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1.
J Biomol Struct Dyn ; : 1-16, 2021 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-34766874

RESUMO

The increased multidrug resistance in Acinetobacter baumannii (A. baumannii) to the present-day known antibiotics has stimulated academic and industrial efforts globally for the development of novel antibacterial agents. Natural compounds as potential drug leads are gaining significant attention due to their less toxic and more tolerant nature. In the current study, the natural product-based compounds were explored as probable inhibitors of UDP-N-acetylmuramoyl-L-alanine:D-glutamate (MurD) ligase from A.baumannii (AbMurD) to provide a new class of drug leads. The prepared natural library of 3,16,714 compounds from ZINC database was screened into the active site of AbMurD using in silico high-throughput virtual screening which resulted in 100 compounds having high binding affinities. Further screening through flexible molecular docking yielded four potential compounds selected on the basis of estimated binding affinity (ΔG) and favorable protein-ligand interactions. MD simulation of these four compounds under physiological conditions and free binding energy calculations using MM/PBSA (molecular mechanics with Poisson- Boltzmann and surface area solvation) approach revealed three compounds ZINC08879777, ZINC30726863, and ZINC95486217 as potential binders of AbMurD. The calculated physicochemical and ADME properties of these compounds revealed that they can be exploited and modified to improve their binding affinity with the enzyme. Two compounds were purchased and tested against bacterial cell cultures of A. baumannii, Salmonella Typhi, and Staphylococcus aureus to determine their broad-spectrum antibacterial activity. The results suggest that the identified compounds can be exploited as potential herbal leads to target both Gram-positive and Gram-negative pathogens. Communicated by Ramaswamy H. Sarma.

2.
J Biomol Struct Dyn ; : 1-11, 2021 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-34766876

RESUMO

Pathogenic RNA viruses are emerging as one of the major threats and posing challenges to human community. RNA viruses have an exceptionally shorter generation time and easy to adapt in host cells. The recent emergence of SARS-CoV-2, a long RNA virus, has shown us how difficult it is to overcome this kind of pandemic without understanding the viral infection and replication mechanisms. It is essential to comprehend replications of the viral genome, including RNA polymerization and the final capping process. The mRNAs of SARS-CoV-2 coronaviruses are protected at their 5'-ends by cap structure. The cap-like system plays a significant role in viral translational process, viral RNA stability, and scatting in detecting innate immune recognition in host cells. Two coronavirus enzymes, Nsp14 and Nsp16, critically help in the formation of capping and are considered as potential drug targets for antiviral therapy. Natural and herbal medicines have a past record of treating various acute respiratory diseases. In this work, we have exploited 56000 natural compounds to screen potential inhibitors against NSP16. In silico virtual screening, docking and Molecular Dynamics (MD) simulation studies were performed to understand how these potential inhibitors are bound to NSP16. We observed that the most highly screened compound binds to protein molecules with a high dock score, primarily through hydrophobic interactions and hydrogen bonding, as previously reported for NSP16. Compound-13 (2-hydroxy-N-({1-[2-hydroxy-1-(hydroxymethyl)ethyl]piperidin-3-yl}methyl)-5-methylbenzamide) and compound-51 (N-(2-isobutoxybenzyl)-N,2-dimethyl-2,8-diazaspiro[4.5]decane-3-carboxamide) occupied in active site along with good pharmokinetices properties. In conclusion, the selected compounds could be used as a novel therapeutic against SARS-CoV-2.Communicated by Ramaswamy H. Sarma.

3.
Cell Mol Life Sci ; 2021 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-34731254

RESUMO

Since the emergence of the first case of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), the viral genome has constantly undergone rapid mutations for better adaptation in the host system. These newer mutations have given rise to several lineages/ variants of the virus that have resulted in high transmission and virulence rates compared to the previously circulating variants. Owing to this, the overall caseload and related mortality have tremendously increased globally to > 233 million infections and > 4.7 million deaths as of Sept. 28th, 2021. SARS-CoV-2, Spike (S) protein binds to host cells by recognizing human angiotensin-converting enzyme 2 (hACE2) receptor. The viral S protein contains S1 and S2 domains that constitute the binding and fusion machinery, respectively. Structural analysis of viral S protein reveals that the virus undergoes conformational flexibility and dynamicity to interact with the hACE2 receptor. The SARS-CoV-2 variants and mutations might be associated with affecting the conformational plasticity of S protein, potentially linked to its altered affinity, infectivity, and immunogenicity. This review focuses on the current circulating variants of SARS-CoV-2 and the structure-function analysis of key S protein mutations linked with increased affinity, higher infectivity, enhanced transmission rates, and immune escape against this infection.

