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AIMS: The present study investigated the vasorelaxant mechanisms of an oral antidiabetic drug, anagliptin, using phenylephrine (Phe)-induced pre-contracted rabbit aortic rings. METHODS: Arterial tone measurement was performed in rabbit thoracic aortic rings. RESULTS: Anagliptin induced vasorelaxation in a dose-dependent manner. Pre-treatment with the classical voltagedependent K+ (Kv) channel inhibitors 4-aminopyridine and tetraethylammonium significantly decreased the vasorelaxant effect of anagliptin, whereas pre-treatment with the inwardly rectifying K+ (Kir) channel inhibitor Ba2+, the ATP-sensitive K+ (KATP) channel inhibitor glibenclamide, and the large-conductance Ca2+-activated K+ (BKCa) channel inhibitor paxilline did not attenuate the vasorelaxant effect. Furthermore, the vasorelaxant response of anagliptin was effectively inhibited by pre-treatment with the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitors thapsigargin and cyclopiazonic acid. Neither cAMP/protein kinase A (PKA)-related signaling pathway inhibitors (adenylyl cyclase inhibitor SQ 22536 and PKA inhibitor KT 5720) nor cGMP/protein kinase G (PKG)-related signaling pathway inhibitors (guanylyl cyclase inhibitor ODQ and PKG inhibitor KT 5823) reduced the vasorelaxant effect of anagliptin. Similarly, the anagliptin-induced vasorelaxation was independent of the endothelium. CONCLUSIONS: Based on these results, we suggest that anagliptin-induced vasorelaxation in rabbit aortic smooth muscle occurs by activating Kv channels and the SERCA pump, independent of other vascular K+ channels, cAMP/PKA- or cGMP/PKG-related signaling pathways, and the endothelium.
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Voltage-dependent K+ (Kv) channels play an important role in restoring the membrane potential to its resting state, thereby maintaining vascular tone. In this study, native smooth muscle cells from rabbit coronary arteries were used to investigate the inhibitory effect of quetiapine, an atypical antipsychotic agent, on Kv channels. Quetiapine showed a concentration-dependent inhibition of Kv channels, with an IC50 of 47.98 ± 9.46 µM. Although quetiapine (50 µM) did not alter the steady-state activation curve, it caused a negative shift in the steady-state inactivation curve. The application of 1 and 2 Hz train steps in the presence of quetiapine significantly increased the inhibition of Kv current. Moreover, the recovery time constants from inactivation were prolonged in the presence of quetiapine, suggesting that its inhibitory action on Kv channels is use (state)-dependent. The inhibitory effects of quetiapine were not significantly affected by pretreatment with Kv1.5, Kv2.1, and Kv7 subtype inhibitors. Based on these findings, we conclude that quetiapine inhibits Kv channels in both a concentration- and use (state)-dependent manner. Given the physiological significance of Kv channels, caution is advised in the use of quetiapine as an antipsychotic due to its potential side effects on cardiovascular Kv channels.
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Antipsicóticos , Vasos Coronários , Músculo Liso Vascular , Miócitos de Músculo Liso , Bloqueadores dos Canais de Potássio , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Fumarato de Quetiapina , Fumarato de Quetiapina/farmacologia , Animais , Coelhos , Antipsicóticos/farmacologia , Antipsicóticos/toxicidade , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Vasos Coronários/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Masculino , Relação Dose-Resposta a Droga , Potenciais da Membrana/efeitos dos fármacos , Células CultivadasRESUMO
We explored the vasorelaxant effects of ipragliflozin, a sodium-glucose cotransporter-2 inhibitor, on rabbit femoral arterial rings. Ipragliflozin relaxed phenylephrine-induced pre-contracted rings in a dose-dependent manner. Pre-treatment with the ATP-sensitive K+ channel inhibitor glibenclamide (10 µM), the inwardly rectifying K+ channel inhibitor Ba2+ (50 µM), or the Ca2+-sensitive K+ channel inhibitor paxilline (10 µM) did not influence the vasorelaxant effect. However, the voltage-dependent K+ (Kv) channel inhibitor 4-aminopyridine (3 mM) reduced the vasorelaxant effect. Specifically, the vasorelaxant response to ipragliflozin was significantly attenuated by pretreatment with the Kv7.X channel inhibitors linopirdine (10 µM) and XE991 (10 µM), the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitors thapsigargin (1 µM) and cyclopiazonic acid (10 µM), and the cAMP/protein kinase A (PKA)-associated signaling pathway inhibitors SQ22536 (50 µM) and KT5720 (1 µM). Neither the cGMP/protein kinase G (PKG)-associated signaling pathway nor the endothelium was involved in ipragliflozin-induced vasorelaxation. We conclude that ipragliflozin induced vasorelaxation of rabbit femoral arteries by activating Kv channels (principally the Kv7.X channel), the SERCA pump, and the cAMP/PKA-associated signaling pathway independent of other K+ (ATP-sensitive K+, inwardly rectifying K+, and Ca2+-sensitive K+) channels, cGMP/PKG-associated signaling, and the endothelium.
