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Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNa(V)) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNa(V) CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNa(V) CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNa(V) voltage dependencies, and demonstrate that a discrete domain can encode the temperature-dependent response of a channel.
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Proteínas Bacterianas/química , Gammaproteobacteria/metabolismo , Canales de Sodio Activados por Voltaje/química , Secuencia de Aminoácidos , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Desplegamiento Proteico , Alineación de SecuenciaRESUMEN
The ATP-binding cassette subfamily B member 1 (ABCB1), encoding a multidrug transporter referred to as P-glycoprotein (Pgp), plays a critical role in the efflux of xenobiotics in humans and is implicated in cancer resistance to chemotherapy. Therefore, developing high-throughput animal models to screen for Pgp function and bioavailability of substrates and inhibitors is paramount. Here, we generated and validated a zebrafish knockout line of abcb4, a human Pgp transporter homolog. CRISPR/Cas9 genome editing technology was deployed to generate a frameshift mutation in exon 4 of zebrafish abcb4. The zebrafish abcb4 homozygous mutant exhibited elevated accumulation of fluorescent rhodamine 123, a substrate of human Pgp, in the intestine and brain area of embryos. Moreover, abcb4 knockout embryos were sensitized toward toxic compounds such as doxorubicin and vinblastine compared to the WT zebrafish. Immunostaining for zebrafish Abcb4 colocalized in the endothelial brain cells of adult zebrafish. Transcriptome profiling using Gene Set Enrichment Analysis uncovered that the 'cell cycle process,' 'mitotic cell cycles,' and 'microtubule-based process' were significantly downregulated in the abcb4 knockout brain with age. This study establishes and validates the abcb4 knockout zebrafish as an animal model to study Pgp function in vivo. Unexpectedly it reveals a potentially novel role for zebrafish abcb4 in age-related changes in the brain. The zebrafish lines generated here will provide a platform to aid in the discovery of modulators of Pgp function as well as the characterization of human mutants thereof.
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Doxorrubicina , Pez Cebra , Adulto , Animales , Humanos , Pez Cebra/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Exones , Células EndotelialesRESUMEN
Substrate efflux by ATP-binding cassette (ABC) transporters, which play a major role in multidrug resistance, entails the ATP-powered interconversion between transporter intermediates. Despite recent progress in structure elucidation, a number of intermediates have yet to be visualized and mechanistically interpreted. Here, we combine cryogenic-electron microscopy (cryo-EM), double electron-electron resonance spectroscopy and molecular dynamics simulations to profile a previously unobserved intermediate of BmrCD, a heterodimeric multidrug ABC exporter from Bacillus subtilis. In our cryo-EM structure, ATP-bound BmrCD adopts an inward-facing architecture featuring two molecules of the substrate Hoechst-33342 in a striking asymmetric head-to-tail arrangement. Deletion of the extracellular domain capping the substrate-binding chamber or mutation of Hoechst-coordinating residues abrogates cooperative stimulation of ATP hydrolysis. Together, our findings support a mechanistic role for symmetry mismatch between the nucleotide binding and the transmembrane domains in the conformational cycle of ABC transporters and is of notable importance for rational design of molecules for targeted ABC transporter inhibition.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Adenosina Trifosfato/química , Proteínas Bacterianas/metabolismo , Bencimidazoles , Sitios de Unión , Clostridium/metabolismo , Microscopía por Crioelectrón , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
INTRODUCTION: RNF213 mutations have been reported mostly in moyamoya disease (MMD) with varying frequencies across different ethnicities. However, its prevalence in non-MMD adult-onset ischemic stroke is still not well explored. AIMS AND OBJECTIVES: This present study thus aims to screen the most common RNF213 variant (Arg4810Lys, among East Asians) in the Eastern Indian non-MMD ischemic stroke patients and correlate it with long-term progression and prognosis of the patients. The subjects were analyzed for this variant using PCR-RFLP and confirmed using Sanger sequencing method. RESULT AND CONCLUSION: We have identified Arg4810Lys variant among eleven young-onset familial ischemic stroke patients in heterozygous manner. A positive correlation of the variant with positive family history (P = 0.001), earlier age at onset (P = 0.002), and history of recurrent stroke (P = 0.015) was observed. However, the carriers showed better cognitive performances in memory (P = 0.042) and executive function (P = 0.004). Therefore, we can conclude that Arg4810Lys/RNF213 - a pathogenic variant for young-onset familial ischemic stroke with higher incidence of recurrent events unlike in MMD cases, have no additional impact on cognition among Eastern Indians.
