Characterization of P. aeruginosa Glucose 6- Phosphate Isomerase: A Functional Insight via In-Vitro Activity Study

2020 ◽  
Vol 20 (29) ◽  
pp. 2651-2661
Author(s):  
Deekshi Angira ◽  
Nalini Natarajan ◽  
Samir R. Dedania ◽  
Darshan H. Patel ◽  
Vijay Thiruvenkatam
Keyword(s):  
Active Site ◽  
Expression System ◽  
Homology Model ◽  
Competitive Inhibitor ◽  
Membered Ring ◽  
Titration Calorimetry ◽  
Motility Factor ◽  
Differential Scanning Fluorimetry ◽  
Ic50 Values

Background: Glucose-6-phosphate isomerase (G6PI) catalyses the second step in glycolysis in the reversible interconversion of an aldohexose glucose 6-phosphate, a six membered ring moiety to a ketohexose, fructose 6-phosphate five membered ring moiety. This enzyme is of utmost importance due to its multifunctional role like neuroleukin, autocrine motility factor, etc. in various species. G6PI from Pseudomonas aeruginosa is less explored for its moonlighting properties. These properties can be predicted by studying the active site conservation of residues and their interaction with the specific ligand. Methods: Here, we study the G6PI in a self-inducible construct in bacterial expression system with its purification using Ni-NTA chromatography. The secondary structure of pure G6PI is estimated using circular dichroism to further predict the proper folding form of the protein. The bioactivity of the purified enzyme is quantified using phosphoglucose isomerase colorimetric kit with a value of 12.5 mU/mL. Differential scanning fluorimetry and isothermal titration calorimetry were employed to monitor the interaction of G6PI with its competitive inhibitor, erythrose 4-phosphate and calculated the Tm, Kd and IC50 values. Further, the homology model for the protein was prepared to study the interaction with the erythrose 4-phosphate. MD simulation of the complex was performed at 100 ns to identify the binding interactions. Results: We identified hydrogen bonds and water bridges dominating the interactions in the active site holding the protein and ligand with strong affinity. Conclusion : G6PI was successfully crystallized and data has been collected at 6Å. We are focused on improving the crystal quality for obtaining higher resolution data.

scholarly journals Design and Characterisation of Inhibitory Peptides against Bleg1_2478, an Evolutionary Divergent B3 Metallo-β-lactamase

Molecules ◽  
2020 ◽  
Vol 25 (24) ◽  
pp. 5797
Author(s):  
Gayathri Selvaraju ◽  
Thean Chor Leow ◽  
Abu Bakar Salleh ◽  
Yahaya M. Normi
Keyword(s):  
Active Site ◽  
Protein Complexes ◽  
Hypothetical Protein ◽  
Titration Calorimetry ◽  
Binding Properties ◽  
Vitro Assay ◽  
In Silico Docking ◽  
Inhibitory Peptides ◽  
Catalytic Function

Previously, a hypothetical protein (HP) termed Bleg1_2437 (currently named Bleg1_2478) from Bacillus lehensis G1 was discovered to be an evolutionary divergent B3 subclass metallo-β-lactamase (MBL). Due to the scarcity of clinical inhibitors for B3 MBLs and the divergent nature of Bleg1_2478, this study aimed to design and characterise peptides as inhibitors against Bleg1_2478. Through in silico docking, RSWPWH and SSWWDR peptides with comparable binding energy to ampicillin were obtained. In vitro assay results showed RSWPWH and SSWWDR inhibited the activity of Bleg1_2478 by 50% at concentrations as low as 0.90 µM and 0.50 µM, respectively. At 10 µM of RSWPWH and 20 µM of SSWWDR, the activity of Bleg1_2478 was almost completely inhibited. Isothermal titration calorimetry (ITC) analyses showed slightly improved binding properties of the peptides compared to ampicillin. Docked peptide–protein complexes revealed that RSWPWH bound near the vicinity of the Bleg1_2478 active site while SSWWDR bound at the center of the active site itself. We postulate that the peptides caused the inhibition of Bleg1_2478 by reducing or blocking the accessibility of its active site from ampicillin, thus hampering its catalytic function.