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Proteínas Quinases Dependentes de AMP Cíclico , Artéria Femoral , Glucosídeos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Transdução de Sinais , Tiofenos , Vasodilatação , Animais , Coelhos , Artéria Femoral/efeitos dos fármacos , Artéria Femoral/fisiologia , Vasodilatação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Tiofenos/farmacologia , Masculino , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Vasodilatadores/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidoresRESUMO
Plasmodium vivax, the most widespread human malaria parasite, and P. knowlesi, an emerging Plasmodium that infects humans, are the phylogenetically closest malarial species that infect humans, which may induce cross-species reactivity across most co-endemic areas in Southeast Asia. The thrombospondin-related anonymous protein (TRAP) family is indispensable for motility and host cell invasion in the growth and development of Plasmodium parasites. The merozoite-specific TRAP (MTRAP), expressed in blood-stage merozoites, is supposed to be essential for human erythrocyte invasion. We aimed to characterize MTRAPs in blood-stage P. vivax and P. knowlesi parasites and ascertain their cross-species immunoreactivity. Recombinant P. vivax and P. knowlesi MTRAPs of full-length ectodomains were expressed in a mammalian expression system. The MTRAP-specific immunoglobulin G, obtained from immune animals, was used in an immunofluorescence assay for subcellular localization and invasion inhibitory activity in blood-stage parasites was determined. The cross-species humoral immune responses were analyzed in the sera of patients with P. vivax or P. knowlesi infections. The MTRAPs of P. vivax (PvMTRAP) and P. knowlesi (PkMTRAP) were localized on the rhoptry body of merozoites in blood-stage parasites. Both anti-PvMTRAP and anti-PkMTRAP antibodies inhibited erythrocyte invasion of blood-stage P. knowlesi parasites. The humoral immune response to PvMTRAP showed high immunogenicity, longevity, and cross-species immunoreactivity with P. knowlesi. MTRAPs are promising candidates for development of vaccines and therapeutics against vivax and knowlesi malaria.
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Malária Vivax , Malária , Parasitos , Plasmodium , Animais , Humanos , Plasmodium vivax/genética , Parasitos/metabolismo , Merozoítos , Trombospondinas/metabolismo , Plasmodium/metabolismo , Malária/parasitologia , Malária Vivax/parasitologia , Proteínas de Protozoários/metabolismo , Mamíferos/metabolismoRESUMO
The increasing utilization of artificial intelligence algorithms in drug development has proven to be highly efficient and effective. One area where deep learning-based approaches have made significant contributions is in drug repositioning, enabling the identification of new therapeutic applications for existing drugs. In the present study, a trained deep-learning model was employed to screen a library of FDA-approved drugs to discover novel inhibitors targeting JAK2. To accomplish this, reference datasets containing active and decoy compounds specific to JAK2 were obtained from the DUD-E database. RDKit, a cheminformatic toolkit, was utilized to extract molecular features from the compounds. The DeepChem framework's GraphConvMol, based on graph convolutional network models, was applied to build a predictive model using the DUD-E datasets. Subsequently, the trained deep-learning model was used to predict the JAK2 inhibitory potential of FDA-approved drugs. Based on these predictions, ribociclib, topiroxostat, amodiaquine, and gefitinib were identified as potential JAK2 inhibitors. Notably, several known JAK2 inhibitors demonstrated high potential according to the prediction results, validating the reliability of our prediction model. To further validate these findings and confirm their JAK2 inhibitory activity, molecular docking experiments were conducted using tofacitinib-an FDA-approved drug for JAK2 inhibition. Experimental validation successfully confirmed our computational analysis results by demonstrating that these novel drugs exhibited comparable inhibitory activity against JAK2 compared to tofacitinib. In conclusion, our study highlights how deep learning models can significantly enhance virtual screening efforts in drug discovery by efficiently identifying potential candidates for specific targets such as JAK2. These newly discovered drugs hold promises as novel JAK2 inhibitors deserving further exploration and investigation.