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Accidente Cerebrovascular Isquémico , Enfermedad de Moyamoya , Adulto , Humanos , Enfermedad de Moyamoya/epidemiología , Predisposición Genética a la Enfermedad , Adenosina Trifosfatasas/genética , Ubiquitina-Proteína Ligasas/genética , Estudios de Asociación Genética , Mutación/genéticaRESUMEN
Alzheimer disease and Parkinson disease dementia are the 2 most common neurodegenerative diseases have substantial overlap in pathologic, genetic, and clinical manifestation and complex in nature. Here, for the first time, we report an Indian female young patient who presented with clinical manifestation of both Alzheimer disease and Parkinsonism, including dystonia with rapid disease progression. We identified a heterozygous mutation in the ATP-binding cassette transporter A7 gene and double heterozygous mutation in PRKN by whole-exome sequencing. This case is an example of complex etiology of neurodegenerative disorders and highlights the importance of genetic tests, including whole-exome sequencing in complex diseases.
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Enfermedad de Alzheimer , Demencia , Enfermedad de Parkinson , Trastornos Parkinsonianos , Femenino , Humanos , Transportadoras de Casetes de Unión a ATP/genética , Demencia/genética , Secuenciación del Exoma , Mutación/genética , Trastornos Parkinsonianos/genéticaRESUMEN
The small multidrug transporter from Escherichia coli, EmrE, couples the energetically uphill extrusion of hydrophobic cations out of the cell to the transport of two protons down their electrochemical gradient. Although principal mechanistic elements of proton/substrate antiport have been described, the structural record is limited to the conformation of the substrate-bound state, which has been shown to undergo isoenergetic alternating access. A central but missing link in the structure/mechanism relationship is a description of the proton-bound state, which is an obligatory intermediate in the transport cycle. Here we report a systematic spin labeling and double electron electron resonance (DEER) study that uncovers the conformational changes of EmrE subsequent to protonation of critical acidic residues in the context of a global description of ligand-induced structural rearrangements. We find that protonation of E14 leads to extensive rotation and tilt of transmembrane helices 1-3 in conjunction with repacking of loops, conformational changes that alter the coordination of the bound substrate and modulate its access to the binding site from the lipid bilayer. The transport model that emerges from our data posits a proton-bound, but occluded, resting state. Substrate binding from the inner leaflet of the bilayer releases the protons and triggers alternating access between inward- and outward-facing conformations of the substrate-loaded transporter, thus enabling antiport without dissipation of the proton gradient.
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Antiportadores/química , Proteínas de Escherichia coli/química , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Conformación Proteica , Protones , Difracción de Rayos XRESUMEN
Multidrug antiporters of the major facilitator superfamily couple proton translocation to the extrusion of cytotoxic molecules. The conformational changes that underlie the transport cycle and the structural basis of coupling of these transporters have not been elucidated. Here we used extensive double electron-electron resonance measurements to uncover the conformational equilibrium of LmrP, a multidrug transporter from Lactococcus lactis, and to investigate how protons and ligands shift this equilibrium to enable transport. We find that the transporter switches between outward-open and outward-closed conformations, depending on the protonation states of specific acidic residues forming a transmembrane protonation relay. Our data can be framed in a model of transport wherein substrate binding initiates the transport cycle by opening the extracellular side. Subsequent protonation of membrane-embedded acidic residues induces substrate release to the extracellular side and triggers a cascade of conformational changes that concludes in proton release to the intracellular side.