4.
Indian J Ophthalmol ; 69(10): 2710-2716, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34571620

RESUMO

Purpose: To report the association of procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) mutations with bilateral primary congenital glaucoma (PCG) in monozygotic twins and with nondominant juvenile-onset primary open-angle glaucoma (JOAG). Methods: We utilized family-based whole-exome sequencing to detect disease-causing mutations in a pair of monozygotic twins with de-novo PCG and compared its existence in 50 nonfamilial cases of JOAG and 30 healthy controls. To validate the identified mutations, direct Sanger sequencing was performed. For further evaluation of gene expression in the ocular tissues, we performed whole-mount in situ hybridization in zebrafish embryos. Results: We identified a novel missense mutation (c.1925A>G, p.Tyr642Cys) in the PLOD2 gene in the monozygotic twin pair with PCG and another missense mutation (c.1880G>A, p.Arg627Gln) in one JOAG patient. Both mutations identified were heterozygous. Neither the parents of the twins nor the parents of the JOAG patient harbored the mutation and it was probably a de-novo change. The zebrafish in situ hybridization revealed expression of the PLOD2 gene during embryogenesis of the eye. Conclusion: We observed an association of PLOD2 mutations with PCG and with nonfamilial JOAG. This new gene needs to be further investigated for its role in pathways associated with glaucoma pathogenesis.


Assuntos
Dioxigenases , Glaucoma de Ângulo Aberto , Glaucoma , Animais , Exoma , Proteínas do Olho/genética , Glaucoma de Ângulo Aberto/diagnóstico , Glaucoma de Ângulo Aberto/genética , Humanos , Ácidos Cetoglutáricos , Lisina , Mutação , Linhagem , Pró-Colágeno , Sequenciamento Completo do Exoma , Peixe-Zebra
5.
J Biomol Struct Dyn ; : 1-13, 2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34348086

RESUMO

Acinetobacter baumannii is an extremely dangerous multidrug-resistant (MDR) gram-negative pathogen which poses a serious life-threatening risk in immunocompromised patients. Phosphopantothenoyl cysteine synthetase (PPCS) catalyzes the formation of an amide bond between L-cysteine and phosphopantothenic acid (PPA) to form 4'- Phosphopantothenoylcysteine during Coenzyme A (CoA) biosynthesis. CoA is a crucial cofactor for cellular survival and inhibiting its synthesis will result in cell death. Bacterial PPCS differs from eukaryotic PPCS in a number of ways like it exists as a C-terminal domain of a PPCDC/PPCS fusion protein whereas eukaryotic PPCS exists as an independent protein. This difference makes it an attractive drug target. For which a conventional iterative approach of SBDD (structure-based drug design) was used, which began with three-dimensional structure prediction of AbPPCS using PHYRE 2.0. A database of FDA-approved compounds (Drug Bank) was then screened against the target of interest by means of docking score and glide energy, leading to the identification of 6 prominent drug candidates. The shortlisted 6 molecules were further subjected to all-atom MD simulation studies in explicit-solvent conditions (using AMBER force field). The MD simulation studies revealed that the ligands DB65103, DB449108 and DB443210, maintained several H-bonds with intense van der Waals contacts at the active site of the protein with high binding free energies: -11.42 kcal/mol, -10.49 kcal/mol and -10.98 kcal/mol, respectively, calculated via MM-PBSA method. Overall, binding of these compounds at the active site was found to be the most stable and robust highlighting the potential of these compounds to serve as antibacterials.Communicated by Ramaswamy H. Sarma.

6.
Mol Divers ; 25(3): 1439-1460, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34159484

RESUMO

The accumulation of massive data in the plethora of Cheminformatics databases has made the role of big data and artificial intelligence (AI) indispensable in drug design. This has necessitated the development of newer algorithms and architectures to mine these databases and fulfil the specific needs of various drug discovery processes such as virtual drug screening, de novo molecule design and discovery in this big data era. The development of deep learning neural networks and their variants with the corresponding increase in chemical data has resulted in a paradigm shift in information mining pertaining to the chemical space. The present review summarizes the role of big data and AI techniques currently being implemented to satisfy the ever-increasing research demands in drug discovery pipelines.

