Competitive Inhibitor Research Articles

BEACH domain proteins function as cargo-sorting adaptors in secretory and endocytic pathways.

We identify BEACH domain-containing proteins (BDCPs) as novel membrane coat proteins involved in the sorting of transmembrane proteins (TMPs) on the trans-Golgi network and tubular sorting endosomes. The seven typical mammalian BDCPs share a predicted alpha-solenoid-beta propeller structure, suggesting they have a protocoatomer origin and function. We map the subcellular localization of seven BDCPs based on their dynamic colocalization with RAB and ARF small GTPases and identify five typical BDCPs on subdomains of dynamic tubular-vesicular compartments on the intersection of endocytic recycling and post-Golgi secretory pathways. We demonstrate that BDCPs interact directly with the cytosolic tails of selected TMPs and identify a subset of TMPs, whose trafficking to the plasma membrane is affected in cells lacking BDCP. We propose that the competitive binding of BDCPs and clathrin coat adaptors to the cytosolic tails of TMPs, followed by their clustering to distinct subdomains of secretory/recycling tubules function as a mechanism for sorting of TMPs in pleomorphic tubular-vesicular compartments that lack a clathrin coat.

Inhibitory effects of phenylpropionamides from the hemp seed husk on tyrosinase

Tyrosinase is an enzyme involved in melanin production. To identify inhibitors of this enzyme in natural products, extracts and fractions of hemp seed husk were screened for tyrosinase inhibition. Phenylpropionamides 1–9, which were isolated from the ethyl acetate fraction, had inhibitory effects; compounds 3 and 4 had the greatest inhibitory effects. Cannabisin A (3) was a non-competitive and cannabisin B (4) was a competitive inhibitor. The binding site and interaction on tyrosinase of each inhibitor were calculated by computational chemistry. Potential inhibitor 3 decreased the melanin content and tyrosinase activity in B16F10 melanoma cells. Therefore, cannabisin A (3) has potential as a tyrosinase inhibitor.

A Deep Dive into PDE5 Inhibition: Innovative Discoveries via Virtual Screening

Background: PDE5 inhibitors have had a surge in popularity over the last decade owing to their efficacy in the treatment of erectile dysfunction, coronary vasculopathy, and pulmonary arterial hypertension. These inhibitors exhibit competitive binding with phosphodiesterase type 5 and inhibit the hydrolysis of cyclic guanosine monophosphate, hence elevating the levels of cGMP in smooth muscle cells and prolonging the duration of an erection. However, due to production costs and side effects, further research is needed to discover new PDE5 inhibitors. Objective: The study aimed to identify potent PDE5 inhibitors by employing the extensive application of computer-aided drug design. Methods: Three different databases, named Million Molecules Database, Natural Product Database, and NCI Database, have been screened, which has been followed by filtering based on various druglikeness rules, docking, ADME, toxicity, consensus molecular docking, and 100 ns molecular dynamics simulation. Results: Three compounds (ZINC05351336, ZINC12030898, and ZINC17949426) have exhibited stable-binding characteristics at the active site of PDE5, demonstrating a robust binding affinity. These molecules have been found to possess drug-like capabilities, effective ADME features, low toxicity, and high stability. Conclusion: The study has delved into the realm of PDE5 inhibitors, which have been found to be effective in treating erectile dysfunction, but high production costs and side effects necessitate new ones. Through computer-aided drug design and screening, three compounds have been identified with promising binding characteristics, drug-appropriate properties, effective ADME profiles, minimal toxicity, and stability, making them potential candidates for future PDE5 inhibitors

Competitive inhibition of family GH11 Aspergillus fumigatus endo-xylanase A by Oryza sativa xylanase inhibitor protein: Investigating key interface residues and non-covalent interactions

