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Sequence alignment of HPR with other ribonucleases. The sequences of ribonucleases were taken from protein data bank their PDB Ids being: HPR, human pancreatic ribonuclease (1DZA); BS-RNase, bovine seminal ribonuclease (1BSR); RNase A: bovine pancreatic ribonuclease (3JW1). The Swiss-Prot ID of DPR, Douc Pancreatic Ribonuclease is Q8SPN4.1. In the parenthesis is given the percent amino acid sequence similarity between different ribonucleases with respect to HPR. The secondary structures are shown at the top of sequences as, α-helices in brown filled box and β-strands in green filled arrow while the loop residues as black line. The identical residues are shown in orange and the conserved cysteine residues are highlighted in light blue. The active site residues, His12 and His119 are shaded in green while the residues under investigation, Gln28, Gly38 and Arg39 in the current study are highlighted in red.
Source publication
Human pancreatic ribonuclease (HPR), a member of RNase A superfamily, has a high activity on double stranded (ds) RNA. By virtue of this activity HPR appears to be involved in the host-defense against pathogenic viruses. To delineate the mechanism of dsRNA cleavage by HPR, we have investigated the role of glutamine 28 and arginine 39 of HPR in its...
Citations
... In the most highly activated forms of Cas13a, k cat of several hundred per sec were observed, which is very high compared to the trans-nuclease activity observed for Cas12a, but within the range for other ribonucleases, including pancreatic ribonucleases in the RNase A family (EC 4.1.1.18): k cat of 323 s À1 for bovine (Moussaoui et al., 2007) and 2317 s À1 for human (Rehman et al., 2011) enzymes. For Cas13a, k cat for polyuridine trans-substrates was as much as 30 times higher than for RNaseAlert, indicating once bound, the former are more rapidly cleaved and released than the latter, and the use of RNaseAlert does not accurately record the full catalytic potential of Cas13a and may be suboptimal for diagnostics. ...
Bacterial CRISPR systems provide acquired immunity against invading nucleic acids by activating RNA-programmable RNases and DNases. Cas13a and Cas12a enzymes bound to CRISPR RNA (crRNA) recognize specific nucleic acid targets, initiating cleavage of the targets as well as non-target (trans) nucleic acids. Here, we examine kinetics of single-turnover target and multi-turnover trans-nuclease activities of both enzymes. High-turnover, non-specific Cas13a trans-RNase activity is coupled to rapid binding of target RNA. By contrast, low-turnover Cas12a trans-nuclease activity is coupled to relatively slow cleavage of target DNA, selective for DNA over RNA, indifferent to base identity, and preferential for single-stranded substrates. Combining multiple crRNA increases detection sensitivity of targets, an approach we use to quantify pathogen DNA in samples from patients suspected of Buruli ulcer disease. Results reveal that these enzymes are kinetically adapted to play distinct roles in bacterial adaptive immunity and show how kinetic analysis can be applied to CRISPR-based diagnostics.
... Retrieval and preparation of ligands/reference drugs Gasteiger partial charges were added, rotatable bonds were defined, and the energies were minimized using MMFF94 forcefield 28,29 . ...
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Recently, the emergence and dissemination of SARS-CoV-2 has caused high mortality and enormous economic loss. In the fight against COVID-19, the rapid development of new drug molecules is the need of hour. However, the conventional approaches of drug development is time consuming and expensive in nature. In this study, we have adopted an alternative approach to identify lead molecules from natural sources using high throughput virtual screening approach. Ligands from natural compounds library from Selleck Inc (L1400) have been screened to evaluate their ability to bind and inhibit the main protease (M<sup>pro</sup> or 3CL<sup>pro</sup>) of SARS-CoV-2, which is a potential drug target. We found that Kaempferol, Quercetin, and Rutin were able to bind at the substrate binding pocket of 3CL<sup>pro</sup> with high affinity (10<sup>5</sup>-10<sup>6</sup> M<sup>-1</sup>) and interact with the active site residues such as His41 and Cys145 through hydrogen bonding and hydrophobic interactions. In fact, the binding affinity of Rutin was much higher than Chloroquine (1000 times) and Hydroxychloroquine (100 times) and was comparable to that of the reference drug Remdesivir, which is in clinical trials to treat COVID-19 patients. The results suggest that natural compounds such as flavonoids have the potential to be developed as novel inhibitors of SARS-CoV-2 with a comparable potency as that of Remdesivir. However, their clinical usage on COVID-19 patients is a subject of further investigations and clinical trials.