Dabigatran Inhibits Both Clot-Bound and Fluid Phase Thrombin In Vitro: Effects Compared to Heparin and Hirudin.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3998-3998 ◽  
Author(s):  
Joanne van Ryn ◽  
Norbert Hauel ◽  
Lisa Waldmann ◽  
Wolfgang Wienen
Keyword(s):  
Active Site ◽  
Platelet Rich Plasma ◽  
Fluid Phase ◽  
Irreversible Inhibitor ◽  
Thrombin Inhibition ◽  
Ic50 Values ◽  
Artificial Surface ◽  
In Vitro Effects ◽  
Targeted Inhibition

Abstract Thrombin bound to fibrin either in an existing thrombus or on an artificial surface is thought to be protected from inhibition by heparin/ATIII. Thus, this surface can remain “active” despite heparin / LMWH therapy. As long as anticoagulant levels are maintained, fluid phase thrombin inhibition is sufficient to prevent progression of thrombosis. However, during times of trough levels or upon discontinued therapy, this active thrombin could initiate further prothrombotic events. Dabigatran is a reversible, univalent, direct inhibitor of thrombin. Considering its specificity and selectivity for the active site, it is to be expected that it can inhibit thrombin equally, both when bound to a clot and in fluid phase. Thus, this study investigated the ability of dabigatran to inhibit thrombin bound to fibrin, or thrombin in plasma as compared to heparin and hirudin in vitro. Clot-bound thrombin: Clots were generated in human platelet rich plasma by adding CaCl2 and incubating 2 hrs at 37°C (1). Clots were then washed to remove trapped FPA. Washed clots were then transferred to 0.5 ml platelet poor plasma (PPP) containing either dabigatran, heparin, hirudin or buffer and incubated at 37°C. Fluid-phase thrombin: Thrombin (20 pM) was added to PPP containing either dabigatran, heparin, hirudin or buffer and incubated for 1 hr at 37°C. Fibrinopeptide A (FPA) Measurement: Uncleaved fibrinogen was precipitated and fibrin formation was measured as FPA release using ELISA. The IC50 (concentration required to inhibit FPA release by 50%) in fluid phase and clot-bound conditions was calculated using nonlinear curve-fitting. Dabigatran, hirudin and heparin inhibited fluid and clot-bound thrombin with increasing concentrations. Dabigatran inhibited both clot-bound and fluid phase thrombin with similar affinity, IC50 values of 200 nM and 186 nM, respectively. The IC50 of heparin-induced inhibition of clot-bound thrombin was 98.7 nM and in fluid phase 8.4 nM. This is consistent with published results indicating that heparin is not as efficient in inhibiting thrombin bound to a clot as in the fluid phase. Hirudin is a potent, irreversible inhibitor of thrombin and in this study it inhibited both free and bound thrombin equally in low nM ranges, the IC50 of clot-bound thrombin was 0.59 nM and fluid phase thrombin 1.63 nM. The IC50 values were converted into a ratio of clot-bound vs fluid phase thrombin to allow a more direct comparison and are listed in the table. Dabigatran Heparin Hirudin Ratio IC50 Clot-bound: Fluid Phase IIa 1.07 11.76 0.36 Dabigatran is equally effective in inhibiting both clot-bound and fluid phase thrombin. These data are consistent with targeted inhibition of the active site of thrombin by a small molecule, irrespective of whether thrombin is bound via the exosite to fibrin or is present as free enzyme in plasma. In contrast, heparin is less effective in inhibiting of clot-bound than fluid phase thrombin, consistent with its mechanism of thrombin inhibition via ATIII. Hirudin more potently inhibits clot-bound than free thrombin at lower concentrations, at higher concentrations inhibition of fluid phase thrombin is favoured. Thus these data suggest that dabigatran could also be effective in clinical situations where surface-bound thrombin could play a role, such as during catheterization procedures or the treatment of established thrombosis.