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Inteligência Artificial , Reposicionamento de Medicamentos , Simulação de Acoplamento Molecular , Reprodutibilidade dos Testes , Redes Neurais de ComputaçãoRESUMO
Malaria eradication efforts in resource-limited areas require a rapid, economical, and accurate tool for detecting of the low parasitemia. The malaria rapid diagnostic test (mRDT) is the most suitable for on-site detection of the deadliest form of malaria, Plasmodium falciparum. However, the deletions of histidine rich protein 2 and 3 genes are known to compromise the effectiveness of mRDT. One of the approaches that have been explored intensively for on-site diagnostics is the loop-mediated isothermal amplification (LAMP). LAMP is a one-step amplification that allows the detection of Plasmodium species in less than an hour. Thus, this study aims to present a new primer set to enhance the performance of a colorimetric LAMP (cLAMP) for field application. The primer binding regions were selected within the A-type of P. falciparum 18S rRNA genes, which presents a dual gene locus in the genome. The test result of the newly designed primer indicates that the optimal reaction condition for cLAMP was 30 minutes incubation at 65°C, a shorter incubation time compared to previous LAMP detection methods that typically takes 45 to 60 minutes. The limit of detection (LoD) for the cLAMP using our designed primers and laboratory-grown P. falciparum (3D7) was estimated to be 0.21 parasites/µL which was 1,000-fold higher than referencing primers. Under optimal reaction condition, the new primer sets showed the sensitivity (100%, 95% CI: 80.49-100%) and specificity (100%, 95% CI: 94.64-100%) with 100% (95% CI: 95.70-100%) accuracy on the detection of dried blood spots from Malawi (n = 84). Briefly, the newly designed primer set for P. falciparum detection exhibited high sensitivity and specificity compared to referenced primers. One great advantage of this tool is its ability to be detected by the naked eye, enhancing field approaches. Thus, this tool has the potential to be effective for accurate early parasite detection in resource-limited endemic areas.
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Malária Falciparum , Malária , Humanos , Plasmodium falciparum/genética , Colorimetria , Sensibilidade e Especificidade , Malária/parasitologia , Malária Falciparum/diagnóstico , Malária Falciparum/parasitologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Diagnóstico Molecular/métodosRESUMO
Clonorchis sinensis is commonly found in East Asian countries. Clonorchiasis is prevalent in these countries and can lead to various clinical symptoms. In this study, we used overlap extension polymerase chain reaction (PCR) and the Xenopus laevis oocyte expression system to isolate a cDNA encoding the choline transporter of C. sinensis (CsChT). We subsequently characterized recombinant CsChT. Expression of CsChT in X. laevis oocytes enabled efficient transport of radiolabeled choline, with no detectable uptake of arginine, α-ketoglutarate, p-aminohippurate, taurocholate, and estrone sulfate. Influx and efflux experiments showed that CsChT-mediated choline uptake was time- and sodium-dependent, with no exchange properties. Concentration-dependent analyses of revealed saturable kinetics consistent with the Michaelis-Menten equation, while nonlinear regression analyses revealed a Km value of 8.3 µM and a Vmax of 61.0 pmol/oocyte/h. These findings contribute to widen our understanding of CsChT transport properties and the cascade of choline metabolisms within C. sinensis.