7.
Sci Rep ; 11(1): 10957, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34040057

RESUMO

Multiple myeloma (MM) is a plasma cell malignancy with diverse clinical phenotypes and molecular heterogeneity not completely understood. Differentially expressed genes (DEGs) and miRNAs (DEMs) in MM may influence disease pathogenesis, clinical presentation / drug sensitivities. But these signatures overlap meagrely plausibly due to complexity of myeloma genome, diversity in primary cells studied, molecular technologies/ analytical tools utilized. This warrants further investigations since DEGs/DEMs can impact clinical outcomes and guide personalized therapy. We have conducted genome-wide meta-analysis of DEGs/DEMs in MM versus Normal Plasma Cells (NPCs) and derived unified putative signatures for MM. 100 DEMs and 1,362 DEGs were found deranged between MM and NPCs. Signatures of 37 DEMs ('Union 37') and 154 DEGs ('Union 154') were deduced that shared 17 DEMs and 22 DEGs with published prognostic signatures, respectively. Two miRs (miR-16-2-3p, 30d-2-3p) correlated with survival outcomes. PPI analysis identified 5 topmost functionally connected hub genes (UBC, ITGA4, HSP90AB1, VCAM1, VCP). Transcription factor regulatory networks were determined for five seed DEGs with ≥ 4 biomarker applications (CDKN1A, CDKN2A, MMP9, IGF1, MKI67) and three topmost up/ down regulated DEMs (miR-23b, 195, let7b/ miR-20a, 155, 92a). Further studies are warranted to establish and translate prognostic potential of these signatures for MM.


Assuntos
Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , MicroRNAs/genética , Mieloma Múltiplo/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Biologia Computacional , Mineração de Dados , Conjuntos de Dados como Assunto , Estudo de Associação Genômica Ampla , Humanos , Plasmócitos/metabolismo , Prognóstico , Mapas de Interação de Proteínas
8.
J Inorg Biochem ; 220: 111461, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33882424

RESUMO

Lactoperoxidase (LPO) is a mammalian heme peroxidase which catalyzes the conversion of thiocyanate (SCN¯) and iodide (I-) by hydrogen peroxide (H2O2) into antimicrobial hypothiocyanite (OSCN¯) and hypoiodite (IO-). The prosthetic heme group is covalently attached to LPO through two ester linkages involving conserved glutamate and aspartate residues. On the proximal side, His351 is coordinated to heme iron while His 109 is located in the substrate binding site on the distal heme side. We report here the first structure of the ternary complex of LPO with iodide (I-) and H2O2 at 1.77 Å resolution. LPO was crystallized with ammonium iodide and the crystals were soaked in the reservoir solution containing H2O2. Structure determination showed the presence of an iodide ion and a H2O2 molecule in the substrate binding site. The iodide ion occupied the position which is stabilized by the interactions with heme moiety, His109, Arg255 and Glu258 while H2O2 was held between the heme iron and His109. The presence of I- in the distal heme cavity seems to screen the positive charge of Arg255 thus suppressing the proton transfer from H2O2 to His109. This prevents compound I formation and allows trapping of a stable enzyme-substrate (LPO-I--H2O2) ternary complex. This stable geometrical arrangement of H2O2 in the distal heme cavity of LPO is similar to that of H2O2 in the structure of the transient intermediate of the palm tree heme peroxidase. The biochemical studies showed that the catalytic activity of LPO decreased when the samples of LPO were preincubated with ammonium iodide.

