Competitive Inhibitor Research Articles

Odontogenic differentiation of dental pulp stem cells by glycogen synthase kinase-3β inhibitory peptides

BackgroundTo investigate the effects of peptide-based substrate competitive inhibitors of GSK-3β (GSK-3βi) on promoting odontogenic differentiation of human dental pulp stem cells (hDPSCs).MethodsThe biocompatibility and proliferation of hDPSCs treated with GSK-3βi peptides (pS9, LRP 6a, L803, and L803-mts) were evaluated using the tetrazolium reduction assay and cell counting kit-8 assay, respectively. The differentiation of hDPSCs following peptide treatment was determined using the alkaline phosphatase assay (ALP), calcium mineralization (alizarin red staining), and quantification of mRNA expression of differentiation markers via quantitative real-time polymerase chain reaction. The accumulation of β-catenin in the nucleus of GSK3-βi-treated hDPSCs was determined using immunofluorescence staining. The effect of peptide treatment on hDPSC migration was characterized using the transwell assay.ResultsAll tested concentrations of the peptides were found to be biocompatible with the hDPSCs, with no significant difference compared to the control (p > 0.05). The peptides had no effect on the proliferation of hDPSCs compared to the control (p > 0.05). However, all the tested peptides significantly increased ALP activity and calcium deposition in a dose-dependent manner (p < 0.05). Specifically, L803-mts showed significantly greater ALP activity and mineralization compared to the other peptides and the controls (p < 0.05). Additionally, L803-mts showed a significant increase (p < 0.05) in the expression of DSPP, DMP-1, Runx-2, along with increased protein expression of DSPP and DMP-1 compared to the control. Furthermore, it enhanced the nuclear translocation of β-catenin and increased the chemotactic migratory potential of hDPSCs.ConclusionsL803-mts, a peptide-based substrate competitive inhibitor of GSK-3β, enhanced the odontogenic differentiation of hDPSCs by activating the Wnt signaling pathway.

Corticosterone rapidly modulates dorsomedial hypothalamus serotonin and behavior in an estrogen- and progesterone-dependent manner in adult female rats: potential role of organic cation transporter 3 (OCT3).

Previous studies have shown that corticosterone rapidly alters extracellular serotonin (5-hydroxytryptamine; 5-HT) concentrations in the dorsomedial hypothalamus (DMH) of adult male rats, suggesting a role for corticosterone actions in the DMH in regulation of physiological and behavioral responses. Whether or not corticosterone also rapidly alters extracellular serotonin concentrations in the DMH of female rats, and the dependence of this effect on ovarian hormones, is not known. To determine the effects of 17β-estradiol (E2), progesterone (P), and corticosterone on extracellular concentrations of serotonin in the DMH, corticosterone and/or P were delivered into the DMH of ovariectomized rats via reverse microdialysis in E2-primed rats. Combined, but not separate, delivery of corticosterone and P into the DMH rapidly and transiently increased extracellular 5-HT concentrations, a result that was dependent upon circulating E2. This effect of corticosterone on DMH 5-HT was replicated by local perfusion of the organic cation transporter 3 (OCT3) competitive inhibitor normetanephrine. Intra-DMH infusions of either corticosterone or normetanephrine also reversibly suppressed lordosis responses in E2+P-primed females. These results suggest that ovarian hormones in combination with corticosterone modulate OCT3-mediated 5-HT clearance in the DMH, potentially representing an adaptive mechanism that allows sexually receptive females to respond rapidly to acute stressors.

Breakthrough Advances in Beta-Lactamase Inhibitors: New Synthesized Compounds and Mechanisms of Action Against Drug-Resistant Bacteria

Beta-lactam drugs hold a central place in the antibacterial arsenal, and the production of beta-lactamases by drug-resistant bacteria has severely compromised the effectiveness of nearly all available beta-lactams. Therefore, in the face of the increasing threat of drug resistance, the combined use of beta-lactamase inhibitors (BLIs) with beta-lactam antibiotics is crucial for treating infections caused by drug-resistant bacteria. Hence, the development of BLIs has always been a hot topic in the field of medicinal chemistry. In recent years, significant progress has been made in screening active drugs by enhancing the affinity of inhibitors for enzymes and the stability of their complexes, based on the design concept of competitive inhibitors. Here, we review the effects and mechanisms of newly synthesized beta-lactamase inhibitors on various BLIs in recent years, to provide ideas for the development of subsequent beta-lactamase inhibitors.