Glycoside hydrolase (GH) family 11 Aspergillus fumigatus endo-xylanase A (AfXylA11) was recombinantly expressed in Pichia pastoris X33 and secreted into culture medium. Hydrolysis of beechwood xylan by recombinant AfXylA11 (reAfXylA11) predominantly generated xylooligosaccharides (XOs), chiefly comprising xylotriose (X2) and xylotetraose (X3). These hydrolysates demonstrated significant antioxidant capabilities. Competitive inhibition of reAfXylA11 was observed in the presence of the Oryza sativa xylanase inhibitor protein (OsXIP), with an inhibition constant (Ki) of 120.98 nM. Molecular dynamics (MD) simulations and conformational analyses of the AfXylA11-OsXIP complex revealed that the extended loop (Lα4β5, K150-V158) of OsXIP penetrated the active groove of AfXylA11, thereby obstructing xylan from interacting with catalytic sites. The R153 at the bottom of the U-shaped loop, was found to form durable hydrogen bonds with E197 (acid/base catalyst) and Y97 of AfXylA11. Additionally, OsXIP established stable interactions with the “cord” and “thumb” regions of AfXylA11 during MD simulations. The binding free energy of the AfXylA11-OsXIP complex was determined to be -74.3 ± 7.6 kcal/mol, with electrostatic interactions and van der Waals forces being the predominant contributors to enzyme-inhibitor interaction. Further MD simulations demonstrated that the mutation of Y116A or S120A in the “cord” region of AfXylA11 significantly reduced its binding affinity to OsXIP by weakening interface residue interactions. Delving into the conformational dynamics of the AfXylA11-OsXIP complex yields insights into the molecular mechanisms dictating their competitive inhibition. This investigation offers useful basis for engineering mutant xylanases that resist to the inhibitory protein yet retain high catalytic efficacy.

New insights into EGCG retards the digestion of wheat starch by α-amylase in ternary system: Comparison with binary systems.

This study was to investigate the mechanism of the action of epigallocatechin gallate (EGCG) on α-amylase in the ternary simulated system and explore the changes in enzyme structure during the digestion process. Enzymatic kinetics, fluorescence spectroscopy, surface hydrophobicity, fluorescence microscopy, and molecular docking were used to compare (in the presence and absence of EGCG) the structural changes of α-amylase and α-amylase-starch complex, as well as the binding characteristics among EGCG and the α-amylase and starch. The results showed that EGCG had a significant inhibitory effect on α-amylase, and it exhibited a coexistence of competitive and anti-competition inhibition type, and predominantly competitive inhibition. In the ternary and binary systems, the inhibitory mechanisms of EGCG on α-amylase were distinct. In the ternary system, EGCG preferably bound to α-amylase to form α-amylase-EGCG binary complexes rather than α-amylase-starch-EGCG ternary complexes, and altered the structure of α-amylase, leading to unfolding of the enzyme's secondary structure and exposing more non-catalytic site aromatic amino acids.

Molecular Docking and Molecular Dynamics Simulation of New Potential JAK3 Inhibitors.

JAK3 kinase inhibitor has become an effective means to treat tumors and autoimmune diseases. In this study, molecular docking and molecular dynamics simulation were used to study the theoretical interaction mechanism between 1-phenylimidazolidine-2-one molecules and JAK3 protein. The results of molecular docking showed that the six 1-phenylimidazolidine-2-one derivatives obtained by virtual screening were bound to the ATP pocket of JAK3 kinase, which were competitive inhibitors of ATP, and were mainly bound to the pocket through hydrogen bonding and hydrophobic interaction. Further, MM/GBSA based on molecular dynamics simulation sampling was used to calculate the binding energy between six molecules and the JAK3 kinase protein. Subsequently, the binding energy was decomposed into the contribution of each amino acid residue, of which Leu905, Lys855, Asp967, Leu956, Tyr904, and Val836 were the main energycontributing residues. Among them, the molecule numbered LCM01415405 can interact with the specific amino acid Arg911 of JAK3 kinase, suggesting that the molecule may be a selective JAK3 kinase inhibitor. The root-mean-square fluctuation (RMSF) of JAK3 kinase pocket residues during molecular dynamics simulation showed that the combination of six new potential small molecule inhibitors with JAK3 kinase could reduce the flexibility of JAK3 kinase pocket residues. These findings reveal the mechanism of 1-phenylimidazolidine-2-one derivatives on JAK3 protein and provide a relatively solid theoretical basis for the development and structural optimization of JAK3 protein inhibitors.