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Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Protones , Antiportadores/metabolismo , Proteínas Bacterianas/química , Transporte Biológico , Concentración de Iones de Hidrógeno , Ligandos , Proteínas de Transporte de Membrana/química , Conformación Proteica , Estabilidad ProteicaRESUMEN
Electrochemical sensors offer promising prospects for real-time pollutant monitoring. In this study, copper oxide-dispersed graphitic carbon nanofibers (CuO-CNFs) grown via chemical vapour deposition were employed as a robust platform for detecting a variety of environmental pollutants. This array-based sensor adeptly identifies three different classes of analytes, i. e., antibiotics (chloramphenicol (CP) and tylosin tartrate (TT)), heavy metals (cadmium (Cd) and lead (Pb)), and pesticides (quinalphos (QP) and imidacloprid (IP)). Electron collection is facilitated by a glassy carbon electrode, while various physico-electrochemical methods delve into the properties of CuO-CNFs. The CuO-CNF-modified GCE array rapidly discerns (<15â sec) a broad linear range: 1-20â ppm for CP, 1-13.33â ppm for TT, 0.66-11.66â ppm for Cd, 20-33.33â ppm for Pb, 1.6-11.6â ppm for QP, and 5-25â ppm for IP, boasting quantification limits of 1.0, 1.0, 0.66, 20.0, 1.6, and 5.0â ppm for CP, TT, Cd, Pb, QP, and IP, respectively. Notably, this sensor achieves simultaneous identification of mixed analytes, including CP and TT, Cd and Pb, and QP and IP, within real tap water. Furthermore, the electrochemical sensor exhibits robustness; heightened sensitivity, selectivity, and stability; a swift response; and impressive reproducibility in detecting CP, TT, Cd, Pb, QP, and IP within aqueous samples. Consequently, this array-based electrochemical sensor has emerged as a rapid and simultaneous detection tool for diverse pollutant residues in surface and groundwater samples.
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Antibacterianos , Carbono , Cobre , Técnicas Electroquímicas , Metales Pesados , Nanofibras , Plaguicidas , Nanofibras/química , Metales Pesados/análisis , Metales Pesados/química , Cobre/química , Cobre/análisis , Plaguicidas/análisis , Antibacterianos/análisis , Antibacterianos/química , Técnicas Electroquímicas/métodos , Carbono/química , Electrodos , Contaminantes Químicos del Agua/análisis , Límite de DetecciónRESUMEN
NorM of the multidrug and toxic compound extrusion (MATE) family of transporters couples the efflux of a broad range of hydrophobic molecules to an inward Na⺠gradient across the cell membrane. Several crystal structures of MATE transporters revealed distinct substrate binding sites leading to differing models of the mechanism of ion-coupled substrate extrusion. In the experiments reported here, we observed that a spin-labeled derivative of daunorubicin, Ruboxyl, is transported by NorM from Vibrio cholerae. It is therefore ideal for characterizing mechanistically relevant binding interactions with NorM and directly addressing the coupling of ion and drug binding. Fluorescence and electron paramagnetic resonance experiments revealed that Ruboxyl binds to NorM with micromolar affinity and becomes immobilized upon binding, even in the presence of Naâº. Using double electron-electron resonance spectroscopy, we determined that Ruboxyl binds to a single site on the periplasmic side of the protein. The presence of Na⺠did not translocate the substrate to a second site as previously proposed. These experiments surprisingly show that Na⺠does not affect the affinity or location of the substrate binding site on detergent-solubilized NorM, thus suggesting that additional factors beyond simple mutual exclusivity of binding, such as the presence of a Na⺠gradient across the native membrane, govern Naâº-drug coupling during antiport.
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Antiportadores/metabolismo , Proteínas Bacterianas/metabolismo , Sodio/metabolismo , Sitios de Unión/efectos de los fármacos , Daunorrubicina/análogos & derivados , Daunorrubicina/metabolismo , Daunorrubicina/farmacología , Escherichia coli/efectos de los fármacos , Unión Proteica , Sodio/farmacología , Marcadores de Spin , Vibrio cholerae/químicaRESUMEN
The nucleocapsid component of SARS-CoV2 is involved in the viral genome packaging. GammaP.1(Brazil) and the 20 C-US(USA) variants had a high frequency of the P80R and P67S mutations respectively in the RNA-binding domain of the nucleocapsid. Since RNA-binding domain participates in the electrostatic interactions with the viral genome, the study of the effects of proline substitutions on the flexibility of the protein will be meaningful. It evinced that the trajectory of the wildtype and mutants was stable during the simulation and exhibited distinct changes in the flexibility of the protein. Moreover, the beta-hairpin loop region of the protein structures exhibited high amplitude fluctuations and dominant motions. Additionally, modulations were detected in the drug binding site. Besides, the extent of correlation and anti-correlation motions involving the protruding region, helix, and the other RNA binding sites differed between the wildtype and mutants. The secondary structure analysis disclosed the variation in the occurrence pattern of the secondary structure elements between the proteins. Protein-ssRNA interaction analysis was also done to detect the amino acid contacts with ssRNA. R44, R59, and Y61 residues of the wildtype and P80R mutant exhibited different duration contacts with the ssRNA. It was also noticed that R44, R59, and Y61 of the wildtype and P80R formed hydrogen bonds with the ssRNA. However in P67S, residues T43, R44, R45, R40, R59, and R41 displayed contacts and formed hydrogen bonds with ssRNA. Binding free energy was also calculated and was lowest for P67S than wildtype andP80R. Thus, proline substitutions influence the structure of the RNA-binding domain and may modulate viral genome packaging besides the host-immune response.Communicated by Ramaswamy H. Sarma.