Overview of preclinical and phase II clinical studies on Pegmolesatide’s long-term erythropoiesis stimulating effect via EPOR-mediated signal transduction

IntroductionAnemia is a prevalent complication of chronic kidney disease (CKD), primarily due to insufficient erythropoietin (EPO). Pegmolesatide (by Hansoh Pharma) is currently the only marketed long-acting EPO mimetic peptide (EMP) for the treatment of anemia in both dialysis and non-dialysis CKD patients. This paper aimed to explore the long-acting erythropoiesis stimulating molecular mechanism of Pegmolesatide.MethodsIn vitro assays were utilized to assess Pegmolesatide erythropoietin receptor (EPOR) affinity, competitive binding, cell proliferation/survival, apoptosis, cell surface receptor expression, and signal transduction. Pharmacokinetics (PK) and Pharmacodynamics (PD) parameters were evaluated in BALB/c mice following single administration. Furthermore, two Phase II clinical trials in dialysis and non-dialysis chronic kidney disease (CKD) patients with anemia, respectively CTR20140533 and CTR20140539, assessed PK-PD and safety following repeated administration.ResultsIn vitro Pegmolesatide demonstrated enhanced binding stability and prolonged residency at EPOR, surpassing erythropoiesis-stimulating agents (ESAs) rHuEPO and Darbepoetin. This sustained EPOR binding facilitated heightened endogenous EPOR expression post-drug withdrawal, maintaining downstream signal transduction pathways (JAK2/STAT5, ERK1/2 MAPK) for erythropoiesis. Pegmolesatide promoted UT-7 cell proliferation & survival and suppressed apoptosis. Following a single 0.08 mg/kg dose of Pegmolesatide in BALB/c mice, reticulocyte count, red blood cells, hemoglobin, and hematocrit persisted at elevated levels 4-6 days after administration. In the two clinical Phase II studies dose-dependent increases in hemoglobin and prolonged response duration were independently observed. Pegmolesatide showed significant PK-PD dual prolongation effects and was well tolerated. Adverse events were mild and manageable, with no reports of severe anaphylaxis.DiscussionPreclinical and clinical evidence signifies that Pegmolesatide is a unique, potent PEGylated EPO-memetic peptide (EMP) with a prolonged PD efficacy and PK half-life and a good safety-tolerability profile. To elucidate further, future studies will address the endocytosis, intracellular degradation, and ligand release of EPOR subsequent to Pegmolesatide binding, thereby supplementing our understanding of the molecular mechanism at play.Trial registration: Phase IIa clinical study of Pegmolesatide on renal anemia, CTR20140533. Initial Public Notice 14 Jan 2015, http://www.chinadrugtrials.org.cn/clinicaltrials.searchlistdetail.dhtml. Phase II clinical study of Pegmolesatide on renal anemia, CTR20140539. Initial Public Notice 26 Jan 2015, http://www.chinadrugtrials.org.cn/clinicaltrials.searchlistdetail.dhtml.

Patient-specific therapeutic benefit of MuSK agonist antibody ARGX-119 in MuSK myasthenia gravis passive transfer models.

Muscle-specific kinase (MuSK) orchestrates the establishment and maintenance of neuromuscular synapses. Autoantibodies targeting MuSK cause myasthenia gravis (MG), a disease characterized by skeletal muscle weakness. MuSK autoantibodies are predominantly IgG4 which are bispecific, functionally monovalent antibodies that are antagonists of MuSK signaling. We hypothesized that bivalent MuSK agonist antibodies can rescue MuSK MG. Here, we investigated whether ARGX-119, a MuSK frizzled-like domain agonist antibody, can ameliorate disease in passive transfer models induced by polyclonal patient IgG4. ARGX-119 improved survival and muscle weakness in a mouse model induced by one patient material, but not by three others. Patient-specific efficacy could not be explained by titer or competition for ARGX-119 binding, but rather correlated with the presence of MuSK activating antibodies in some patients. This first proof of concept of a MuSK agonist in a clinically relevant MuSK MG model forms a starting point for therapeutic studies toward ARGX-119 efficacy in neuromuscular diseases.