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... Site II is much smaller in size comparatively and accommodates the binding of hydrophobic drugs due to the predominate hydrophobic residues present. [13][14][15][16] A study conducted by Ping Li, according to the free drug theory it states that in an in-vivo system, the drug interchanges from proteinbound to unbound states through reversible rapid equilibrium processes. When the drug is distributed to the circulatory blood-stream, only the free unbound drug can permeate through the tissue to the active or target site, which produces its effect and, after some time reaches its half-life. ...
The accuracy in Anti-tuberculous drug assaying was supported by computational modeling using a 2 and 3-dimensional thermodynamic binding affinity prediction of each drug during multiple drug co-administration regimens. The United States Food and Drug Administration has highlighted the need for extensive research to improve the recovery during analytical drug method development, where the recovery affects the slope of the calibration curve. Here we focused on the drug-protein binding variation that affects the extrapolation of the patient sample drug concentration from the slope of the calibration curve. The binding constants calculated at a physiological temperature from the fluorescence spectroscopy data were as follows: Rifampicin 5.379 X 102 M-1 (moderate affinity), Isoniazid 9.285 M-1 (low affinity), 25-Desacetyl Rifampicin 3.156 M-1 (low affinity), Ethambutol 3.443 M-1 (low affinity) and Pyrazinamide 3.076 X 102 M-1 (moderate affinity). These drugs Gibbs free energy were below zero, indicating spontaneous binding reactions. Rifampicin a non-polar weak acid with a higher affinity indicating the most stable complex formation with albumin as opposed to soluble Isoniazid due to it being polar and an ionized form to be easily excreted in the urine resulting in low levels of detection. This will affect the bioavailability and accuracy of the assay levels for patients experiencing hyper and hypoalbuminemia with related competition and induction processes of the enzymes. These complications are apparent where larger numbers of patients are involved in clinical trials, bioequivalence and bioavailability studies with varying protein levels that may be more crucial for drugs with a narrow therapeutic index.
... Site II is much smaller in size comparatively and accommodates the binding of hydrophobic drugs due to the predominate hydrophobic residues present. [13][14][15][16] A study conducted by Ping Li, according to the free drug theory it states that in an in-vivo system, the drug interchanges from protein-bound to unbound states through reversible rapid equilibrium processes. When the drug is distributed to the circulatory blood-stream, only the free unbound drug can permeate through the tissue to the active or target site, which produces its effect and, after some time reaches its half-life. ...
The accuracy in Anti-TB drug assaying was supported by computational modeling using a 2 and 3-dimensional thermodynamic binding affinity prediction of each drug during multiple drug co-administration regimens. The United States Food and Drug Administration has highlighted the need for extensive research to improve the recovery during analytical drug method development, where the recovery affects the slope of the calibration curve. Here we focused on the drug-protein binding variation that affects the extrapolation of the patient sample drug concentration from the slope of the calibration curve. The binding constants calculated at a physiological temperature from the fluorescence spectroscopy data were as follows: Rifampicin 5.379 X 102 M-1 (moderate affinity), Isoniazid 9.285 M-1 (low affinity), 25-Desacetyl Rifampicin 3.156 M-1 (low affinity), Ethambutol 3.443 M-1 (low affinity) and Pyrazinamide 3.076 X 102 M-1 (moderate affinity). These drugs Gibbs free energy were below zero, indicating spontaneous binding reactions. Rifampicin a non-polar weak acid with a higher affinity indicating the most stable complex formation with albumin as opposed to soluble Isoniazid due to it being polar and an ionized form to be easily excreted in the urine resulting in low levels of detection. This will affect the bioavailability and accuracy of the assay levels for patients experiencing hyper and hypoalbuminemia with related competition and induction processes of the enzymes. These complications are apparent where larger numbers of patients are involved in clinical trials, bioequivalence and bioavailability studies with varying protein levels that may be more crucial for drugs with a narrow therapeutic index.
