms providing a reference for the clinical prescription of polyphyllin H and its source herbs. The effects of polyphyllin H were evaluated in pooled human liver microsomes using probe substrates of CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 to determine their activities. The Lineweaver-Burk was used to model the inhibition, and a time-dependent inhibition experiment was performed to understand the characteristics of the inhibition. Polyphyllin H significantly suppressed the activity of CYP1A2, 2D6, and 3A4 with IC50 values of 6.44, 13.88, and 4.52 μM, respectively. The inhibition of CYP1A2 and 2D6 was best fitted with a competitive model, yielding the inhibition constant (Ki) values of 3.18 and 6.77 μM, respectively. The inhibition of CYP3A4 was fitted with the non-competitive model with the Ki value of 2.38 μM. Moreover, the inhibition of CYP3A4 was revealed to be time-dependent with the inhibition parameters inhibition constant (KI) and inactivation rate constant (Kinact) values of 2.26 μM-1 and 0.045 min-1. Polyphyllin H acted as a competitive inhibitor of CYP1A2 and 2D6 and a non-competitive and time-dependent inhibitor of CYP3A4.

Valtrate Suppresses TNFSF14-Mediated Arrhythmia After Myocardial Ischemia-Reperfusion by Inducing N-linked Glycosylation of LTβR to Regulate MGA/MAX/c-Myc/Cx43.

Myocardial ischemia-reperfusion (MIR)-induced arrhythmia remains a major cause of death in patients with cardiovascular diseases. The reduction of Cx43 has been known as a major inducer of arrhythmias after MIR, but the reason for the reduction of Cx43 remains largely unknown. The aim of this study was to find the key mechanism underlying the reduction of Cx43 after MIR and to screen out an herbal extract to attenuate arrhythmia after MIR. The differentially expressed genes in the peripheral blood mononuclear cell (PBMCs) after MIR were analyzed using the data from several gene expression omnibus data sets, followed by the identification in PBMCs and the serum of patients with myocardial infarction. Tumor necrosis factor superfamily protein 14 (TNFSF14) was increased in PBMCs and the serum of patients, which might be associated with the injury after MIR. The toxic effects of TNFSF14 on cardiomyocytes were investigated in vitro . Valtrate was screened out from several herbal extracts. Its protection against TNFSF14-induced injury was evaluated in cardiomyocytes and animal models with MIR. Recombinant TNFSF14 protein not only suppressed the viability of cardiomyocytes but also decreased Cx43 by stimulating the receptor LTβR. LTβR induces the competitive binding of MAX to MGA rather than the transcriptional factor c-Myc, thereby suppressing c-Myc-mediated transcription of Cx43. Valtrate promoted the N-linked glycosylation modification of LTβR, which reversed TNFSF14-induced reduction of Cx43 and attenuated arrhythmia after MIR. In all, valtrate suppresses TNFSF14-induced reduction of Cx43, thereby attenuating arrhythmia after MIR.

BPP43_05035 is a Brachyspira pilosicoli cell surface adhesin that weakens the integrity of the epithelial barrier during infection

ABSTRACT The anaerobic spirochete Brachyspira causes intestinal spirochetosis, characterized by the intimate attachment of bacterial cells to the colonic mucosa, potentially leading to symptoms such as diarrhea, abdominal pain, and weight loss. Despite the clinical significance of Brachyspira infections, the mechanism of the interaction between Brachyspira and the colon epithelium is not known. We characterized the molecular mechanism of the B. pilosicoli-epithelium interaction and its impact on the epithelial barrier during infection. Through a proteomics approach, we identified BPP43_05035 as a candidate B. pilosicoli surface protein that mediates bacterial attachment to cultured human colonic epithelial cells. The crystal structure of BPP43_05035 revealed a globular lipoprotein with a six-bladed beta-propeller domain. Blocking the native BPP43_05035 on B. pilosicoli, either with a specific antibody or via competitive inhibition, abrogated its binding to epithelial cells, which required cell surface-exposed N-glycans. Proximity labeling and interaction assays revealed that BPP43_05035 bound to tight junctions, thereby increasing the permeability of the epithelial monolayer. Extending our investigation to humans, we discovered a downregulation of tight junction and brush border genes in B. pilosicoli-infected patients carrying detectable levels of epithelium-bound BPP43_05035. Collectively, our findings identify BPP43_05035 as a B. pilosicoli adhesin that weakens the colonic epithelial barrier during infection.