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The ATP-binding cassette subfamily B member 1 (ABCB1), encoding a multidrug transporter referred to as P-glycoprotein (Pgp), plays a critical role in the efflux of xenobiotics in humans and is implicated in cancer resistance to chemotherapy. Therefore, developing high throughput animal models to screen for Pgp function and bioavailability of substrates and inhibitors is paramount. Here, we generated and validated a zebrafish knockout line of abcb4 , a human Pgp transporter homolog. CRISPR/Cas9 genome editing technology was deployed to generate a frameshift mutation in exon 4 of zebrafish abcb4 . The zebrafish abcb4 homozygous mutant exhibited elevated accumulation of fluorescent rhodamine 123, a substrate of human Pgp, in the intestine and brain area of embryos. Moreover, abcb4 knockout embryos were sensitized toward toxic compounds such as doxorubicin and vinblastine compared to the WT zebrafish. Immuno-staining for zebrafish Abcb4 colocalized in the endothelial brain cells of adult zebrafish. Transcriptome profiling using Gene Set Enrichment Analysis (GSEA) uncovered that the 'cell cycle process,' 'mitotic cell cycles,' and 'microtubule-based process' were significantly downregulated in the abcb4 knockout brain with age. This study establishes and validates the a bcb4 knockout zebrafish as an animal model to study Pgp function in vivo. Unexpectedly it reveals a potentially novel role for zebrafish abcb4 in age-related changes in the brain. The zebrafish lines generated here will provide a platform to aid in the discovery of modulators of Pgp function as well as the characterization of human mutants thereof.
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Epitopes are the cornerstones for the development of rational vaccine design strategies. Conventionally, epitopes are used by chemical conjugation with the carrier protein. This chapter describes our computational epitope grafting methodology to identify the preferential grafting site in a carrier protein/scaffold. We have used the mota epitope as an example, as it was already experimentally validated by an independent group. In this chapter, we have provided sufficient details to enable the wet experimentalist to employ this computational methodology in their research objective. Scripts/programs are extensively described in this chapter and freely accessible through the provided link.
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Proteínas Portadoras , Biología Computacional , Epítopos , Epítopos de Linfocito T , Epítopos de Linfocito BRESUMEN
BACKGROUND: T cell exhaustion and activation markers are helpful in determining the therapies and predicting the overall survival in pancreatic cancer (PC) patients. PURPOSE: In this systematic review, we have addressed two questions, how do these markers differ in their expression levels in PC patients and healthy individual and correlating the expression level of these markers with the cancer stage. METHODS: The systematic review was registered with Prospective Register of Systematic Reviews (PROSPERO) with registration number "CRD42022246780." All the included articles were obtained from three databases, PubMed, MEDLINE, and Cochrane, published from January 2010 to 26th May 2022. Two independent reviewers followed the PRISM protocol and reviewed and extracted data from the included articles. RESULTS: PD-1 and CTLA-4 were the most studied markers in this field. A clear elevation in the expression of PD-1, CTLA-4, TIM-3, LAG-3, and TIGIT was found in most of the studies. CD69, CD25, and HLA-DR expression was found to be upregulated after chemotherapy and immunotherapy. CD25 was the only marker analyzed against cancer progression, in a single study. No study compared the expression of exhaustion and activation markers (except CD69) with the cancer progression of the tumor stage. CONCLUSION: Since the exhaustion markers are upregulated in patients, single or multiple markers can be targeted in immunotherapies. Knowledge of the dynamics of these markers at various cancer stages will help in determining the right immunotherapy for pancreatic cancer patients. Stage-wise comparison could also be made possible by developing in vitro models.