... The preparation of the proteins/ligands, generation of receptor grid, and docking were performed on AutoDock 4.2 as described recently [27]. The SDF files of 3-oxolupenal and katononic acid were retrieved from PubChem database bearing PubChem CIDs 11,848,142 and 9,981,416, respectively. ...
Nuxia oppositifolia is traditionally used in diabetes treatment in many Arabian countries; however, scientific evidence is lacking. Hence, the present study explored the antidiabetic and antioxidant activities of the plant extracts and their purified compounds. The methanolic crude extract of N. oppositifolia was partitioned using a two-solvent system. The n-hexane fraction was purified by silica gel column chromatography to yield several compounds including katononic acid and 3-oxolupenal. Antidiabetic activities were assessed by α-amylase and α-glucosidase enzyme inhibition. Antioxidant capacities were examined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) scavenging assays. Further, the interaction between enzymes (α-amylase and α-glucosidase) and ligands (3-oxolupenal and katononic acid) was followed by fluorescence quenching and molecular docking studies. 3-oxolupenal and katononic acid showed IC50 values of 46.2 μg/mL (101.6 µM) and 52.4 μg/mL (119.3 µM), respectively against the amylase inhibition. 3-oxolupenal (62.3 µg/mL or 141.9 μM) exhibited more potent inhibition against α-glucosidases compared to katononic acid (88.6 µg/mL or 194.8 μM). In terms of antioxidant activity, the relatively polar crude extract and n-butanol fraction showed the greatest DPPH and ABTS scavenging activity. However, the antioxidant activities of the purified compounds were in the low to moderate range. Molecular docking studies confirmed that 3-oxolupenal and katononic acid interacted strongly with the active site residues of both α-amylase and α-glucosidase. Fluorescence quenching results also suggest that 3-oxolupenal and katononic acid have a good affinity towards both α-amylase and α-glucosidase enzymes. This study provides preliminary data for the plant’s use in the treatment of type 2 diabetes mellitus.
... Secondary structure measurement of protein using CD provided information that whether the purified protein is folded, and to checks its conformation or stability (Alam Khan et al. 2009Khan et al. , 2010Greenfield 2006;Rehman et al. 2011;Singh et al. 2015). In addition, analysis of CD spectra helps to estimate the secondary structure composition of a protein (Anwer et al. 2014(Anwer et al. , 2015Haque et al. 2015;Khan et al. 2016;Rahaman et al. 2015). ...
... Far-UV CD spectra of AGP in the presence and absence of different concentrations of Cu/Zn metallo-drugs were recorded using Applied Photophysics spectropolarimeter (Chirascan) as reported earlier [42]. The instrument was regularly calibrated with D-10-camphorsulfonic acid. ...
Drug-binding and interactions with plasma proteins strongly affect their efficiency of delivery, hence considered as a key factor in determining the overall pharmacological action. Alpha-1-acid glycoprotein (AGP), a second most abundant plasma protein in blood circulation, has unique drug binding ability and involved in the transportation of various compounds. Here, we have investigated the mechanism of interaction between AGP and potential Cu/Zn metallo-drugs of benzimidazole derived organic motifs (CuL2 and ZnL2, where L is Schiff base ligand) by applying integrated spectroscopic, biophysical techniques and computational molecular docking analyses. We found that both the metallo-drugs (CuL2 and ZnL2) were bound at the central cavity of AGP interacting with the residues of lobe I, lobe II as well as lobe III. The binding of metallo-drugs to AGP occurs in 1:1 M ratios. Hydrogen bonding, electrostatic and hydrophobic interactions played a significant role in stabilizing the AGP-metallo-drug complexes. Binding affinities of both the metallo-drugs towards AGP at 298 K were of the order of 104-105 M-1, corresponding to Gibbs free energy of stabilization of approximately -5.50 to -6.62 kcal mol-1. Furthermore, the spectroscopic investigation by circular dichroism and synchronous fluorescence analyses suggest conformational changes in AGP upon the binding of metallic compounds.