Nanomolar Activity of Coumarin-3-thiosemicarbazones Targeting Trypanosoma cruzi Cruzain and T. brucei Cathepsin-L-like Protease

Trypanosoma cruzi (T. cruzi) and Trypanosoma brucei (T. brucei) urgently demand innovative drug development due to their impact on public health worldwide. Their cysteine proteases, Cruzain (CRZ) and T. brucei Cathepsin L-like protease (TbrCATL) are established drug targets for these parasites. In this study, our coumarin-3-thiosemicarbazones demonstrated nanomolar IC50 values (22-698 nM) toward these proteases. Against T. cruzi amastigotes and T. brucei trypomastigotes, LASF-01 displayed a promising result. Herein, MCG-02, the most potent TbrCATL inhibitor, underwent comprehensive analyses, including cytotoxicity assessments and in vitro tests. Molecular dynamics (MD) simulations and a multiscale Quantum Mechanics/Quantum Mechanics (QM/QM) approach were used to generate insights into their binding modes. These suggested that MCG-02 could be a reversible, competitive covalent inhibitor. Further, confirmatory assays were experimentally performed changing different parameters to prove its efficacy. Additionally, the predicted pharmacokinetic profile showed that there is no violation of the Lipinski rule of five. Notably, coumarin-3-thiosemicarbazone hybrids emerged as promising candidates for designing highly active inhibitors against CRZ and TbrCATL. Overall, the integration of in silico and experimental approaches enhanced our understanding regarding the binding modes of MCG-02, which were experimentally corroborated, providing valuable insights for future drug development.

Development of hot melt extruded Polycaprolactone (PCL) matrices for an oral ultra-long-lasting delivery of Galantamine for Alzheimer’s disease therapy

Galantamine Hydrobromide (GAL) is a tertiary alkaloid, acting as a competitive and reversible cholinesterase inhibitor. It is indicated for the symptomatic treatment of patients from mild to moderate dementia level of the Alzheimer’s disease (AD) type. For treatment of mild to moderate AD, GAL is generally combined with other drugs. The purpose of this work was development and characterization of hot melt extruded (HME) matrices based on polycaprolactone (PCL) for GAL oral ultra-long-lasting delivery. PCL is biodegradable polymer, biocompatibility, water-insoluble and excellent for HME process. From HME drug and polymer mixtures arms from 1 % up to 50 wt-% of GAL were obtained. The raw materials and the as-obtained HME arms were characterized by particle size distribution (PSD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), wide angle x-ray scattering (WAXS) and dissolution test. Our findings showed that GAL possesses crystalline form I polymorphic, it is thermodynamically stable, and remains their particle and crystalline form even after HME process. Crystallization temperature of PCL increases with GAL content, indicating strong heterogeneous nucleating effect of drug on polymer crystallization process. SEM morphological analysis indicates continuous polymeric phase on surface. However, higher amounts of GAL (50/50 wt-%) presented clusters and heterogeneity, especially in extruded cross-section. It may have influenced the drug release property, decreasing the sustained release ability, due to the transport of medium molecules through the bulk extrudates by GAL soluble clusters. The diffractograms (WAXS) showed patterns peaks of GAL and PCL, indicating that GAL remains its crystalline structure and PCL recrystallized. In the dissolution test, PCL/GAL 80/20 and 90/10 achieved 100% with over seven days and profiles that can be tuned by varying GAL concentration. Finally, we demonstrate the feasibility of using HME process based on the mixture of PCL and GAL as proposals for oral therapies for conditions of AD. This is the first report showing PCL-GAL polymer matrices that achieves several-days drug release.

Enhancing immunotherapy efficacy in oral cancer through AKB-9778-mediated vascular normalization.