A novel chitosan/quercetin-based film enables non-enzymatic detection of glucose: Electrochemical and fluorescent behavior

In this study, a novel functional hydrogel film was synthesized using natural polymer chitosan and quercetin as raw materials. Fourier transform infrared (FT-IR) spectra and X-ray photoelectron spectroscopy (XPS) analysis indicate that phenylboronic acid modified carbon nanodots (PBA-CDs) were successfully prepared via a simple microwave-assisted hydrothermal method. The experimental results demonstrate that a dual electrochemical and fluorescence detection of glucose can be realized based on the boronate ester bonding between chitosan/quercetin (Chit0/Quer) films and PBA-CDs. The addition of PBA-CDs hinders the electron transfer between the phenolic hydroxyl groups of quercetin and the redox mediators, and inhibits the intramolecular rotation of quercetin, which leads to a decreased electrical signal and enhanced fluorescence intensity. After the addition of glucose, the peak redox current of the Chit0/Quer hydrogel film is restored due to the competitive binding of glucose to the boronate ester, resulting in a dual-input logic circuit for the qualitative detection of glucose. In addition, the fluorescence intensity of the hydrogel decreases with increasing glucose concentration, and the concentration of glucose can be quantitatively detected by detecting the change in fluorescence intensity. Besides, the sensor has better stability and selectivity for biomarkers. Finally, we demonstrate that the electrochemical and fluorescence sensing strategies can be used for the detection of glucose in real beverage samples. In conclusion, based on the redox activity of quercetin and the competitive binding of boronate ester bonds, we explored a new strategy for enzyme-free electrochemical and fluorescence sensing for the detection of glucose, which is expected to be applied in biomedicine, food industry and other fields.

Green preparation and isolation of carrageenan oligosaccharides and assessment of their ability to inhibit melanogenesis

An efficient method was developed for generating carrageenan oligosaccharides, a high-value material with health benefits for humans. The study used microwave-assisted mild acid hydrolysis of κ-carrageenan to achieve complete liquefaction. Under optimal reaction conditions, the yield of κ-carrageenan disaccharide (KCO2) was 22.90% ± 0.80%, with a total sugar product recovery rate of approximately 90%, while retaining the sulfate groups. Different degrees of polymerization (DP) carrageenan oligosaccharides with 4-sulfate-D-galactose (D-G4S) as the non-reducing end were obtained, with purities of over 90% for both KCO2 and κ-carrageenan tetrasaccharide (KCO4). KCO2 was identified as the oligosaccharide with the highest tyrosinase inhibitory activity. Enzyme kinetics studies revealed that KCO2 exhibited typical reversible competitive inhibition, with an IC50 of 1.09 ± 0.04 mg/mL and a Ki value of 0.16 ± 0.01 mg/mL. Molecular docking showed that KCO2 primarily interacted with key His residues at the tyrosinase active site through its sulfate groups. Molecular dynamics (MD) simulations indicated that the KCO2-tyrosinase complex structure was stable. These findings show that the carrageenan oligosaccharides generated by this strategy could be used as tyrosinase inhibitors in the food, cosmetic, and nutraceutical fields.

Generating and validating renewable affimer protein binding reagents targeting SH2 domains.