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BACKGROUND: The main objective of this study was to determine the results of the cell-based assay (CDC-XM and FC-XM), and correlate with the results of solid phase assay (L-SAB). METHODS: In this retrospective study, 350 prospective renal transplant recipients were tested for the presence of HLA antibodies by CDC-XM, FC-XM and L-SAB screening with their corresponding donor. RESULTS: T-cell-FC-XM showed a sensitivity of 71.43% and a specificity of 91.50% for detecting class I L-SAB (+), while B-cell-FCXM showed a sensitivity of 94.94% and a specificity of 61.99% for detecting class II L-SAB (+). On the other hand, T-CDC-XM showed a sensitivity of 32.14% and a specificity of 98.64% for detecting class I L-SAB (+), while B-CDC-XM showed a sensitivity of 44.30% and a specificity of 94.83% for detecting class II L-SAB (+). In this study, the results indicated that DSA class I MFI value of 2845 and above significantly (p ≤0.001) correlated with T-cell-FC-XM positivity, while MFI value of 4585 and above (p ≤0.001) showed strong predictive accuracy of a positive T-cell-CDC-XM. However, DSA class II MFI cut-off of 1988 and above significantly (p ≤0.001) correlated with B-cell-FC-XM positivity, while MFI value of 5986 and above (p ≤0.001) showed strong predictive accuracy of a positive B-cell-CDC-XM. CONCLUSIONS: Our study showed that CDC-XM has poor sensitivity, while FC-XM has poor specificity to detect DSA. L-SAB has good correlation with T-cell-FC-XM (p < 0.0001) but not with B-cell-FC-XM (P = 0.31). DSA strength >2845 and > 1988 significantly correlated with T-cell-FC-XM positivity and B-cell-FC-XM positivity, respectively. While, a MFI value of >4585 and > 5986 significantly correlated with T-cell-CDC-XM positivity and B-cell-CDC-XM positivity, respectively. These MFI cut-off values could serve as a surrogate marker for CDC-XM and FC-XM tests and may help in resolving the limitations of cell-based techniques. In conclusion, we found that L-SAB is more sensitive and specific than CDC-XM and FC-XM and therefore may be used as a test of choice.
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Trasplante de Riñón , Anticuerpos , Citometría de Flujo/métodos , Rechazo de Injerto/diagnóstico , Prueba de Histocompatibilidad/métodos , Isoanticuerpos , Estudios Prospectivos , Estudios RetrospectivosRESUMEN
Post-stroke cognitive impairment (PSCI) is a clinical outcome in around 30% of post-stroke survivors. BDNF is a major gene in this regard. It is regulated by circadian rhythm. The circadian genes are correlated with stroke timings at molecular level. However, studies suggesting the role of these on susceptibility to PSCI are limited. We aim here to determine: (a) genetic risk variants in circadian clock genes, BDNF and (b) dysregulation in expression level of CLOCK, BMAL1, and BDNF that may be associated with PSCI. BDNF (rs6265G/A, rs56164415C/T), CLOCK (rs1801260T/C, rs4580704G/C), and CRY2 (rs2292912C/G) genes variants were genotyped among 119 post-stroke survivors and 292 controls from Eastern part of India. In addition, we analyzed their gene expression in Peripheral blood Mononuclear cells (PBMC) from 15 PSCI cases and 12 controls. The mRNA data for BDNF was further validated by its plasma level through ELISA (n = 38). Among the studied variants, only rs4580704/CLOCK showed an overall association with PSCI (P = 0.001) and lower Bengali Mini-Mental State Examination (BMSE) score. Its 'C' allele showed a correlation with attention deficiency. The language and memory impairments showed association with rs6265/BDNF, while the 'CC' genotype of rs2292912/CRY2 negatively influenced language and executive function. A significant decrease in gene expression for CLOCK and BDNF in PBMC (influenced by specific genotypes) of PSCI patients was observed than controls. Unlike Pro-BDNF, plasma-level mBDNF was also lower in them. Our results suggest the genetic variants in CLOCK, CRY2, and BDNF as risk factors for PSCI among eastern Indians. At the same time, a lowering expression of CLOCK and BDNF genes in PSCI patients than controls describes their transcriptional dysregulation as underlying mechanism for post-stroke cognitive decline.