Tumor vasculature exhibit numerous abnormal features distinct from those of healthy vessels, potentially advancing tumor development by establishing an aberrant microenvironment. Therefore, vascular normalization has proven to be an effective tactic for substantially enhancing treatment efficacy across multiple tumors. However, the methods to attain vascular normalization may vary among tumor types. VE-PTP, expressed exclusively in vascular endothelial cells (ECs) and acting as a critical suppressor of vascular maturity and functionality, is upregulated in oral cancer due to hypoxia. In this study, we explored the effect and mechanism of AKB-9778, a competitive inhibitor of VE-PTP, in promoting vascular normalization of oral cancer. Initially, we showed that AKB-9778 can slow down tumor progression by fostering vascular normalization in a murine OSCC model. This was evidenced by improvements in vessel density, pericyte coverage, local hypoxia, and vascular permeability. RNA sequencing additionally indicated that the ECs of tumor vasculature exhibit abnormal alterations in adhesion molecules, and AKB-9778 treatment might facilitate vascular normalization by modulating lipid metabolism pathways, especially HSD17B7-regulated steroid biosynthesis. AKB-9778 treatment significantly up-regulated the HSD17B7 expression, thereby restoring the lipid content in tumor ECs. Moreover, this restoration of lipid metabolism mediated by HSD17B7 was associated with improved adhesion molecule expression and vascular normalization, facilitating immune cell infiltration and contributing to AKB-9778's anti-tumor effects. Finally, we verified the effects and safety of combined AKB-9778 treatment on improving the efficacy of anti-PD-1 immunotherapy. In summary, this study revealed the mechanism and potential application of AKB-9778-induced vascular normalization in patients with oral cancer.

Celecoxib Enhances Oxidative Muscle Fibre Formation and Improves Muscle Functions Through Prokr1 Activation in Mice.

Muscle diseases are serious challenges to human health. Prokineticin receptor 1 (PROKR1) has emerged as a potential target to improve muscle function through increasing oxidative muscle fibres, but there are no clinically applicable synthetic PROKR1 agonists. Drugs with biological properties of prokineticin 2 (PK2) were discovered through connectivity map (CMap) analysis. Their effects on PROKR1 were evaluated using molecular docking, PROKR1 signalling and competitive binding assays. Pregnant dams were fed diets containing varying celecoxib concentrations (0, 500, 1000 and 1500 ppm) from gestation day 5 through weaning. Offspring were given high-fat diets (HFD) from weaning until 20 weeks old, and body composition, insulin resistance, energy expenditure, exercise performance and histological analysis of muscle tissues were evaluated. Celecoxib, with a connectivity score of 64.19 to PK2 and a docking score of -9.0 to PROKR1, selectively activated Gs signalling at 4 μM of EC50 and increased NR4A2 protein levels by 1.6-fold (p < 0.01) in PROKR1-overexpressing cells. It competitively inhibited PK2 binding to PROKR1 and reduced cAMP accumulation. In murine and human myotubes, celecoxib increased Prokr1 protein levels by 1.8-fold (p < 0.05), pCreb by 1.5-fold (p < 0.05) and Nr4a2 by 1.3-fold (p < 0.05). It also elevated Myh7 index by 2.2-fold (p < 0.0001), mitochondrial content by 1.6-fold (p < 0.001) and fatty acid oxidation (FAO) activity by 4.1-fold (p < 0.05). Offspring exposed to celecoxib during pre- and postnatal muscle development exhibited activated Prokr1 signalling, enhanced oxidative muscle fibre formation and improved muscle phenotype despite HFD. At weaning, both male and female offspring showed dose-dependent increases in lean mass (> 9.35%, p < 0.001) and grip strength (< 18.0%, p < 0.01). At 12 weeks old, mice displayed a dose-dependent decrease in weight loss (> 13.3%, p < 0.05), increased lean mass (> 16.2%, p < 0.05), improved insulin resistance (> 70.4%, p < 0.0001), energy expenditure (> 173%, p < 0.0001) and grip strength (> 23.5%, p < 0.001). Celecoxib also increased Myh7-positive muscle fibre composition (> 10.8%, p < 0.05) and mitochondrial mass (> 32.8%, p < 0.05) in the gastrocnemius and soleus muscles, accompanied by significant Prokr1 signalling activation. These effects persisted in both male and female mice at 20 weeks old. Celecoxib shows promise as a PROKR1 agonist and clinically applicable exercise mimetic for the treatment of muscular disorders.

Developing multifaceted drug synergistic therapeutic strategy against neurological disorders.