Despite SH2 domains, being pivotal in protein interactions linked to various diseases like cancer, we lack specific research tools for intracellular assays. Understanding SH2-mediated interactions and creating effective inhibitors requires tools which target individual protein domains. Affimer reagents exhibit promise, yet their potential against the extensive SH2 domain family remains largely unexplored. Our study aimed to bridge this gap by identifying Affimer reagents that selectively bind to 22 out of 41 SH2 domains. These reagents enabled a medium-throughput screening approach resembling siRNA studies, shedding light on their functionality. Notably, select Affimers demonstrated the ability to curtail the nuclear translocation of pERK, with Grb2 being a prominent target. Further analyses revealed that these Grb2-specific Affimer reagents displayed competitive inhibition with impressive metrics: IC50s ranging from 270.9 nM to 1.22 µM, together with low nanomolar binding affinities. Moreover, they exhibited the ability to pull down endogenous Grb2 from cell lysates, illustrating their efficacy in binding the Grb2 SH2 domain. This comprehensive assessment underscores the potential of Affimer reagents as domain-specific inhibitors. Their viability for medium/high-throughput phenotypic screening presents a promising avenue via which to identify and characterize potential drug targets within the SH2 domain family.

Structure-based discovery of a new type of scaffold compound as binding competitors for protein-bound Uremic Toxins.

Protein-bound uremic toxins (PBUTs) are the main cause of uremia, but traditional hemodialysis is ineffective in removing them because of their strong ability to bind to human serum albumin (HSA), highlighting the need for new treatments. In this study, first, structure-based docking was used to screen a diverse library of 200,376 virtual compounds against the active sites I and II. After two rounds of docking screening, 3944 candidate molecules were obtained. Second, 23 candidate molecules were obtained after ADMET prediction and toxicity analysis. Five candidate molecules were finally obtained after visual analysis and MM-PBSA calculations. We subsequently assessed their competitive displacement efficiency through a microdialysis experiment, and the results revealed that ZINC000008791789, ZINC000012297018, and ZINC000012296493 are promising binding competitors for PBUTs, as they have higher dialysis efficiency than the optimal displacer LA, approximately double the dialysis efficiency. The other two molecules, ZINC000031161007 and ZINC000004090361, although less efficient than LA, still outperformed the control group. Notably, four of them shared the same molecular scaffold, and three of them contained a flavonoid group. These findings provide a foundation for the development of more effective PBUT binding competitors, potentially benefiting uremia patients in the future.

Cinnamaldehyde ameliorates diabetes-induced biochemical impairments and AGEs macromolecules in a pre-clinical model of diabetic nephropathy.

Cinnamaldehyde, has various therapeutic potentials including glucose-lowering effect, and insulinotropic effect; however, its glycation inhibitory mechanism is not known yet. In this study, we explored the effects of cinnamaldehyde for its AGEs inhibitory mechanism in a streptozotocin-complete Freund's adjuvant (STZ-CFA) induced diabetic nephropathy (DN) rat model. Pre-clinical DN model was developed by the administration of multiple low doses of STZ-CFA in rats, mainly characterized by abnormal blood parameters and nephrotic damages. Diabetes-related systemic profile and histopathological hallmarks were evaluated using biochemical assays, microscopic imaging, immunoblot, and real-time PCR analyses, supported by cinnamaldehyde-albumin interaction assessed using STD-NMR and in silico site-directed interactions in the presence of glucose. Cinnamaldehyde-treatment significantly reversed DN hallmarks, fasting blood glucose (FBG), serum insulin, glycated hemoglobin (HbA1c), urinary microalbumin, and creatinine contrasted to non-treated DN rats and aminoguanidine, a positive reference advanced glycation end products (AGEs) inhibitor. The pathological depositions of AGEs, receptor for advanced glycation end products (RAGE), and carboxymethyl lysine (CML), and transcriptional levels of AGE-RAGE targeted immunomodulatory factors (IL1β, TNF-α, NF-κB, TGF-β) were significantly improved in cinnamaldehyde treated rats as compared to aminoguanidine. Cinnamaldehyde post-treatment improved pancreatic pathology and systemic glycemic index (0.539 ± 0.01 vs. 0.040 ± 0.001, P < 0.001) in DN rats. Subsequently, in silico profiling of cinnamaldehyde defined the competitive binding inhibition with glucose in AGE and RAGE receptors that was further confirmed by in vitro STD-NMR analysis. These findings suggest potential role of cinnamaldehyde in reversing STZ-induced diabetic nephropathic impairments; therefore, appears promising candidate for further pharmacological explorations towards diabetes-associated complications.