scholarly journals DESIGN, SYNTHESIS AND COX1/2 INHIBITORY ACTIVITY OF NEW QUINAZOLINE-5-ONE DERIVATIVES

2017 ◽  
Vol 13 (1) ◽  
pp. 5923-5931
Author(s):  
Ahmed S. Aboraia ◽  
Mohammed A. Hara ◽  
Mostafa H. Abdelrahman ◽  
Mohamed M. Amin ◽  
Osama I. El-Sabbaghab
Keyword(s):  
Active Site ◽  
Docking Study ◽  
Selectivity Index ◽  
Inhibition Assay ◽  
Inhibitor Concentration ◽  
Design Synthesis ◽  
Ic50 Values ◽  
Cox 2 ◽  
Cox 1

A new series of 1-(4-Acetylphenyl)-7,7-dimethyl-3-(substitutedphenyl)-1,2,3,4,7,8-octahydroquinazolin-5(6H)-ones (6-15) were synthesized and tested against COX-1 and COX-2 enzymes. Those compounds exhibited strong interaction at the COX-2 binding site and poor interaction at the COX-1 active site. Subjected to in vitro cyclooxygenase COX-1/COX-2 inhibition assay; most of the compounds especially compounds 6, 7, 12, 13, and 16 exhibited potent anti-inflammatory effects, selective COX-2 inhibition, with half-maximal inhibitor concentration (IC50) values of 0.22–1.42 μM and selectivity index (SI) values of 6.16–14.18 compared with celecoxib (IC50 = 0.05 μM and COX-2 SI: 296), diclofenac (IC50 = 0.8 μM and COX-2 SI: 4.87), and indomethacin (IC50 = 0.49 μM and COX-2 SI: 0.08) as reference drugs. Docking study has been carried out to confirm the binding affinity and selectivity of the most active compound (compound 6) to COX-2 enzyme.

Active site characterization and activity of the human aspartyl (asparaginyl) β-hydroxylase

Metallomics ◽  
2021 ◽  
Vol 13 (10) ◽  
Author(s):  
Jenna M Greve ◽  
Andrew M Pinkham ◽  
Zechariah Thompson ◽  
J A Cowan
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Catalytic Domain ◽  
Negative Impact ◽  
Enzyme Stability ◽  
Titration Calorimetry ◽  
Cellular Biochemistry ◽  
Biophysical Approach

Abstract Human aspartyl/asparaginyl beta-hydroxylase (HAAH) is a member of the superfamily of nonheme Fe2+/α-ketoglutarate (αKG) dependent oxygenase enzymes with a noncanonical active site. HAAH hydroxylates epidermal growth factor (EGF) like domains to form the β-hydroxylated product from substrate asparagine or aspartic acid and has been suggested to have a negative impact in a variety of cancers. In addition to iron, HAAH also binds divalent calcium, although the role of the latter is not understood. Herein, the metal binding chemistry and influence on enzyme stability and activity have been evaluated by a combined biochemical and biophysical approach. Metal binding parameters for the HAAH active site were determined by use of isothermal titration calorimetry, demonstrating a high-affinity regulatory binding site for Ca2+ in the catalytic domain in addition to the catalytic Fe2+ cofactor. We have analyzed various active site derivatives, utilizing LC-MS and a new HPLC technique to determine the role of metal binding and the second coordination sphere in enzyme activity, discovering a previously unreported residue as vital for HAAH turnover. This analysis of the in vitro biochemical function of HAAH furthers the understanding of its importance to cellular biochemistry and metabolic pathways.

Use of a Homology Model of the PIM-1 Kinase To Identify Variant Flavonoids Having Selective Inhibitory Activity Against PIM-1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2566-2566 ◽  
Author(s):  
Michael B. Lilly ◽  
Sheldon Holder ◽  
Marina Zemskova ◽  
Jonathan Neidigh
Keyword(s):  
Small Molecule ◽  
Kinase Inhibitors ◽  
Small Molecule Inhibitors ◽  
Homology Model ◽  
Binding Energies ◽  
Kinase Assay ◽  
Ic50 Values ◽  
In Vitro Kinase Assay ◽  
Threonine Kinases