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Clonorchis sinensis , Animais , Clonorchis sinensis/genética , Proteínas de Membrana Transportadoras/metabolismo , Transporte Biológico , Colina/metabolismoRESUMO
γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades γ-aminobutyric (GABA) in the brain. GABA is an important inhibitory neurotransmitter that plays important neurological roles in the brain. Therefore, GABA-AT is an important drug target that regulates GABA levels. Novel and potent drug development to inhibit GABA-AT is still a very challenging task. In this study, we aimed to devise novel and potent inhibitors against GABA-AT using computer-aided drug design (CADD) tools. Since the crystal structure of human GABA-AT was not yet available, we utilized a homologous structure derived from our previously published paper. To identify highly potent compounds relative to vigabatrin, an FDA-approved drug against human GABA-AT, we developed a pharmacophore analysis protocol for 530,000 Korea Chemical Bank (KCB) compounds and selected the top 50 compounds for further screening. Preliminary biological analysis was carried out for these 50 compounds and 16 compounds were further assessed. Subsequently, molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations were carried out. In the results, four predicted compounds, A07, B07, D08, and H08, were found to be highly potent and were further evaluated by a biological activity assay to confirm the results of the GABA-AT activity inhibition assay.
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4-Aminobutirato Transaminase , Vigabatrina , Humanos , Simulação de Acoplamento Molecular , Ácido gama-Aminobutírico/metabolismo , Simulação de Dinâmica Molecular , Fosfato de Piridoxal/metabolismoRESUMO
The zoonotic malaria parasite Plasmodium knowlesi is an important public health concern in Southeast Asia. Invasion of host erythrocytes is essential for parasite growth, and thus, understanding the repertoire of parasite proteins that enable this process is vital for identifying vaccine candidates and how some species are able to cause zoonotic infection. Merozoite surface protein 1 (MSP1) is found in all malaria parasite species and is perhaps the most well-studied as a potential vaccine candidate. While MSP1 is encoded by a single gene in P. falciparum, all other human infective species (P. vivax, P. knowlesi, P. ovale, and P. malariae) additionally encode a divergent paralogue known as MSP1P, and little is known about its role or potential functional redundancy with MSP1. We, therefore, studied the function of P. knowlesi merozoite surface protein 1 paralog (PkMSP1P), using both recombinant protein and CRISPR-Cas9 genome editing. The recombinant 19-kDa C-terminus of PkMSP1P (PkMSP1P-19) was shown to bind specifically to human reticulocytes. However, immunoblotting data suggested that PkMSP1P-19-induced antibodies can recognize PkMSP1-19 and vice versa, confounding our ability to separate the properties of these two proteins. Targeted disruption of the pkmsp1p gene profoundly impacts parasite growth, demonstrating for the first time that PkMSP1P is important in in vitro growth of P. knowlesi and likely plays a distinct role from PkMSP1. Importantly, the MSP1P KO also enabled functional characterization of the PkMSP1P-19 antibodies, revealing clear immune cross-reactivity between the two paralogues, highlighting the vital importance of genetic studies in contextualizing recombinant protein studies.
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Malária Falciparum , Malária Vivax , Malária , Plasmodium knowlesi , Vacinas , Humanos , Proteína 1 de Superfície de Merozoito/genética , Plasmodium knowlesi/genética , Plasmodium knowlesi/metabolismo , Malária/parasitologia , Eritrócitos/parasitologia , Anticorpos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Caffeic acid (CA) derivatives have been reported to exert anti-inflammatory activities in various inflammatory conditions. However, the impact of CA methyl ester (CAME) on the inflammatory response in vascular endothelial cells has not been thoroughly elucidated. In the present study, the aim was to understand how CAME can reduce inflammation in human umbilical vein endothelial cells (HUVECs), which were challenged with lipopolysaccharide (LPS), and elucidate its mechanisms. CAME significantly attenuated LPS-induced TNF-α and IL-1ß release. Furthermore, CAME inhibited cyclooxygenase 2 expression and consequent secretion of prostaglandin E2. CAME also suppressed LPS-stimulated inducible nitric oxide synthase expression. In addition, CAME significantly enhanced the expression of heme oxygenase-1 (HO-1) and nuclear factor erythroid-derived 2-related factor 2 (Nrf2) phosphorylation in the absence or presence of LPS stimulation in HUVECs. CAME also significantly suppressed LPS-induced NF-κB phosphorylation and inhibitor of κB phosphorylation and degradation. In conclusion, the present results provide clear evidence that CAME exerts its anti-inflammatory activities by increasing HO-1/Nrf2-mediated cytoprotection and inhibiting NF-κB-mediated pro-inflammatory pathways in HUVECs.