The novel mechanism on the inhibition of superoxide anion generating from xanthine oxidase in the presence of myricetin.

To gain an in-depth understanding of the process of myricetin (MY) inhibiting the generation of •O2- from Xanthine Oxidase (XOD) at a novel perspective, the inhibiting pathway is necessary to be re-elaborated through inhibitory type, thermodynamic analysis and molecular simulation. The results demonstrated that MY is indeed a potent inhibiting agent for •O2- producing from XOD, with the maximum value of 33.78 % in the inhibition of •O2-. Furthermore, the deprotonated form (De-MY) of MY was examined to be an effective agent for interacting with XOD, with the measurement of •O2- in productions from MY, XOD@MY and XAN-XOD@MY, as comparing to the XAN-XOD@ALL. On the inhibition kinetics, De-MY was witnessed to be on a mixed-type inhibition, with more competitive inhibition. In the thermodynamic analysis, the Stern-Volmer plots for De-MY obtained at 298 K, 303 K and 308 K, were polynomial-fitted to form curves approaching the X axis via the fluorescence quenching of XOD, which was against the reported with good linearity. It indicated that the interaction between De-MY and XOD occurred at the range of MY concentrations from 0 to 0.025 (×10-5 mol L-1), is dominated by a static quenching procedure, ascribing to the appearance of accessible interaction with corresponding f values less than 1. As well, via combining the synchronous fluorescence data, Molecular dynamics simulations showed that MY binding to the Trp 283, ARG-60 and Tyr 58 with H-binding interactions is an available access to block the transfer of free electrons from FADH2 to O2. Therefore, in this work, successfully re-elaborating the inhibition mechanism of •O2- generating from XOD by MY is benefited for MY in the future applications.

In silico studies on nicotinamide analogs as competitive inhibitors of nicotinamidase in methicillin-resistant Staphylococcus aureus.

Nicotinamidase/PncA is a member of the hydrolase enzyme family, catalyzing the de-amidation of nicotinamide (NM) to nicotinic acid (NA) via salvage pathway. Products are fed into Preiss-Handler pathway for NAD+ biosynthesis which is an important enzyme cofactor and crucial for redox balance in microorganisms. Pathogens like methicillin-resistant Staphylococcus aureus (MRSA) are NAD+ auxotroph and rely on their host environment for NAD+ precursors to synthesize NAD+. Mutations in nicotinamidase/PncA have been reported to be associated with resistance to pyrazinamide (PZA), a front-line anti-tubercular drug, underlying its importance as an important link in NAD+ biosynthesis network in pathogenic organisms such as MRSA. The conserved features of PncA and essentiality of salvage route in MRSA and the absence of this enzyme in humans and other eukaryotes are attractive options to explore therapeutics against this target. In this work, we have screened novel substrate analogs from the PubChem database using virtual screening approaches employing fingerprint tanimoto-based 2D similarity search against Staphylococcus aureus PncA (SaPncA). Identified compounds were further assessed using molecular dynamics simulations to investigate conformational stability and structural integrity. We propose two analogs, namely L28 and L33 with greater stability, favorable binding and strong binding free energies in MM-PBSA calculations. The strategy could provide an important clue in developing similar compound scaffolds as potent drug-like molecules against MRSA and other pathogenic species harboring this enzyme. Smaller scaffolds of these molecules could be attractive options for fragment-based derivatization for inhibitor discovery.

Computational Model-Assisted Development of a Nonenzymatic Fluorescent Glucose-Sensing Assay.