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Disfunción Cognitiva , Accidente Cerebrovascular , Humanos , Leucocitos Mononucleares , Factor Neurotrófico Derivado del Encéfalo/genética , Disfunción Cognitiva/etiología , Disfunción Cognitiva/genética , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/genética , Factores de Riesgo , Variación GenéticaRESUMEN
BACKGROUND: In this retrospective study, our aim was to find the effect of leucodepleted (LD) blood transfusions on the formation of anti-HLA-antibodies when compared to non-leucodepleted (non-LD) transfusions using Luminex-based method. METHODS: In this study, Luminex single antigen bead assay (L-SAB) and HLA typing were performed on 310 patients. Test positivity rates (as MFI - Mean florescence intensity) were analyzed according to the different sensitization events and gender. RESULTS: Of the 310 patients included in the study, 58.06% (180) patients were male and 41.93% (130) were female. The average age of the patients was 42.86 (±12.37) years. In this study, test positivity rates were significantly lower in the patients who received LD RBC units than in those who received non-LD RBC units (28.43% = 29 of 102 Vs 55.22% = 74 of 134, p < 0.05). In our study, transfusion combined with a history of pregnancy had higher number of significant HLA antibodies compared to cases where transfusion was the only sensitization event (81.81% = 18/22 Vs 39.71% = 85/214, p < 0.05). In addition, anti-HLA-antibodies-MFI were significantly (p < 0.01) higher in non-LD patients compared to LD patients. CONCLUSION: Patients who received LD RBC units had a significantly lower rate of transfusion-associated alloimmunization compared to those who received non-LD RBC units. Multiparous women had a high risk for transfusion-related alloimmunization compared to both nulliparous women and male patient. Furthermore, class I-anti-HLA-antibodies (HLA-B and HLA-A + B) were significantly associated with pregnancy sensitization and/or blood transfusion as a single sensitization.
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Transfusión Sanguínea , Antígenos HLA , Reacción a la Transfusión , Estudios Retrospectivos , Humanos , Masculino , Femenino , Transfusión Sanguínea/métodos , Antígenos HLA/metabolismo , Leucocitos , Isoanticuerpos/metabolismoRESUMEN
CDRI 99/411 is a potent 1,2,4-trioxane anti-malarial candidate compound of the Central Drug Research Institute, India. This study aimed to conduct comprehensive in vitro metabolic investigations of CDRI 99/411 to corroborate its preclinical investigations. Preliminary in vitro metabolic investigations were performed to assess the metabolic stability [in vitro half-life (t(1/2) ) and in vitro hepatic intrinsic clearance (Cl(int) )] of CDRI 99/411 in male Sprague-Dawley rat and human liver microsomes using validated high-performance liquid chromatography with photodiode array detector. The observed in vitro t(1/2) of the compound in rat and human liver microsomes was 13 min with in vitro Cl(int) 130.7±25.0 µL/min/mg and 19 min with in vitro Cl(int) 89.3 ± 17.40 µL/min/mg. These observations suggested moderate metabolic degradation and in vitro Cl(int) with insignificant difference (p>0.05) in the metabolic stability profile in rat and human. Hence, in vitro metabolic investigations were performed with rat liver microsomes. It was observed that CDRI 99/411 exhibited sigmoidal kinetics. At nonlinear regression (r ≥ 0.99) EC(50) and Hill slope values were 17 µm and 1.50, respectively. The metabolism of CDRI 99/411 was primarily mediated by CYP3A2 and was inferred by CYP reaction phenotyping with known potent inhibitors. Two metabolites of CDRI 99/411 were detected which were undetectable on incubation with 1-aminobenzotriazole and ketoconazole.