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BACKGROUND: The Plasmodium vivax merozoite restrictively invades immature erythrocytes, suggesting that its ligand(s) might interact with corresponding receptor(s) that are selectively abundant on reticulocytes to complete the invasion. Finding the ligandâreceptor interaction involved in P. vivax invasion is critical to vivax malaria management; nevertheless, it remains to be unraveled. METHODS: A library of reticulocyte receptors and P. vivax ligands were expressed by a HEK293E mammalian cell expression system and were then used to screen the interaction using enzyme-linked immunosorbent assay (ELISA). A flow cytometry-based erythrocyte binding assay and bio-layer interferometry experiment were further utilized to cellularly and quantitatively identify the ligandâreceptor interaction, respectively. RESULTS: Plasmodium vivax merozoite-specific thrombospondin-related anonymous protein (PvMTRAP) was found to interact with human CD36 using systematic screening. This interaction was specific at a molecular level from in vitro analysis and comparable to that of P. vivax Duffy binding protein (PvDBP) and Duffy antigen receptor for chemokines (DARC) (KD: 37.0 ± 1.4 nM and 7.7 ± 0.5 nM, respectively). Flow cytometry indicated that PvMTRAP preferentially binds to reticulocytes, on which CD36 is selectively present. CONCLUSIONS: Human CD36 is selectively abundant on reticulocytes and is able to interact specifically with PvMTRAP, suggesting that it may function as a ligand and receptor during the invasion of reticulocytes by P. vivax.
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Malária Vivax , Plasmodium vivax , Animais , Humanos , Reticulócitos , Ligantes , Merozoítos , Trombospondinas , MamíferosRESUMO
Machine learning algorithms have been increasingly applied in drug development due to their efficiency and effectiveness. Machine learning-based drug repurposing can contribute to the identification of novel therapeutic applications for drugs with other indications. The current study used a trained machine learning model to screen a vast chemical library for new JAK2 inhibitors, the biological activities of which were reported. Reference JAK2 inhibitors, comprising 1911 compounds, have experimentally determined IC50 values. To generate the input to the machine learning model, reference compounds were subjected to RDKit, a cheminformatic toolkit, to extract molecular descriptors. A Random Forest Regression model from the Scikit-learn machine learning library was applied to obtain a predictive regression model and to analyze each molecular descriptor's role in determining IC50 values in the reference data set. Then, IC50 values of the library compounds, comprised of 1,576,903 compounds, were predicted using the generated regression model. Interestingly, some compounds that exhibit high IC50 values from the prediction were reported to possess JAK inhibition activity, which indicates the limitations of the prediction model. To confirm the JAK2 inhibition activity of predicted compounds, molecular docking and molecular dynamics simulation were carried out with the JAK inhibitor reference compound, tofacitinib. The binding affinity of docked compounds in the active region of JAK2 was also analyzed by the gmxMMPBSA approach. Furthermore, experimental validation confirmed the results from the computational analysis. Results showed highly comparable outcomes concerning tofacitinib. Conclusively, the machine learning model can efficiently improve the virtual screening of drugs and drug development.