9.
Comput Struct Biotechnol J ; 19: 1896-1906, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33897984

RESUMO

Antibiotic resistance poses a major threat to public health. More effective ways of the antibiotic prescription are needed to delay the spread of antibiotic resistance. Employment of sequencing technologies coupled with the use of trained neural network algorithms for genotype-to-phenotype prediction will reduce the time needed for antibiotic susceptibility profile identification from days to hours. In this work, we have sequenced and phenotypically characterized 171 clinical isolates of Escherichia coli and Klebsiella pneumoniae from Norway and India. Based on the data, we have created neural networks to predict susceptibility for ampicillin, 3rd generation cephalosporins and carbapenems. All networks were trained on unassembled data, enabling prediction within minutes after the sequencing information becomes available. Moreover, they can be used both on Illumina and MinION generated data and do not require high genome coverage for phenotype prediction. We cross-checked our networks with previously published algorithms for genotype-to-phenotype prediction and their corresponding datasets. Besides, we also created an ensemble of networks trained on different datasets, which improved the cross-dataset prediction compared to a single network. Additionally, we have used data from direct sequencing of spiked blood cultures and found that AMR-Diag networks, coupled with MinION sequencing, can predict bacterial species, resistome, and phenotype as fast as 1-8 h from the sequencing start. To our knowledge, this is the first study for genotype-to-phenotype prediction: (1) employing a neural network method; (2) using data from more than one sequencing platform; and (3) utilizing sequence data from spiked blood cultures.

10.
J Biochem Mol Toxicol ; 35(7): e22785, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33860986

RESUMO

Azilsartan is found to be more potent than other angiotensin receptor blockers in reducing blood pressure. However, its effect on the heart following myocardial infarction remains to be established. For the first time, we investigated the peroxisome proliferator-activated receptor-γ (PPAR-γ) agonistic and cardioprotective properties of azilsartan. Computational modeling studies of interactions between azilsartan and PPAR-γ revealed azilsartan as an agonist of PPAR-γ and showed the mechanism of azilsartan in cardioprotection. Our study compared the cardioprotective potential of telmisartan to that of azilsartan in a murine model of myocardial ischemia-reperfusion injury by comparing their antioxidant, ant apoptotic, anti-inflammatory, mitogen-activated protein kinase (MAPK)-modulating ability, and PPAR-γ agonistic activity. Male Wistar rats were grouped into four to receive vehicle (dimethyl sulfoxide [0.05%] 2 ml/kg) telmisartan (10 mg/kg p.o.), azilsartan (10 mg/kg p.o.) or azilsartan with specific PPAR-γ blocker, GW 9662 for 28 days. Ischemia was induced for 45 min on the 29th day followed by 60 min of reperfusion. Telmisartan and azilsartan pretreatment significantly nearly normalized cardiac parameters and preserved structural changes. Both drugs inhibited oxidative burst, inflammation, as well as cell death by modulating apoptotic protein expression along with reduction in 4',6-diamidino-2-phenylindole/terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. An increment in pro-survival kinase ERK paralleled with a reduction in p38 and JNK was also revealed by MAPK pathway studies, after administration of these drugs. Interestingly, the aforementioned changes induced by both drugs were reversed by administration of the specific PPAR-γ antagonist, GW9662. However, we found that azilsartan upregulated PPAR-γ to a lesser extent as compared to telmisartan and the latter may be preferred in hypertensive patients at risk of myocardial infarction.


Assuntos
Benzimidazóis/farmacologia , Cardiotônicos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica , Miocárdio , Oxidiazóis/farmacologia , Telmisartan/farmacologia , Animais , Modelos Animais de Doenças , Masculino , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Miocárdio/patologia , Ratos , Ratos Wistar
11.
3 Biotech ; 11(2): 109, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33552834

RESUMO

The pandemic COVID-19 was caused by a novel Coronavirus-2 (SARS-CoV-2) that infects humans through the binding of glycosylated SARS-CoV-2 spike 2 protein to the glycosylated ACE2 receptor. The spike 2 protein recognizes the N-terminal helices of the glycosylated metalloprotease domain in the human ACE2 receptor. To understand the susceptibility of animals for infection and transmission, we did sequence and structure-based molecular interaction analysis of 16 ACE2 receptors from different mammalian species with SARS-CoV-2 spike 2 receptor binding domain. Our comprehensive structure analysis revealed that the natural substitution of amino acid residues Gln24, His34, Phe40, Leu79 and Met82 in the N-terminal α1 and α2 helices of the ACE2 receptor results in loss of crucial network of hydrogen-bonded and hydrophobic interactions with receptor binding domain of SARS-CoV-2 spike protein. Another striking observation is the absence of N-glycosylation site Asn103 in all mammals and many species, lack more than one N-linked glycosylation site in the ACE2 receptor. Based on the loss of crucial interactions and the absence of N-linked glycosylation sites we categorized Felis catus, Equus caballus, Panthera tigris altaica, as highly susceptible while Oryctolagus cuniculus, Bos Tauras, Ovis aries and Capra hircus as moderately susceptible species for infection. Similarly, the E. asinus, Bubalus bubalis, Canis lupus familiaris, Ailuropoda melaleuca and Camelus dromedarius are categorized as low susceptible with Loxodonta Africana, Mus musculus, Sus scrofa and Rattus rattus as least susceptible species for SARS-CoV-2 infection. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-020-02599-2.