Drug synergism can alter the ultimate biological effects and bioavailability of phytoconstituents. Acetylcholinesterase (AChE) inhibitors as symptomatic drugs are potent therapeutic regimen for neurodegenerative diseases. In this context, this study characterized the synergistic antioxidant, anti-inflammatory and anti-AChE effects of the selected phytochemicals including standard drugs followed by enzyme kinetics, structure-based ligands screening and molecular dynamics simulation study. The synergistic interactions were evaluated through Isoradiation and Synergy finder 3.0 methods. The combinations of Quercetin (QCT), Folic acid (FA), and Swertiamarin (SWT) with specific reference drugs were studied. The combinations of SWT+GA (Gallic acid) and FA+GA at 1:1 (γ:0.10 & 0.08, respectively) showed the significant synergistic antioxidant effect via ABTS assay. Further, in combination, QCT+SWT showed the maximum synergistic effect (γ: 0.02-0.13) in anti-inflammatory assay. Moreover, the combinations QCT, FA, and SWT with reference drug, Donepezil (DP), illustrated potent synergistic activity as anti-AChE in 1:1 proportion (γ: 0.18). The interaction pattern of phytochemicals significantly exhibited synergism (γ<1) depicting their optimum activity in combinations compared to individual components. Enzyme kinetics evaluation showed the competitive binding of SWT with AChE as of donepezil. All the parameters of ADMET study proposed the QCT and SWT as acceptable oral drug molecules. Computational docking study revealed that QCT and SWT with lowest RMSD (1.096, 2.104) and lowest docking score (-9.831, -7.435kcal/mol) showed maximum binding efficacy. Furthermore, molecular simulation study depicted the stability of protein-ligand complexes. These findings provide novel insight in the development of dietary treatment based on their synergistic effects for neurological disorders as optimum alternative therapeutic agents.

Key site residues of Cnaphalocrocis medinalis odorant-binding protein 13 CmedOBP13 involved in interacting with rice plant volatiles.

Odorant binding proteins (OBPs) play key roles in the insect olfactory system by assisting the neuronal response to hydrophobic odor molecules, understanding their interaction with ligands will facilitate the virtual screening of behaviorally active compounds in insects. Here, we successfully cloned and confirmed CmedOBP13, an antennae-biased OBP from the rice leaffolder Cnaphalocrocis medinalis, as a secreted protein. Recombinant CmedOBP13 was obtained using the Escherichia coli system, and its binding affinities to 35 volatile compounds emitted by rice plants and three sex pheromone components from female moths were assessed by a competitive binding assay. The results revealed that CmedOBP13 exhibited binding affinity to 23 rice volatiles, while no binding affinity for sex pheromone components. Furthermore, the stability of its conformation was found to be dependent on the pH level. Finally, the interaction between CmedOBP13 and odorants was predicted and confirmed by molecular docking and mutation functional assays, respectively. The combination of multiple hydrophobic residues created an adequate hydrophobic setting for ligands, and three residues (Glu13, Arg34, and Tyr115) might form hydrogen bonds with 15 odorants. Single mutations of Glu13, Arg34, Leu72, and Tyr115 diminished the binding affinities of CmedOBP13 to corresponding odorants, respectively. These findings provided valuable insights into the mode of action of CmedOBP13 interacting with the volatiles of rice plants and will guide the screening of behaviorally active compounds against C. medinalis in future.

Synthesis and in vitro evaluation of novel compounds and discovery of a promising iodine-125 radioligand for purinergic P2X7 receptor (P2X7R).

The purinergic P2X ligand-gated ion channel 7 receptor (P2X7R) plays a critical role in various inflammatory processes and other diseases. Fast determination of compounds P2X7R binding potency and discovery of a promise PET radiotracer for imaging P2X7R require a P2X7R suitable radioligand for radioactive competitive binding assay. Herein, we designed and synthesized thirteen new P2X7R ligands and determined the in vitro binding potency. The fluorescence screening assay identified the iodide compound 1c with high potency and specificity toward P2X7R with an IC50 of 0.25±0.05nM. Therefore, 1c was 125I-labeled to afford [125I]1c with a good radiochemical yield (44±12%, n=3) and high radiochemical purity (>95%). Radioligand saturation binding assay showed that [125I]1c specifically bound to human P2X7R with high affinity (Kd=1.68nM and Bmax=94 fmol/mg). A radioactive high throughput binding assay using [125I]1c for our new compounds demonstrated that the imidazole compounds 1b, 1c, and 1d exhibited high inhibition for >70%, while the analogues of GSK314181A exhibited low inhibition for <35%. In addition, our radioligand competitive binding assays using [125I]1c demonstrated that 1b, 1c, and 1d have high potency with IC50 values of 7.91±0.22, 7.06±1.68, and 7.16±0.41 nM toward P2X7R, respectively.Together, compounds 1b, 1c, and 1d are highly potent for P2X7R, and [125I]1c has great potential to be a radioligand for screening P2X7R binding potency of the new compounds and investigating the P2X7R expression in animal models of human disease.