Deep-red fluorescence was implemented in this fully injectable, nonenzymatic glucose biosensor design to allow for better light penetration through the skin, particularly for darker skin tones. In this work, a novel method was developed to synthesize Cy5.5 labeled mannose conjugates (Cy5.5-mannobiose, Cy5.5-mannotriose, and Cy5.5-mannotetraose) to act as the fluorescent competing ligand in a competitive binding assay with the protein Concanavalin A acting as the recognition molecule. Using fluorescence anisotropy (FA) data, a computational model was developed to determine optimal concentration ratios of the assay components to allow for sensitive glucose measurements within the physiological range. The model was experimentally validated by measuring the glucose response via FA of the three Cy5.5-labeled mannose conjugates synthesized with Cy5.5-mannotetraose demonstrating the most sensitive response to glucose across the physiological range. The developed method may be broadly applied to a vast range of commercially available fluorescent dyes and opens up opportunities for glucose measurements using nonenzymatic assays.

Abstract 4134148: Reduced BMPR2 in Monocytes De-represses HERV-K and Sustains Inflammation in Pulmonary Arterial Hypertension

Background&amp;Rationale: Human endogenous retroviral (HERV) elements are embedded in 8% of our genome. They remain quiescent but can be transiently activated by viral infection to induce an interferon (IFN) response. In pulmonary arterial hypertension (PAH), HERV-K expression is elevated in myeloid cells, and thus can promote inflammation observed in pulmonary arteries (PAs) with progressive obstructive remodeling. PAH is linked to a mutation or a decrease in BMPR2 expression which our laboratory has recently shown in monocytes (MONO). HERVs are silenced by SUMOylated (S) TRIM28 (KAP1, a methylase) and SPEN (a histone deacetylase) factors that are also sequestered by the lnc RNA XIST which is increased in females to inactivate the extra X-chromosome. When TRIM28 is phosphorylated (P) by DNA-PK, it loses methylase activity and can act as a transcription factor. Hypothesis: Reduced BMPR2 in MONO results in DNA-PK mediated P-TRIM28 causing demethylation of XIST targets (X-related genes) and HERV-K thus increasing their expression. Competitive SPEN binding to the increased XIST also de-represses HERV-K . Approach: We used THP-1 MONOs with BMPR2 shRNA and induced pluripotent stem cell (i) MONOs derived from BMPR2 mutant PAH patients and assessed HERV-K , XIST , IFNs and related our findings to DNA-PK–P-TRIM28 and sequestration of SPEN. Results: XIST and HERV-K mRNA were significantly elevated in THP-1 MONOs with shBMPR2 vs. shControl and in PAH-iMONO vs. control iMONO based on gender. Reduced BMPR2 also increased IFN genes. These findings were consistent with nuclear DNA-PK and P-TRIM28 shown to bind in affinity purification mass spectrometry analysis. Bisulfite conversion assay demonstrated decreased methylation of HERV-K in BMPR2 -deficient MONOs attributed to a reduction in the methylase S-TRIM28. RNA-IP in THP-1 MONOs with shBMPR2 vs. shControl indicated increased binding of XIST with TRIM28 and SPEN, suggesting a competitive reduction in binding to HERV-K. Conclusions: Loss of BMPR2 increases XIST and HERV-K in MONO related to DNA-PK induced P-TRIM28 and reduced methylase S-TRIM28. Increased HERV-K may also be a function of the increase in XIST sequestering its deacetylase SPEN. These molecular features underlie a chronic HERV-K -IFN inflammatory response in PAH. They may explain the female predisposition to PAH, because the increase in XIST in females heightens the propensity to activate HERV-K -IFN signaling and chronic inflammation in PAs infiltrated by MONO.