Discovery and Functional Characterization of a Potent, Selective, and Metabolically Stable PROTAC of the Protein Kinases DYRK1A and DYRK1B.

Small-molecule-induced protein degradation has emerged as a promising pharmacological modality for inactivating disease-relevant protein kinases. DYRK1A and DYRK1B are closely related protein kinases that are involved in pathological processes such as neurodegeneration, cancer development, and adaptive immune homeostasis. Herein, we report the development of the first DYRK1 proteolysis targeting chimeras (PROTACs) that combine a new ATP-competitive DYRK1 inhibitor with ligands for the E3 ubiquitin ligase component cereblon (CRBN) to induce ubiquitination and subsequent proteasomal degradation of DYRK1A and DYRK1B. The lead compound (DYR684) promoted fast, efficient, potent, and selective degradation of DYRK1A in cell-based assays. Interestingly, an enzymatically inactive splicing variant of DYRK1B (p65) resisted degradation. Compared to competitive kinase inhibition, targeted degradation of DYRK1 by DYR684 provided improved suppression of downstream signaling. Collectively, our results identify DYRKs as viable targets for PROTAC-mediated degradation and qualify DYR684 as a useful chemical probe for DYRK1A and DYRK1B.

Biochemical Amenability in Fabry Patients Under Chaperone Therapy-How and When to Test?

Current recommendations for Fabry disease include α-galactosidase A (AGAL) activity measurements to assess the biochemical response in migalastat-treated patients. Owing to contradictory data from laboratories, we aimed to analyze why AGAL activity measures from dried blood spots (DBS) often fail to detect migalastat-mediated enzymatic activity increases in treated patients. 43 patients with 58 visits under migalastat were consecutively recruited. Enzymatic AGAL activities were measured from DBS and peripheral blood mononuclear cells (PBMCs). Migalastat concentrations in sera were determined using modified serum-mediated inhibition assays to assess Cmax and serum half-life. Results were set in relation to the time of last migalastat intake and blood sampling to assess an optimal timepoint for AGAL activity measures. DBS-based AGAL activity measurements of 21 (42.0%) amenable patients were below the limit of detection. Serum samples from migalastat-treated patients showed significant AGAL inhibition, depending on the time between migalastat intake and blood sampling (r2 = 0.8140, p < 0.0001). Migalastat concentrations were determined in serum samples confirming a Cmax at 3 h and a serum half-life of 4 h. At 24 h after intake, migalastat clearance was significantly associated with renal function (r2 = 0.3135, p = 0.0102). Enzymatic AGAL activities were higher in samples from DBS and PBMCs 24 h after migalastat intake (both p < 0.05). The optimal time for enzymatic AGAL activity measurement in migalastat-treated patients appears to be 24 h after the last migalastat intake. Since migalastat is a competitive inhibitor of AGAL, enzymatic AGAL activity measurements should be better performed from PBMCs to reduce migalastat-mediated interferences.

Targeting DNM1L/DRP1-FIS1 axis inhibits high-grade glioma progression by impeding mitochondrial respiratory cristae remodeling.