Biochemical and Structural Studies of Protein Tyrosine Phosphatase PTP-PEST (PTPN12) in Search of Small Molecule Inhibitors.

PTP-PEST (also known as PTPN12) regulates cellular signaling and transduction pathways by dephosphorylating its substrate. PTP-PEST is considered an important drug target owing to its involvement in cancer progression and myocardial injury. Till now only a few inhibitors are currently being studied in the inhibition of PTP-PEST, majorly belonging to the class of metal-based drugs. In this study, we aimed to investigate small molecules that could potentially inhibit PTP-PEST for further development of PTP-PEST inhibitors. As an approach, we used an in silico molecular docking technique to screen an in-house synthesized molecular library. Further, we validated the docking results by invitro inhibition screening of the best molecules using the purified catalytic domain of human PTP-PEST, which was over-expressed in E.coli. We identified a myo-inositol based derivative, J1-65, which binds to PTP-PEST and results in the competitive inhibition of the protein. Further, we confirmed this protein-ligand binding using binding affinity studies based on protein thermal shift assay and in silico molecular dynamic simulations. Our efforts to discover a novel scaffold for inhibiting hPTP-PEST mark a crucial stride in laying the groundwork for the future development of selective PTP-PEST inhibitors.

ATP Binding and Inhibition of Intrinsically Disordered Protein Interactions.

Recent studies have shown that ATP at high physiological concentrations (>5 mM) can inhibit liquid-liquid phase separation (LLPS) driven by interactions between intrinsically disordered proteins (IDPs). However, the mechanism underlying such inhibitory effect still remains elusive. Here, we used all-atom molecular dynamics simulation to study the interaction of ATP with two typical IDPs (i.e., FUS PLD and RGG domain of hnRNP G), and its impacts on IDP interactions. ATP exhibits a considerable tendency to bind to both IDPs and effectively inhibits their interactions. For the RGG domain, Arg residues are critical for both ATP binding and IDP interactions. The inhibitory effect of ATP is largely attributed to its competitive binding mode to Arg residues. Similar competitive binding of ATP is also observed in FUS PLD. Both ATP binding and the PLD interaction share the residues including Gln, Ser, and Tyr residues, while the competition is rather modest due to the abundance of these residues in the sequence. Interestingly, ATP undergoes considerable diffusion on the surface of PLD, which is an order of magnitude faster than the evolution of the contact area of PLDs. The temporal separation of these two processes remarkably promotes the inhibitory effect of ATP on PLD interaction. Given the representativeness of these two IDPs, competitive binding may serve as a general mechanism underlying ATP inhibition on IDP interactions at high physiological levels.

Studying interaction effects on toxicokinetics in zebrafish combining experimental and modelling approaches.

Humans and wildlife are exposed to a complex mixture of anthropogenic chemicals of which only a few have been subjected to regulations. Chemical risk assessment is currently based on evaluating single chemicals, which is costly, time-consuming, and neglect toxicokinetic and toxicodynamic mixture effects. This study focused on interaction effects on the absorption, distribution, metabolism and excretion (ADME) processes of selected chemicals representing potential modulators of these processes. Adult female zebrafish (Danio rerio) were exposed to selected mixture of 11 chemicals and bioconcentration factors (BCFs) on tissue level were determined for 9 of them: bisphenol A (BPA), bisphenol AF (BPAF), bisphenol Z (BPZ), triclosan, tribromophenol, pentachlorophenol, heptafluorobutyric acid (PFBA), perfluorobutanesulfonic acid (PFBS), and perfluorooctanesulfonic acid (PFOS). Comparison of BCFs of bisphenols obtained from single chemical exposure experiments versus the current study revealed no statistically significant differences (p>0.05), implying no mixture effects on kinetics of bisphenols at investigated concentrations. The same conclusion was reached using two physiologically based kinetic (PBK) models, developed for individual bisphenols and per- and polyfluoroalkyl substances (PFAS), showing good model fit for BPA, BPZ, BPAF, and PFOS. To simulate exposure scenarios where kinetic interaction effects may occur through competitive protein binding in blood, a new PBK model was developed. Simulations where zebrafish were dosed with BPA and BPZ, individually, and combined with varying levels of PFOS, showed that competitive binding to serum proteins alter tissue levels of bisphenols when levels of PFOS exceeded 1μg/L. This indicates that chemicals acting in concert could perturb ADME but only at higher levels or in complex mixtures.