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Antimaláricos/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Compuestos Heterocíclicos/metabolismo , Microsomas Hepáticos/metabolismo , Compuestos de Espiro/metabolismo , Animales , Antimaláricos/análisis , Antimaláricos/farmacocinética , Citocromo P-450 CYP3A/metabolismo , Inhibidores del Citocromo P-450 CYP3A , Compuestos Heterocíclicos/análisis , Compuestos Heterocíclicos/farmacocinética , Humanos , Cetoconazol/farmacología , Cinética , Masculino , Metaboloma , Dinámicas no Lineales , Ratas , Ratas Sprague-Dawley , Compuestos de Espiro/análisis , Compuestos de Espiro/farmacocinética , Triazoles/farmacologíaRESUMEN
Our objective was to identify the molecule which can inhibit SARS-CoV-2 main protease and can be easily procured. Natural products may provide such molecules and can supplement the current custom chemical synthesis-based drug discovery for this objective. A combination of docking approaches, scoring functions, classical molecular dynamic simulation, binding pose metadynamics, and free energy perturbation calculations have been employed in this study. Theaflavin digallate has been observed in top-scoring compounds after the three independent virtual screening simulations of 598435 compounds (unique 27256 chemical entities). The main protease-theaflavin digallate complex interacts with critical active site residues of the main protease in molecular dynamics simulation independent of the explored computational framework, simulation time, initial structure, and force field used. Theaflavin digallate forms approximately three hydrogen bonds with Glutamate166 of main protease, primarily through hydroxyl groups in the benzene ring of benzo(7)annulen-6-one, along with other critical residues. Glu166 is the most critical amino acid for main protease dimerization, which is necessary for catalytic activity. The estimated binding free energy, calculated by Amber and Schrodinger MMGBSA module, reflects a high binding free energy between theaflavin digallate and main protease. Binding pose metadynamics simulation shows the highly persistent H-bond and a stable pose for the theaflavin digallate-main protease complex. Using method control, experimental controls, and test set, alchemical transformation studies confirm high relative binding free energy of theaflavin digallate with the main protease. Computational molecular interaction suggests that theaflavin digallate can inhibit the main protease of SARS-CoV-2.
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COVID-19 , SARS-CoV-2 , Humanos , Simulación de Dinámica Molecular , Proteasas 3C de Coronavirus , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/químicaRESUMEN
Background/Purpose: To study the clinical characteristics, outcomes, and the incidence of postpartum diabetes in women with overt diabetes and GDM. Methods: This prospective cohort observational study was done on women with GDM diagnosed after the first trimester, attending the tertiary care hospitals for 2.3 years. Comparison of clinical variables and maternal and neonatal outcomes were recorded in women with overt diabetes and GDM. The postpartum glycemic status was determined at 1 and 6 months. Results: There were 32(17.9%) women with overt diabetes and 146(82.1%) women with GDM. Women with overt diabetes were older, had a higher frequency of pre-pregnancy and maternal obesity, family history of diabetes, previous history of GDM, polycystic ovarian syndrome (PCOS), signs of insulin resistance, and need for insulin treatment than women with GDM. The prevalence of gestational hypertension and frequency of spontaneous abortion and large for gestational age (LGA) neonates were significantly higher in women with overt diabetes than in GDM, despite comparable glycemic goals. Postpartum diabetes at 1 and 6 months was significantly higher in women with overt diabetes than in those with GDM. Conclusion: It is critical to identify women with overt diabetes in pregnancy because of increased risk of adverse maternal and neonatal outcomes, and rapid progression to postpartum diabetes, despite achieving optimal glycemic goals.
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BACKGROUND AND AIMS: The aim of the study was to evaluate the maternal and neonatal outcomes in women with recurrent gestational diabetes mellitus (GDM), compared to women with GDM. METHODS: This prospective observational cohort study was done on multiparous women with GDM attending the two tertiary care hospitals. Subjects were divided into two groups, recurrent GDM and GDM. Demographics, clinical variables, and maternal and neonatal outcomes were recorded between the two groups. The postpartum glycemic status was determined at six months. RESULTS: There were 36 (20.2%) women with recurrent GDM and 142 (79.8%) women with GDM. Women with recurrent GDM were older (32.4 ± 6.2 versus 29.8 ± 5.6 years), had higher frequency of obesity, and insulin resistance than women with GDM. Women with recurrent GDM had poor glycemia at diagnosis as compared to GDM. Although the glycemic goals achieved were comparable but women with recurrent GDM have increased frequency of gestational hypertension, preeclampsia, and need for cesarean section. Women with recurrent GDM significantly had higher frequency of large for gestational age (LGA) and macrosomic neonates. Postpartum diabetes at six months was significantly higher in women with recurrent GDM. CONCLUSION: Women with recurrent GDM are at increased risk of adverse maternal and perinatal outcomes despite achieving optimal glycemic goals and also at the most significant risk of postpartum diabetes.