The dynamics of mitochondrial respiratory cristae (MRC) and its impact on oxidative phosphorylation (OXPHOS) play a crucial role in driving the progression of high-grade glioma (HGG). However, the underlying mechanism remains unclear. In the present study, we employed machine learning-based transmission electron microscopy analysis of 7141 mitochondria from 54 resected glioma patients. Additionally, we conducted bioinformatics analysis and multiplex immunohistochemical (mIHC) staining of clinical glioma microarrays to identify key molecules involved in glioma. Subsequently, we modulated the expression levels of mitochondrial dynamic-1-like protein (DNM1L/DRP1), and its two receptors, mitochondrial fission protein 1 (FIS1) and mitochondrial fission factor (MFF), via lentiviral transfection to further investigate the central role of these molecules in the dynamics of glioblastoma (GBM) cells and glioma stem cells (GSCs). We then evaluated the potential impact of DNM1L/DRP1, FIS1, and MFF on the proliferation and progression of GBM cells and GSCs using a combination of CCK-8 assay, Transwell assay, Wound Healing assay, tumor spheroid formation assay and cell derived xenograft assay employing NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt (NCG) mouse model. Subsequently, we validated the ability of the DNM1L/DRP1-FIS1 axis to remodel MRC structure through mitophagy by utilizing Seahorse XF analysis technology, mitochondrial function detection, MRC abundance detection and monitoring dynamic changes in mitophagy. Our findings revealed that compared to low-grade glioma (LGG), HGG exhibited more integrated MRC structures. Further research revealed that DNM1L/DRP1, FIS1, and MFF played pivotal roles in governing mitochondrial fission and remodeling MRC in HGG. The subsequent validation demonstrated that DNM1L/DRP1 exerts a positive regulatory effect on FIS1, whereas the interaction between MFF and FIS1 demonstrates a competitive inhibition relationship. The down-regulation of the DNM1L/DRP1-FIS1 axis significantly impaired mitophagy, thereby hindering the remodeling of MRC and inhibiting OXPHOS function in glioma, ultimately leading to the inhibition of its aggressive progression. In contrast, MFF exerts a contrasting effect on MRC integrity, OXPHOS activity, and glioma progression. This study highlights that the DNM1L/DRP1-FIS1 axis stabilizes MRC structures through mitophagy in HGG cells while driving their OXPHOS activity ultimately leading to robust disease progression. The inhibition of the DNM1L/DRP1-FIS1 axis hinders MRC remodeling and suppresses GBM progression. We propose that down-regulation of the DNM1L/DRP1-FIS1 axis could be a potential therapeutic strategy for treating HGG.

Lipase Enzyme Kinetics with the Addition of Avocado Seed Extract as an Inhibitor in Olive Oil

Free fatty acids are compounds resulting from the hydrolysis of triglycerides catalyzed by the enzyme lipase. Free fatty acids harm health if they enter the body in large amounts. Avocado seed extract can reduce free fatty acid levels by inhibiting lipase activity. This research is a type of true-experimental research that aims to determine the effect of variations in substrate concentration on lipase enzyme activity with the addition of avocado seed extract and to determine the impact of the addition of avocado seed extract as an inhibitor on lipase enzyme kinetics. Lipase activity was determined based on the amount of oleic acid formed using Cu acetate pyridine reagent by Uv-Vis spectrophotometry at a wavelength of 715 nm. Lipase enzyme kinetics were determined using the Lineweaver-Burk curve equation, a transformed form of the Michaelis-Menten equation. The results showed that 60% substrate concentration was the most effective in reducing lipase enzyme activity by 60%. In addition, the addition of avocado seed extract proved to act as a competitive inhibitor, with the KM value decreasing from 0.014 M to 0.004 M and Vmaks remaining relatively stable at 23.2 µM. This shows that avocado seed extract effectively reduces lipase enzyme activity, which has implications for reducing FFA levels in olive oil. Thus, this study concludes that avocado seed extract has great potential as a natural inhibitor agent in reducing lipase enzyme activity.

A weekly 4-methylpyrazole treatment attenuates the development of non-obese metabolic dysfunction-associated steatotic liver disease (MASLD) in male mice: Role of JNK.

4-methylpyrazole (4MP, fomepizole) is a competitive inhibitor of alcohol dehydrogenase (ADH) preventing the metabolism of ethylene glycol and methanol, respectively, into their toxic metabolites. 4MP seems also to possess a potential in the treatment of intoxication from other substance, for example, acetaminophen, and to modulate JNK-dependent signalling. Here, we determined if a treatment with 4MP once weekly affects the development of diet-induced non-obese metabolic dysfunction-associated steatotic liver disease (MASLD) in C57BL/6 mice. Male C57BL/6 mice (6-8 weeks old, n = 7-8/group) were pair-fed either a liquid control diet (C) or a liquid sucrose-, fat- and cholesterol-rich diet (SFC) for 8 weeks while being concomitantly treated with 4MP (50 mg/kg bw i.p.) or vehicle once a week. Liver damage, inflammatory markers and glucose tolerance were assessed. Moreover, in endotoxin-challenged J774A.1 cells pretreated with 4MP, pro-inflammatory markers were assessed. The concomitant treatment of SFC-fed mice with 4MP attenuated the increase in JNK phosphorylation and pro-inflammatory markers like IFNγ, IL-6 and 3-nitrotyrosine protein adducts in liver tissue found in vehicle-treated SFC-fed mice, while not affecting impairments of glucose tolerance or the increase in portal endotoxin levels. Moreover, a pretreatment of endotoxin-stimulated J774A.1 cells with 4MP significantly attenuated the increases in JNK phosphorylation and pro-inflammatory mediators like IL-6 and Mcp1. Taken together, our results suggest that a treatment with 4MP once weekly attenuates the activation of JNK and dampens the development of non-obese MASLD in mice.