12.
Arch Biochem Biophys ; 701: 108786, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33548211

RESUMO

DNA Gyrase is a type II topoisomerase that utilizes the energy of ATP hydrolysis for introducing negative supercoils in DNA. The protein comprises two subunits GyrA and GyrB that form a GyrA2GyrB2 heterotetramer. GyrB subunit contains the N-terminal domain (GBNTD) for ATPase activity and the C-terminal domain (GBCTD) for interaction with GyrA and DNA. Earlier structural studies have revealed three different conformational states for GBNTD during ATP hydrolysis defined as open, semi-open, and closed. Here we report, the three-dimensional structure of a new transient closed conformation of GBNTD from Salmonella Typhi (StGBNTD) at 1.94 Å resolution. Based on the structural analysis of this transient closed conformation, we propose the role of protein in the mechanism of ATP hydrolysis. We further explored the effect of pH on ATPase activity and structural stability of the GBNTD using CD and fluorescence spectroscopy at varying pH environment. Kinetic parameters obtained from the ATPase assay were correlated with its secondary and tertiary structure at their respective pH environment. The protein possessed maximum ATPase activity and structural stability at optimum pH 8. At acidic pH, a remarkable decrease in both enzymatic activity and structural stability was observed whereas at alkaline pH there was no significant change. The structural analysis of StGBNTD reveals the role of polar interactions in stabilizing the overall dimeric conformation of the protein.


Assuntos
Adenosina Trifosfatases/química , DNA Girase/química , Salmonella typhi/enzimologia , Adenosina Trifosfatases/genética , Cristalografia por Raios X , DNA Girase/genética , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Domínios Proteicos , Salmonella typhi/genética
13.
Biochimie ; 182: 152-165, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33417980

RESUMO

The quest for effective anticancer therapeutics continues to be extensively pursued. Over the past century, several drugs have been developed, however, a majority of these drugs have a poor therapeutic index and increased toxicity profile. Hence, there still exists ample opportunity to discover safe and effective anticancer drugs. Aurora Kinase B (AurB), a member of the Aurora kinase family and a key regulator of mitotic cell division, is found to be frequently overexpressed in a variety of human cancers and has thus emerged as an attractive target for the design of anticancer therapeutics. In the present study, a structure-based scaffold hopping approach was utilized to modify the heterocyclic moiety of (S)-3-(3-aminopyrrolidine-1-carbonyl)-4,11-dihydroxy-2-methylanthra [2,3-b]furan-5,10-dione (anthrafuran 1) to generate a series of heteroarene-fused anthraquinone derivatives, which were then subjected to virtual screening for the identification of potential AurB inhibitors. The obtained hits were subsequently synthesized and evaluated by using a combination of in silico and biophysical techniques for elucidating their in vitro binding and inhibition activity with recombinantly expressed AurB. Four identified hits presented an improved binding profile as compared to their parent analog anthrafuran 1. One derivative, anthrathiophene 2 demonstrated excellent in vitro inhibition of AurB (7.3 µM).


Assuntos
Antraquinonas , Aurora Quinase B , Inibidores de Proteínas Quinases , Antraquinonas/síntese química , Antraquinonas/química , Antraquinonas/farmacologia , Aurora Quinase B/antagonistas & inibidores , Aurora Quinase B/química , Aurora Quinase B/metabolismo , Linhagem Celular Tumoral , Humanos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia
14.
J Biol Inorg Chem ; 26(1): 149-159, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33427997