Identification of new 1,2,3-Triazole analogues of sulfanilamide as inhibitors of the carbonic anhydrase II enzyme: Comprehensive in-vitro and in-vivo analyses.

Carbonic anhydrases (CAs) play a vital role in various physiological processes by catalyzing the reversible hydration of CO2 into HCO3-, hence maintaining the fluid and pH balance. Overexpression of carbonic anhydrases II (CA II) is associated with diseases, such as glaucoma, and epilepsy; therefore, it is considered as an important clinical target. Therapeutically used CA inhibitors exhibit several undesirable effects; therefore, there is an urgent need to identify new, safe, and effective inhibitors of the CAs. Keeping in view the importance of CA II inhibition, a library of new 1,3-disubstituted-1,2,3-triazole analogues of sulfanilamide is synthesized via Click chemistry, starting from sulfanilamide azide and different substituted propargyl ethers, incorporating benzyl and heteroarylmethyl moieties. The new derivatives showed significant CA II inhibitory activity (IC50 ranging between 0.19 and 0.66 μM) when compared with the standard inhibitor, acetazolamide (0.13 ± 0.01 μM). Among all, compounds 16 and 17 showed the most potent activity (IC50 = 0.19 μM) followed by compounds 23, and 18 (IC50 = 0.24 ± 0.014 and 0.26 ± 0.04 μM, respectively). Kinetics studies showed that all compounds are competitive inhibitors of bCA II enzyme (Ki ranging between 0.14 and 0.68 μM). Additionally, molecular docking studies revealed that all compounds formed network of interactions with the active site residues of the bCA II enzyme. All compounds were found to be non-toxic against BJ Human fibroblast cells. From in-vivo studies, we found that CA activity was significantly inhibited by the intraperitoneal administration of compounds 16 and 17 for up to 5 h. In conclusion, new 1,2,3-triazole analogues of sulfanilamide were identified as good CA II inhibitors.

A FRET-based competitive binding assay using coumestrol and the ligand-binding domain of human estrogen receptor alpha tagged with mTurquoise2 efficiently expressed in E. coli with ethanol.

The estrogen receptor (ER) is a nuclear receptor and one of the most extensively researched targets in the study of endocrine-disrupting chemicals (EDCs). Many biosensors and bioassays for estrogenic EDCs use the ligand-binding domain of human ERα (LBD-hERα) as a biological recognition element. However, the LBD-hERα is poorly stable and difficult to produce as a functional LBD-hERα in the E. coli expression system. In this study, we efficiently expressed the functional LBD-hERα tagged with the cyan fluorescent protein, mTurquoise2 (LBD-hERα-mTq2) by the addition of ethanol (3%) to E. coli suspension during protein expression (> 40 times more compared to without ethanol). We found that ethanol not only promoted the proper folding of LBD-hERα-mTq2, but also prevented the proteolysis of poorly folded recombinant proteins. We established a FRET-based binding assay between a fluorescent estrogen, coumestrol, and the LBD-hERα-mTq2, in which the formation of the complex exhibits a significant degree of FRET. A subsequent competitive binding assay with diethylstilbestrol demonstrates that our system successfully functions as a simple and reliable bioassay to detect estrogenic EDCs.

P-1073. Developing Vaccines and Antibodies Targeting Conserved Peptidoglycan, Lipoteichoic Acid, and Lipopolysaccharide Epitopes to Combat Antimicrobial Resistance