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Reposicionamento de Medicamentos , Inibidores de Janus Quinases , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Janus Quinase 2 , Aprendizado de Máquina , Inibidores de Janus Quinases/farmacologiaRESUMO
Artificial intelligence algorithms have been increasingly applied in drug development due to their efficiency and effectiveness. Deep-learning-based drug repurposing can contribute to the identification of novel therapeutic applications for drugs with other indications. The current study used a trained deep-learning model to screen an FDA-approved drug library for novel COX-2 inhibitors. Reference COX-2 data sets, composed of active and decoy compounds, were obtained from the DUD-E database. To extract molecular features, compounds were subjected to RDKit, a cheminformatic toolkit. GraphConvMol, a graph convolutional network model from DeepChem, was applied to obtain a predictive model from the DUD-E data sets. Then, the COX-2 inhibitory potential of the FDA-approved drugs was predicted using the trained deep-learning model. Vismodegib, an anticancer agent that inhibits the hedgehog signaling pathway by binding to smoothened, was predicted to inhibit COX-2. Noticeably, some compounds that exhibit high potential from the prediction were known to be COX-2 inhibitors, indicating the prediction model's liability. To confirm the COX-2 inhibition activity of vismodegib, molecular docking was carried out with the reference compounds of the COX-2 inhibitor, celecoxib, and ibuprofen. Furthermore, the experimental examination of COX-2 inhibition was also carried out using a cell culture study. Results showed that vismodegib exhibited a highly comparable COX-2 inhibitory activity compared to celecoxib and ibuprofen. In conclusion, the deep-learning model can efficiently improve the virtual screening of drugs, and vismodegib can be used as a novel COX-2 inhibitor.
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The cell-traversal protein for ookinetes and sporozoites (CelTOS), expressed on the surface of ookinetes and sporozoitesin Plasmodium species, is a promising malaria vaccine candidate. CelTOS is essential for parasite invasion into mosquito midgut and human hepatocytes, thereby contributing to malaria transmission and disease pathogenesis. This study explores the genetic diversity, polymorphisms, haplotypes, natural selection, phylogenetic analysis, and epitope prediction in the full-length Plasmodium knowlesi CelTOS gene in clinical samples from Sarawak, Malaysian Borneo, and long-term laboratory strains from Peninsular Malaysia and the Philippines. Our analysis revealed a high level of genetic variation in the PkCelTOS gene, with a nucleotide diversity of π ~ 0.021, which was skewed towards the 3' end of the gene. This level of diversity is double that observed in PfCelTOS and 20 times that observed in PvCelTOS from worldwide clinical samples. Tests of natural selection revealed evidence for positive selection within clinical samples. Phylogenetic analysis of the amino acid sequence of PkCelTOS revealed the presence of two distinct groups, although no geographical clustering was observed. Epitope prediction analysis identified two potential epitopes (96AQLKATA102 and 124TIKPPRIKED133) using the IEDB server and one epitope (125IKPPRIKED133) by Bcepred server on the C' terminal region of PkCelTOS protein. Both the servers predicted a common epitope region of nine amino acid length (IKPPRIKED) peptide, which can be studied in the future as a potential candidate for vaccine development. These findings shed light on the genetic diversity, polymorphism, haplotypes, and natural selection within PkCelTOS in clinical samples and provide insights about its future prospects as a potential candidate for P. knowlesi malaria vaccine development.
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Gamma-aminobutyric acid (GABA) transaminase-also called GABA aminotransferase (GABA-AT)-deficiency is a rare autosomal recessive disorder characterized by a severe neonatal-infantile epileptic encephalopathy with symptoms such as seizures, hypotonia, hyperreflexia, developmental delay, and growth acceleration. GABA transaminase deficiency is caused by mutations in GABA-AT, the enzyme responsible for the catabolism of GABA. Mutations in multiple locations on GABA-AT have been reported and their locations have been shown to influence the onset of the disease and the severity of symptoms. We examined how GABA-AT mutations influence the structural stability of the enzyme and GABA-binding affinity using computational methodologies such as molecular dynamics simulation and binding free energy calculation to understand the underlying mechanism through which GABA-AT mutations cause GABA-AT deficiency. GABA-AT 3D model depiction was carried out together with seven individual mutated models of GABA-AT. The structural stability of all the predicted models was analyzed using several tools and web servers. All models were evaluated based on their phytochemical values. Additionally, 100 ns MD simulation was carried out and the mutated models were evaluated using RMSD, RMSF, Rg, and SASA. gmxMMPBSA free energy calculation was carried out. Moreover, RMSD and free energy calculations were also compared with those obtained using online web servers. Our study demonstrates that P152S, Q296H, and R92Q play a more critical role in the structural instability of GABA-AT compared with the other mutated models: G465R, L211F, L478P, and R220K.