RESUMO

Lactoperoxidase, a heme-containing glycoprotein, catalyzes the oxidation of thiocyanate by hydrogen peroxide into hypothiocyanite which acts as an antibacterial agent. The prosthetic heme moiety is attached to the protein through two ester linkages via Glu258 and Asp108. In lactoperoxidase, the substrate-binding site is formed on the distal heme side. To study the effect of physiologically important potassium ion on the structure and function of lactoperoxidase, the fresh protein samples were isolated from yak (Bos grunniens) colostrum and purified to homogeneity. The biochemical studies with potassium fluoride showed a significant reduction in the catalytic activity. Lactoperoxidase was crystallized using 200 mM ammonium nitrate and 20% PEG-3350 at pH 6.0. The crystals of LPO were soaked in the solution of potassium fluoride and used for the X-ray intensity data collection. Structure determination at 2.20 Å resolution revealed the presence of a potassium ion in the distal heme cavity. Structure determination further revealed that the propionic chain attached to pyrrole ring C of the heme moiety, was disordered into two components each having an occupancy of 0.5. One component occupied a position similar to the normally observed position of propionic chain while the second component was found in the distal heme cavity. The potassium ion in the distal heme cavity formed five coordinate bonds with two oxygen atoms of propionic moiety, Nε2 atom of His109 and two oxygen atoms of water molecules. The presence of potassium ion in the distal heme cavity hampered the catalytic activity of lactoperoxidase.


Assuntos
Lactoperoxidase/metabolismo , Potássio/metabolismo , Animais , Sítios de Ligação , Biocatálise , Cálcio/química , Cálcio/metabolismo , Bovinos , Colostro/enzimologia , Cristalografia por Raios X , Heme/química , Heme/metabolismo , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Lactoperoxidase/química , Potássio/química , Ligação Proteica
15.
Protein J ; 40(1): 8-18, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33389415

RESUMO

Lactoperoxidase (LPO) is a heme containing oxido-reductase enzyme. It is secreted from mammary, salivary, lachrymal and mucosal glands. It catalyses the conversion of thiocyanate into hypothiocyanate and halides into hypohalides. LPO belongs to the superfamily of mammalian heme peroxidases which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). The heme prosthetic group is covalently linked in LPO through two ester bonds involving conserved residues Glu258 and Asp108. It was isolated from colostrum of yak (Bos grunniens), purified to homogeneity and crystallized using ammonium iodide as a precipitating agent. The crystals belonged to monoclinic space group P21 with cell dimensions of a = 53.91 Å, b = 78.98 Å, c = 67.82 Å and ß = 92.96°. The structure was determined at 1.55 Å resolution. This is the first structure of LPO from yak. Also, this is the highest resolution structure of LPO determined so far from any source. The structure determination revealed that three segments (Ser1-Cys15), (Thr117-Asn138) and (Cys167-Leu175) were disordered and formed one surface of LPO structure. In the substrate binding site, the iodide ions were observed in three subsites which are formed by (1) heme moiety and residues, Gln105, Asp108, His109, Phe113, Arg255, Glu258, Phe380 and Phe381, (2) residues, Asn230, Lys232, Pro236, Cys248, Phe254, Phe381 and Pro424 and (3) residues, Ser198, Leu199 and Arg202. The structure determination also revealed that the side chain of Phe254 was disordered. It was observed to adopt two conformations in the structures of LPO.


Assuntos
Aminoácidos/química , Compostos de Amônio/química , Heme/química , Peróxido de Hidrogênio/química , Lactoperoxidase/química , Aminoácidos/metabolismo , Compostos de Amônio/metabolismo , Animais , Sítios de Ligação , Bovinos , Colostro/química , Cristalização , Cristalografia por Raios X , Feminino , Expressão Gênica , Heme/metabolismo , Peróxido de Hidrogênio/metabolismo , Lactoperoxidase/genética , Lactoperoxidase/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Especificidade por Substrato
16.
J Biomol Struct Dyn ; 39(17): 6649-6659, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-32741313

RESUMO

The recent outbreak of the SARS-CoV-2 virus leading to the disease COVID 19 has become a global pandemic that is spreading rapidly and has caused a global health emergency. Hence, there is an urgent need of the hour to discover effective drugs to control the pandemic caused by this virus. Under such conditions, it would be imperative to repurpose already known drugs which could be a quick and effective alternative to discovering new drugs. The main protease (Mpro) of SARS-COV-2 is an attractive drug target because of its essential role in the processing of the majority of the non-structural proteins which are translated from viral RNA. Herein, we report the high-throughput virtual screening and molecular docking studies to search for the best potential inhibitors against Mpro from FDA approved drugs available in the ZINC database as well as the natural compounds from the Specs database. Our studies have identified six potential inhibitors of Mpro enzyme, out of which four are commercially available FDA approved drugs (Cobicistat, Iopromide, Cangrelor, and Fortovase) and two are from Specs database of natural compounds (Hopeaphenol and Cyclosieversiodide-A). While Cobicistat and Fortovase are known as HIV drugs, Iopromide is a contrast agent and Cangrelor is an anti-platelet drug. Furthermore, molecular dynamic (MD) simulations using GROMACS were performed to calculate the stability of the top-ranked compounds in the active site of Mpro. After extensive computational studies, we propose that Cobicistat and Hopeaphenol show potential to be excellent drugs that can form the basis of treating COVID-19 disease.Communicated by Ramaswamy H. Sarma.