... CD spectra were observed at HSA to 2,4-TZD molar ratios of 1:5 and 1:10 at a constant HSA concentration of 4 µM. The CD spectra were recorded over the range 200-250 nm and mean residual ellipticity (MRE) values were calculated using relation 9 [17], ...
Thiazolidinedione derivatives (TZDs) have attracted attention because of their pharmacological effects. For example, certain TZDs have been reported to ameliorate type II diabetes by binding and activating PPARs (peroxisome proliferator-activated receptors). Nonetheless, no information is available on the interaction between the heterocyclic 2, 4-thiazolidinedione (2,4-TZD) moiety and serum albumin, which could affect the pharmacokinetics and pharmacodynamics of TZDs. In this study, we investigated the binding of 2,4-TZD to human serum albumin (HSA). Intrinsic fluorescence spectroscopy revealed a 1:1 binding stoichiometry between 2,4-TZD and HSA with a binding constant (Kb) of 1.69 ± 0.15 × 103 M−1 at 298 K. Isothermal titration calorimetry studies showed that 2,4-TZD/HSA binding was an exothermic and spontaneous reaction. Molecular docking analysis revealed that 2,4-TZD binds to HSA subdomain IB and that the complex formed is stabilized by van der Waal’s interactions and hydrogen bonds. Molecular dynamics simulation confirmed the stability of the HSA-TZD complex. Further, circular dichroism and 3D fluorescence studies showed that the global conformation of HSA was slightly altered by 2,4-TZD binding, enhancing its stability. The results obtained herein further help in understanding the pharmacokinetic properties of thiazolidinedione.
... The native conformation or the folded state of proteins can be governed by a variety of physical conditions such as pH, temperature, ionic strength, presence or absence of other regulatory proteins [32,55,58,[61][62][63][64]. So, the aberrant change in these conditions may change the conformational stability of the proteins by modifying its secondary, tertiary or quaternary structures [65][66][67][68][69]. ...
Integrin-linked kinase (ILK), a ubiquitously expressed intracellular Ser/Thr protein kinase, plays a major role in the oncogenesis and tumour progression. The conformational stability and unfolding of kinase domain of ILK (ILK193-446) was examined in the presence of increasing concentrations of urea. The stability parameters of the urea-induced denaturation were measured by monitoring changes in [θ]222 (mean residue ellipticity at 222nm), difference absorption coefficient at 292nm (Δε292) and intrinsic fluorescence emission intensity at pH7.5 and 25°C. The urea-induced denaturation was found to be reversible. The protein unfolding transition occurred in the urea concentration range 3.0-7.0M. A coincidence of normalized denaturation curves of optical properties ([θ]222, Δε292 and λmax, the wavelength of maximum emission intensity) suggested that urea-induced denaturation of ILK domain is a two-state process. We further performed molecular dynamics simulation for 100ns to see the effect of urea on structural stability of ILK domain at atomic level. Structural changes with increasing concentrations of urea were analysed, and we observed a significant increase in the root mean square deviation, root mean square fluctuations, solvent accessible surface area and radius of gyration. A correlation was observed between in vitro and in silico studies.
... Secondary structure measurement of protein using CD provided information that whether the purified protein is folded, and to checks its conformation or stability (Alam Khan et al. 2009Khan et al. , 2010Greenfield 2006;Rehman et al. 2011;Singh et al. 2015). In addition, analysis of CD spectra helps to estimate the secondary structure composition of a protein (Anwer et al. 2014(Anwer et al. , 2015Haque et al. 2015;Khan et al. 2016;Rahaman et al. 2015). ...
Rho GTPases activating protein 2 (RGA2) is primarily involved in the modulation of numerous morphological events in eukaryotes. It protects plants by triggering the defense system which restricts the pathogen growth. This is the first report on the isolation, purification and characterization of RGA2 from the stems of Tinospora cordifolia, a medicinal plant. The RGA2 was purified using simple two-step process using DEAE-Hi-Trap FF and Superdex 200 chromatography columns, with a high yield. The purity of RGA2 was confirmed by SDS-PAGE and identified by MALDI-TOF/MS. The purified protein was further characterized for its secondary structural elements using the far-UV circular dichroism measurements. Our purification procedure is simple two-step process with high yield which can be further used to produce RGA2 for structural and functional studies.