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4-Aminobutirato Transaminase , Transaminases , 4-Aminobutirato Transaminase/genética , Transaminases/genética , Transaminases/metabolismo , Mutação , Simulação de Dinâmica Molecular , Ácido gama-Aminobutírico/genéticaRESUMO
Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX1) is an orphan nuclear receptor encoded by the NR0B1 gene. The functional study showed that DAX1 is a physiologically significant target for EWS/FLI1-mediated oncogenesis, particularly Ewing Sarcoma (ES). In this study, a three-dimensional DAX1 structure was modeled by employing a homology modeling approach. Furthermore, the network analysis of genes involved in Ewing Sarcoma was also carried out to evaluate the association of DAX1 and other genes with ES. Moreover, a molecular docking study was carried out to check the binding profile of screened flavonoid compounds against DAX1. Therefore, 132 flavonoids were docked in the predicted active binding pocket of DAX1. Moreover, the pharmacogenomics analysis was performed for the top ten docked compounds to evaluate the ES-related gene clusters. As a result, the five best flavonoid-docked complexes were selected and further evaluated by Molecular Dynamics (MD) simulation studies at 100 ns. The MD simulation trajectories were evaluated by generating RMSD, hydrogen bond plot analysis, and interaction energy graphs. Our results demonstrate that flavonoids showed interactive profiles in the active region of DAX1 and can be used as potential therapeutic agents against DAX1-mediated augmentation of ES after in-vitro and in-vivo evaluations.
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Sarcoma de Ewing , Humanos , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/genética , Receptores do Ácido Retinoico/metabolismo , Proteínas Repressoras/genética , Simulação de Acoplamento MolecularRESUMO
BACKGROUND: Plasmodium vivax (P. vivax) is one of the highly prevalent human malaria parasites. Due to the presence of extravascular reservoirs, P. vivax is extremely challenging to manage and eradicate. Traditionally, flavonoids have been widely used to combat various diseases. Recently, biflavonoids were discovered to be effective against Plasmodium falciparum. METHOD: In this study, in silico approaches were utilized to inhibit Duffy binding protein (DBP), responsible for Plasmodium invasion into red blood cells (RBC). The interaction of flavonoid molecules with the Duffy antigen receptor for chemokines (DARC) binding site of DBP was investigated using a molecular docking approach. Furthermore, molecular dynamic simulation studies were carried out to study the stability of top-docked complexes. RESULTS: The results showed the effectiveness of flavonoids, such as daidzein, genistein, kaempferol, and quercetin, in the DBP binding site. These flavonoids were found to bind in the active region of DBP. Furthermore, the stability of these four ligands was maintained throughout the 50 ns simulation, maintaining stable hydrogen bond formation with the active site residues of DBP. CONCLUSION: The present study suggests that flavonoids might be good candidates and novel agents against DBP-mediated RBC invasion of P. vivax and can be further analyzed in in vitro studies.
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Anti-inflammatory effects of caffeic acid derivatives have been widely reported. However, the effect of caffeic acid methyl ester (CAME) on the anti-allergic effect in mast cells has not been elucidated. The present study was aimed to investigate the anti-allergic properties of CAME and its underlying mechanism. Rat basophilic leukemia (RBL-2H3) cells were incubated withphorbol-12-myristate-13-acetate (PMA) and a calcium ionophore, A23187 to induce mast cell activation. Anti-allergic effect of CAME was examined by measuring cytokine, histamine and ß-hexosaminidase release. Western blotting was conducted to determine cyclooxygenase-2 (COX-2) expression, Mitogen-activated protein kinases (MAPKs) activation and nuclear factor-κB (NF-κB) translocation. CAME significantly suppressed PMA/A23187-induced TNF-α secretion, and ß-hexosaminidase and histamine release in a concentration-dependent manner. Furthermore, CAME significantly attenuated PMA/A23187-induced COX-2 expression and nuclear translocation of NF-κB. CAME significantly suppressed PMA/A23187-induced increased phosphorylation of p38, ERK and JNK RBL-2H3 cells. The results demonstrate that CAME significantly attenuates anti-allergic action by suppressing degranulation of mast cells through the suppression of MAPKs/NF-κB signaling pathway in RBL-2H3 cells.