Assuntos
COVID-19 , Preparações Farmacêuticas , Humanos , Simulação de Acoplamento Molecular , Peptídeo Hidrolases , Inibidores de Proteases , SARS-CoV-2
17.
J Biomol Struct Dyn ; 39(17): 6713-6727, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32741322

RESUMO

The recent outbreak of the SARS-CoV-2 virus leading to the disease COVID 19, a global pandemic has resulted in an unprecedented loss of life and economy worldwide. Hence, there is an urgent need to discover effective drugs to control this pandemic. NSP16 is a methyltransferase that methylates the ribose 2'-O position of the viral nucleotide. Taking advantage of the recently solved structure of NSP16 with its inhibitor, S-Adenosylmethionine, we have virtually screened FDA approved drugs, drug candidates and natural compounds. The compounds with the best docking scores were subjected to molecular dynamics simulations followed by binding free energy calculations using the MM-PBSA method. The known drugs which were identified as potential inhibitors of NSP16 from SARS-CoV-2 included DB02498, DB03909, DB03186, Galuteolin, ZINC000029416466, ZINC000026985532, and ZINC000085537017. DB02498 (Carba-nicotinamide-adenine-dinucleotide) is an approved drug which has been used since the late 1960s in intravenous form to significantly lessen withdrawal symptoms from a variety of drugs and alcohol addicts and it has the best MM-PBSA binding free energy of-12.83 ± 0.52 kcal/mol. The second best inhibitor, Galuteolin is a natural compound that inhibits tyrosinase enzyme with MM-PBSA binding free energy value of -11.21 ± 0.47 kcal/mol. Detailed ligand and protein interactions were analyzed and common residues across SARS-CoV, SARS-CoV-2, and MERS-CoV were identified. We propose Carba-nicotinamide-adenine-dinucleotide and Galuteolin as the potential inhibitors of NSP16. The results in this study can be used for the treatment of COVID-19 and can also form the basis of rational drug design against NSP16 of SARS-CoV-2.


Assuntos
COVID-19 , Preparações Farmacêuticas , Reposicionamento de Medicamentos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , SARS-CoV-2
18.
J Biomol Struct Dyn ; 39(15): 5668-5681, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32643552

RESUMO

SARS-CoV-2 is the causative agent of COVID-19 and has been declared as pandemic disease by World Health Organization. Lack of targeted therapeutics and vaccines for COVID-2019 have triggered the scientific community to develop new vaccines or drugs against this novel virus. Many synthetic compounds and antimalarial drugs are undergoing clinical trials. The traditional medical practitioners widely use Indian medicinal plant Withania somnifera (Ashwagandha) natural constituents, called withanolides for curing various diseases. The main protease (Mpro) of SARS-CoV-2 plays a vital role in disease propagation by processing the polyproteins which are required for its replication. Hence, it denotes a significant target for drug discovery. In the present study, we evaluate the potential of 40 natural chemical constituents of Ashwagandha to explore a possible inhibitor against main protease of SARS-CoV-2 by adopting the computational approach. The docking study revealed that four constituents of Ashwagandha; Withanoside II (-11.30 Kcal/mol), Withanoside IV (-11.02 Kcal/mol), Withanoside V (-8.96 Kcal/mol) and Sitoindoside IX (-8.37 Kcal/mol) exhibited the highest docking energy among the selected natural constituents. Further, MD simulation study of 100 ns predicts Withanoside V possess strong binding affinity and hydrogen-bonding interactions with the protein active site and indicates its stability in the active site. The binding free energy score also correlates with the highest score of -87.01 ± 5.01 Kcal/mol as compared to other selected compounds. In conclusion, our study suggests that Withanoside V in Ashwagandha may be serve as a potential inhibitor against Mpro of SARS-CoV-2 to combat COVID-19 and may have an antiviral effect on nCoV.Communicated by Ramaswamy H. Sarma.