Abstract 4136614: The design of novel therapeutics that target the L-type calcium channel to prevent hypertrophic cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) is an inherited autosomal dominant disease of the sarcomere. Pathogenic features include ventricular hypertrophy, increased myofilament calcium sensitivity, myocardial fibrosis, and diastolic dysfunction. At the level of the myocyte there is cytoskeletal disarray, hypercontractility and altered mitochondrial function. Mitochondrial dysfunction is considered to be a key driver in HCM pathology. Research question: We previously demonstrated that the L-type Ca 2+ channel plays a role in the development of HCM facilitated by a structural-functional communication with mitochondria that can be regulated via the alpha interaction domain (AID) of the channel. In search of a preventative HCM therapy, we explored the efficacy of amino acid peptide variants that correspond to the AID of the cardiac L-type Ca 2+ channel. Methods and Results: Consistent with in silico predictions , competition binding assays confirmed that 4 variant peptides bound with higher affinity to the beta subunit than the AID peptide. In vitro studies confirmed that 3 of the 4 peptides could decrease the characteristic hypermetabolic state in myocytes isolated from a murine model of human HCM ( cTnI-G203S ). In vivo treatment of cTnI-G203S mice with peptide variants prevented the development of HCM and the development of fibrosis, in the absence of alterations in blood pressure, or kidney and liver function or changes in behaviour. Of note, the peptide variants also significantly improved contractile function. Similar effects were measured in αMHC 403/+ mice expressing the MYH6 mutation. Conclusions: Here we describe a first in class therapy that uniquely targets the L-type Ca 2+ channel to modify mitochondrial function and prevent hypertrophic cardiomyopathy. The peptides may be effective for treatment of HCM broadly because the mechanism of action involves the modification of mitochondrial function and impaired energy metabolism that is a common characteristic and driver of the pathology.

Synthesis of arylated tetrahydrobenzo[H]quinoline-3-carbonitrile derivatives as potential hits for treatment of diabetes.

Aim: Quinoline scaffolds are serving as the core structure for numerous antifungal, analgesic, antipyretic, anti-inflammatory drugs as well as have also been investigated for their potential antidiabetic properties. Though further exploration is required in this area as the current antidiabetic agents, such as acarbose, miglitoland voglibose, are associated with several adverse side effects. In this context, arylated tetrahydrobenzo[H]quinoline-3-carbonitrile derivatives were designed and evaluated as potential antidiabetic agents.Materials & methods: A one-pot multicomponent reaction of 6-methoxy-1-tetralone with ethyl cyanoacetate, ammonium acetateand varying aldehydes yielded a range of new arylated tetrahydrobenzo[h]quinoline-3-carbonitrile molecules 1-36.Results: Compounds 2-5, 12, 13, 19 and 32-34 showed excellent inhibition against α-amylase (IC50=3.42-15.14μM) and α-glucosidase (IC50=0.65-9.23μM) enzymes in comparison to the standard acarbose (IC50=14.35μM). In addition, all compounds revealed significant to moderate DPPH radical scavenging activity (SC50=21.30-138.30μM) compared with BHT (SC50=64.40μM). Kinetic studies confirmed competitive inhibition mode, while molecular docking studies comprehend ligands'interaction with enzyme's active sitesand absorption, distribution, metabolism, and excretion analysis confirms that all synthetic derivatives are nontoxic.Conclusion: This research offers a range of lead candidates to become antidiabetic agents after further advanced study.