Abstract Background Antimicrobial resistance (AMR) poses a significant global health threat, driving the urgent need for non-antibiotic therapies such as vaccines and monoclonal antibodies (mAbs). Targeting conserved epitopes on gram-positive and gram-negative bacteria, including mycobacteria will be important for developing strategies against AMR. Key epitopes, including Peptidoglycan (PGN), Lipoteichoic acid (LTA), and Lipopolysaccharide (LPS) are crucial in this context. In this study, we illustrate the efficacy of a mAb directed against ultrapure PGN from Staphylococcus aureus (SA), alongside the capabilities of polyclonal serum antibodies induced by a multi-epitope composite peptide vaccine comprised of PGN, LTA, and LPS epitopes. Methods MAb MD11 was produced from a mouse immunized subcutaneously with ultrapure PGN from SA and the mAb specificity identified using ultrapure PGN and a synthetic PGN peptide in a competitive inhibition assay. Additionally, polyclonal serum antibodies were derived from ICR mice immunized subcutaneously on days 0, 21, and 35 with 20 µg of multi-epitope composite peptide vaccine LPS.PGN.LTA05, formulated with AddaVax™ adjuvant. IgG1 titers to the immunogens and whole bacteria: SA, Staphylococcus epidermidis, Mycobacterium smegmatis, Escherichia coli, B. subtilis, Grp. B Streptococcus and killed Mycobacterium tuberculosis (MTB) were analyzed by ELISA. Results MAb MD11 recognized and opsonized various gram-positive bacteria, including mycobacteria. PGN peptide competitively inhibited binding of MD11 to Ultrapure PGN, indicating specificity of MD11 to PGN epitope. Robust and broad polyclonal serum IgG1 antibodies recognizing both gram-positive and gram-negative bacteria, including mycobacteria were generated by the multi-epitope composite peptide vaccine. Conclusion Anti-PGN mAb MD11 was broadly reactive to gram-positive bacteria. Polyclonal serum antibodies showed broad reactivity across both gram-positive and gram-negative bacteria, including mycobacteria. Developing vaccines and antibodies that target conserved bacterial epitopes may provide important strategies for prevention and treatment of bacterial infections, particularly for immunocompromised individuals, and could offer a useful approach to combat AMR. Disclosures All Authors: No reported disclosures

Clarification of Sugarcane Juice Catalyzed by Magnetic Immobilized Laccase Intensified by Alternating Magnetic Field

In this study, Cu2+-chelated magnetic silicon dioxide nanoparticles were synthesized as carriers for laccase immobilization. The prepared magnetic immobilized laccase was applied in the clarification of sugarcane juice. The optimal conditions for the clarification of sugarcane juice with magnetic immobilized laccase in a shake flask were determined to be as follows: a temperature of 35 °C, pH of 5.5, rotation speed of 150 r/min, and immobilized laccase dosage of 1.0 mg/mL. The sucrose in the sugarcane juice inhibited both free and immobilized laccase. The inhibitory effect was characterized as mixed inhibition, wherein competitive inhibition played a dominant role. An alternating magnetic field was introduced into the catalysis process using magnetic immobilized laccase, and the catechin degradation rate was improved to 77.2% under a magnetic field intensity of 80 Gs and magnetic field frequency of 400 Hz. Under the optimal alternating magnetic field conditions, the treatment time of sugarcane juice was reduced to 20 min when catalyzed by the magnetic immobilized laccase, wherein a decolorization rate of 54.4%, reduction in turbidity of 89.7%, and total phenol degradation rate of 43.4% were achieved. Compared with the shaking condition, the assistance of alternating magnetic fields can shorten the clarifying time, increase the clarifying effect, and enhance the catalyst reusability. These results reveal useful information about the enzymatic treatment of high-sugar juice and provide a potential strategy for juice clarification with magnetic immobilized enzymes.

Designing High-Damping, Optically Clear Ionogels through Competitive Binding for Flexible and Impact-Resistant Applications.

Developing damping materials that are both optically transparent and mechanically robust, while offering broad frequency damping capacity, is a significant challenge─particularly for devices that require protection without compromising visual clarity. Conventional methods often either fail to maintain transparency or involve complex designs that are difficult to implement. Here, we present an ionogel system that integrates a physically cross-linked elastic copolymer network with a viscous ionic liquid. The competitive interactions between the ionic liquid and the polymer network enable fine-tuning of the mechanical stability and damping capacity. The resulting ionogel is transparent and mechanically robust and exhibits excellent damping over a wide frequency range. Remarkably, a thin layer (0.15 mm) absorbs nearly 60% of the impact force and retains its performance after exposure to extreme conditions. This approach offers a straightforward method for designing advanced damping materials that meet both the aesthetic and functional demands of modern technologies.