Assuntos
COVID-19 , Withania , Antivirais/farmacologia , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Extratos Vegetais , Inibidores de Proteases/farmacologia , SARS-CoV-2
19.
Biochim Biophys Acta Gen Subj ; 1865(2): 129807, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33278547

RESUMO

BACKGROUND: The p53, tumor suppressor protein is inactivated upon mutation in the DNA-binding domain and the non-functional protein leads to cancers. The p53Y220C is one of the most frequently observed mutations in p53 with a scope of rescuing the protein function using small molecules. METHODS: Using computational modeling, biophysical, and experimental cell-based studies we tried to understand the molecular basis of Curcumin as a potential small molecule to stabilize p53Y220C mutant and restore its function. The pancreatic adenocarcinomas BxPC-3 p53Y220C mutant cell line was used for cell-based assays to determine the therapeutic potential of Curcumin to restore mutant p53 to function like wild type. RESULTS: Our results showed that the Curcumin binds p53Y220C with Kd = 3.169 ± 0.257 µM and it increases the DNA binding affinity of the mutant by 4-fold with Kd = 851.29 ± 186.27 nM. By Fluorescence, CD, and IR spectroscopy, we could characterize the secondary structural changes and stabilization of the p53Y220C DNA binding domain upon Curcumin binding. By caspase-3 and Annexin V assays, we could demonstrate that Curcumin at 3 µM to 8 µM concentration could initiate p53 mediated apoptosis in BxPC-3 cell line. Based on our experimental studies, we propose a mechanism for the activation of ATM/Chk1 kinases pathways for apoptosis and/or G2/M cell cycle arrest in the BxPC-3 cell line mediated by functionally restored p53Y220C. CONCLUSION: The study indicated that the natural compound Curcumin could rescue mutant p53Y220C in BxPC-3 pancreatic adenocarcinomas cell line to function like wild-type and activate apoptotic pathways.


Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Curcumina/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Proteína Supressora de Tumor p53/genética , Adenocarcinoma/genética , Linhagem Celular Tumoral , Humanos , Simulação de Acoplamento Molecular , Neoplasias Pancreáticas/genética , Mutação Puntual/efeitos dos fármacos
20.
Bioorg Chem ; 104: 104244, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32966903

RESUMO

The emerged resistance in Typhoidal Salmonella has limited the treatment options for typhoid fever. In this scenario, there is a need to find alternate treatment modalities against this pathogen. Amongst the therapeutic agents currently being used to treat enteric fever, quinolones have enjoyed considerable success since past three decades. These drugs act upon DNA gyrase and the acquired resistance is due to mutations at Ser83 and Asp87 of gyrase A subunit. In the present study DNA gyrase enzyme was targeted to seek out potential new inhibitors which are not affected by these mutations. Molecular modelling and docking studies were performed in Schrödinger's molecular modelling software. Homology model of DNA gyrase-DNA complex was built using templates 1AB4 and 3LTN. Molecular dynamic simulations were performed in SPC solvent for 100 ns. Total 17,900,742 drug like molecules were downloaded from ZINC library of chemical compounds. The Glide XP score of the compounds ranged from -5.285 to -13.692. All the ligands bound at the four base pair staggered nick in the DNA binding groove of DNA gyrase enzyme with their aromatic rings intercalating between the bases of two successive nucleotides stabilized by π - π stacking interactions. The binding pocket of DNA gyrase B comprising conserved residues Lys 447, Gly 448, Lys 449, Ile 450, Leu 451, Gln 465 and Val 467 interacts with the ligand molecules through van der Waals interactions. The MIC (minimum inhibitory concentration), MBC (minimum bactericidal concentration) and IC50 of the tested compounds ranged from 500 to 125 mg/L, 750 to 500 mg/L and 100 to 12.5 mg/L, respectively. The selected hits bind to quinolone binding pocket, but their mode of binding and conformation is different to fluoroquinolones, and hence, their binding is not affected by mutations at Ser83 or Asp87 positions. These lead compounds can be further explored as a scaffold to design inhibitors against DNA gyrase to bypass quinolone resistance.


Assuntos
Antibacterianos/farmacologia , DNA Girase/metabolismo , Descoberta de Drogas , Salmonella typhi/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Salmonella typhi/enzimologia , Relação Estrutura-Atividade
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