Nucleic acid binding affinity and antioxidant activity of N-m-Tolyl-4-Chlorophenoxyacetohydroxamicacid.

Hydroxamic acids represent a group of weak organic acids, both naturally occurring and synthetically derived, characterized by the general formula RC(= O)N(R'OH). In this study, we investigated the binding behavior of N-m-tolyl-4-chlorophenoxyaceto hydroxamic acid with calf thymus DNA (ct-DNA) and torula yeast RNA (t-RNA) through a combination of techniques including UV-visible spectroscopy, fluorescence emission analysis, viscometry, and computational simulations using AutoDock4 software. Our findings reveal that the mode of binding between the compound and the nucleic acids is consistent with intercalation. Competitive binding experiments demonstrated that the complex competes effectively with ethidium bromide (EB) for binding to ct-DNA/t-RNA, displacing EB from its binding sites. Additionally, the introduction of the compound into the DNA-EB system resulted in a quenching of fluorescence emission peaks. Analysis of absorption spectra indicated a red shift and hypochromic shift when the compound interacted with DNA, further supporting the intercalative binding mode. The calculated binding constant (Kb) value for the compound is 6.62 × 104M-1 and 5.40 × 103M-1 indicating a strong interaction with ct-DNA and t-RNA respectively. We determined the Stern-Volmer constants for ct-DNA and t-RNA as 9.96 × 104M-1 and 8.13 × 105M-1, respectively. The binding free energy values for ct-DNA/t-RNA were calculated to be -3.741 × 107 and -5.425 × 108kcal/mol, respectively. Viscometric studies corroborated the UV results, showing a continuous increase in relative viscosity of ct-DNA/t-RNA solutions with the addition of the optimal hydroxamic acid concentration. Furthermore, we assessed the antioxidant activity of the compound using DPPH-radical scavenging and β-carotene linoleic acid assays. Gel electrophoresis results demonstrated the compound's remarkable efficacy in preventing DNA damage. Collectively, all experimental evidence supports the conclusion that N-m-tolyl-4-chlorophenoxyaceto hydroxamic acid binds to ct-DNA/t-RNA through an intercalative mechanism, which is consistent with our molecular docking simulations.

Synthesis and biological assessment of novel 4H-chromene-3-carbonitrile derivatives as tyrosinase inhibitors.

Excessive activity of the tyrosinase enzyme during melanogenesis results in hyperpigmentation in the skin. To address this issue, there is a need to develop effective tyrosinase inhibitors as a treatment for hyperpigmentation. In this study, we synthesized some novel 4H-chromene-3-carbonitrile compounds (6a-o) and assessed their inhibitory activities against tyrosinase, comparing them with kojic acid, which is known as a positive control. Compound 6f emerged as the most effective inhibitor, with an IC50 of 35.38 ± 2.12 µM. Kinetic studies of 6f exhibited competitive inhibition, with Ki = 16.15 µM. Molecular docking studies highlighted the importance of π-π stacking and hydrogen bonding interactions within the binding site. Molecular dynamics simulations showed that the R-enantiomer 6f exhibited superior binding stability compared to the S-enantiomer, with a lower standard deviation of RMSD and more persistent interactions with the key active site residues. These findings underscore the potential of the R-enantiomer of compound 6f as a potent tyrosinase inhibitor and provide insights for developing effective treatments for hyperpigmentation and related skin conditions.