EXTH-34. ACETYLATION SUPPRESSION MEETS ONCOLYTIC VIRUSES FOR THE TREATMENT OF PEDIATRIC DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline glioma (DMG) remains a devastating pediatric brain tumor without effective treatment. In a recent clinical trial, we demonstrated the feasibility and efficacy of Delta-24-RGD oncolytic virus combined with radiation therapy in patients with newly diagnosed diffuse intrinsic pontine gliomas (NCT03178032). Because the adenoviral protein E1A binds to P300 and redirects acetylation upon virus infection, we hypothesized that combining oncolytic adenoviruses with P300 inhibitors will cause a remarkable shifting in acetylation and the post-translational landscape of DMG, ultimately enhancing the anti-glioma effect of oncolytic viruses. Western blot analyses showed a longitudinal reduction in H3K27ac (89%) and H3K18ac (99%) in a panel of human (TP54 and SF8628) and murine (24B_1 and 26C_7) DMG cells after Delta-24-RGD infection. In addition, using histone fractionation by high-performance liquid chromatography, we detected an increase in total H3 upon adenovirus infection. Mass spectrometry of histones in the human DIPG cell line, TP54 using middle-down approach illustrated post-translational changes across histones, including hypoacetylation of the H3.2K14 mark. To further modify histone acetylation in DMG during Delta-24-RGD infection, we combined virotherapy with C646, a selective and competitive histone acetyltransferase inhibitor of the E1A-binding p300/CBP complex of proteins. In vitro, the combination of Delta-24-RGD with C646 synergistically increased cell death in a panel of human and murine DIPG cell lines (zip-scores ranging from 6.3-11.3 with zip-scores above 10 being indicative of synergy). Ongoing experiments using a panel of adenoviruses expressing E1A with defective P300 binding or expressing a dominant-negative form of P300 or wild-type P300 are designed to test the extent to which E1A binding of P300 drives hypoacetylation in DMG upon virus infection. In addition, we are performing in vivo experiments to establish the anti-cancer effect of the combination of Delta-24-RGD and P300 inhibitors in clinically relevant animal models of DMG, with a feasible translation to the clinic.

Structure of the yeast ceramide synthase.

Ceramides are essential lipids involved in forming complex sphingolipids and acting as signaling molecules. They result from the N-acylation of a sphingoid base and a CoA-activated fatty acid, a reaction catalyzed by the ceramide synthase (CerS) family of enzymes. Yet, the precise structural details and catalytic mechanisms of CerSs have remained elusive. Here we used cryo-electron microscopy single-particle analysis to unravel the structure of the yeast CerS complex in both an active and a fumonisin B1-inhibited state. Our results reveal the complex's architecture as a dimer of Lip1 subunits bound to the catalytic subunits Lag1 and Lac1. Each catalytic subunit forms a hydrophobic crevice connecting the cytosolic site with the intermembrane space. The active site, located centrally in the tunnel, was resolved in a substrate preloaded state, representing one intermediate in ceramide synthesis. Our data provide evidence for competitive binding of fumonisin B1 to the acyl-CoA-binding tunnel.

Characterization of two glutathione S-transferase genes involved in clothianidin resistance in Bradysia odoriphaga.

Glutathione S-transferase (GST) is a key phase II detoxification enzyme involved in xenobiotics metabolism, and plays a pivotal role in the evolution of resistance to various types of insecticides. However, the specific functions of GST genes in clothianidin resistance remain obscure in Bradysia odoriphaga. Here, a specific GST inhibitor, diethyl maleate (DEM), significantly increased the mortality of Bradysia odoriphaga larvae following exposure to clothianidin, and the activity of GST enzyme in clothianidin-resistant (CL-R) strain of Bradysia odoriphaga was markedly greater than that in the SS strain. Two sigma BoGSTs (BoGSTs1 and BoGSTs2) were markedly overexpressed in the CL-R strain and exhibited a higher abundance in the Malpighian tubules or midgut. Exposure to clothianidin resulted in a significant increased expression of BoGSTs1 and BoGSTs2. The knockdown of BoGSTs1 and BoGSTs2 increased sensitivity of larvae to clothianidin in the resistant strain. Furthermore, overexpression of BoGSTs1 and BoGSTs2 led to a significant increase in Escherichia coli cells tolerance to clothianidin. In vitro metabolic assays indicate that these two GSTs cannot directly metabolize clothianidin and its secondary metabolite desmethyl-clothianidin. Disk diffusion assays and fluorescence competitive binding assays indicated that BoGSTs1 and BoGSTs2 play a critical role in clothianidin resistance by antioxidant activity and non-catalytic binding activity. The docking results showed that BoGSTs1 and BoGSTs2 have strong binding affinity toward clothianidin. Collectively, these findings pinpoint the potential role of BoGSTs1 and BoGSTs2 in conferring insecticide resistance in Bradysia odoriphaga and contribute to our understanding of the underlying mechanisms of insecticide resistance. © 2024 Society of Chemical Industry.