Abstract The pim family of kinases are small cytoplasmic serine/threonine kinases implicated in the development of leukemias, lymphomas, and prostate cancer. Expression of pim-1 and pim-2 is induced by several oncogenes and growth factors. These kinases confer a survival phenotype through modulation of the expression or activity of various bcl-2 family members. To identify prototype small molecule inhibitors of pim-1 we have undertaken a series of experimental and computational studies. Central to our efforts has been the development of a homology model of the PIM-1 protein, using death-associated kinase as a template. After a series of computational refinements using the CFF forcefield, we then further modified our model through experimental approaches. The members of the pim family appear to have a unique, conserved structure to the hinge region, based on its length and the presence of two invariant prolines. This observation suggested that ATP-pocket binding compounds could be identified that would have selectivity for pim-1. We used a liquid phase, ELISA-based in vitro kinase assay to measure IC50 values for a set of 14 flavonoids, a family of known kinase inhibitors. Calculated binding energies for the test flavonoids were then compared with measured IC50 values, and the model giving the highest correlations was then used for further studies. A set of 25 additional flavonoids was then examined for binding energy to the homology model. Our studies predicted that the flavonoids quercetagetin and gossypetin could be potential pim-1 inhibitors. This hypothesis was then tested by in vitro kinase assay. Indeed these two flavonoids were found to be active pim-1 inhibitors with high-nanomolar potency. Quercetagetin was found to be competitive with ATP in the kinase assay. Furthermore the two flavonoids were seen to have a degree of selectivity for pim-1 kinase, compared with related serine-threonine kinases and a tyrosine kinase. IC50 (micromolar) of flavonoids against PIM-1 and other kinases Inhibitor PIM-1 PIM-2 cAMP-dep. kinase c-ABL quercetagetin 0.33 4.0 21 >200 gossypetin 0.430 7.1 250 >200 quercetin 2.0 ND ND 5.7 In contrast the potency and pim-1-selectivity of the related flavonoid quercetin (a promiscuous inhibitor of kinases) was much inferior. SAR analysis of the flavonoid set showed that pim-1 selectivity depended on the presence of three hydrogen bond donors on the flavonoid A ring. Quercetagetin has been tested for biologic effects on factor-dependent FDCP1 cells transfected with a cDNA for human pim-1. These test cells show prolonged, pim-1-dependent survival after removal of IL-3 from the growth medium. This phenotype was completely abolished by treatment with quercetagetin (IC50 = 3-5micromolar final concentration). Flavonoids such as quercetagetin and gossypetin may serve as guides for the development of small molecule inhibitors specific for pim family kinases. Such reagents will be useful for determining the role of constitutive pim-1 expression in the development of leukemias and lymphomas.

scholarly journals Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified through crystallographic screening and computational docking

2021 ◽  
Vol 7 (16) ◽  
pp. eabf8711
Author(s):  
Marion Schuller ◽  
Galen J. Correy ◽  
Stefan Gahbauer ◽  
Daren Fearon ◽  
Taiasean Wu ◽  
...  
Keyword(s):  
Active Site ◽  
Nonstructural Protein ◽  
Adenosine Diphosphate ◽  
Titration Calorimetry ◽  
Conformational Heterogeneity ◽  
Time Resolved ◽  
Computational Docking ◽  
Wide Range ◽  
Differential Scanning Fluorimetry ◽  
Biophysical Techniques

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate–ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

Correlating In Vitro Target-Oriented Screening and Docking: Inhibition of Matrix Metalloproteinases Activities by Flavonoids

Planta Medica ◽  
2017 ◽  
Vol 83 (11) ◽  
pp. 901-911 ◽  
Author(s):  
Lucia Crascì ◽  
Livia Basile ◽  
Annamaria Panico ◽  
Carmelo Puglia ◽  
Francesco Bonina ◽  
...  
Keyword(s):  
Active Site ◽  
Inhibitory Activity ◽  
In Silico ◽  
In Silico Docking ◽  
Lead Compounds ◽  
Good Ability ◽  
Metalloprotease Inhibitors ◽  
Ic50 Values ◽  
Further Development

AbstractMetalloproteases are a family of zinc-containing endopeptidases involved in a variety of pathological disorders. The use of flavonoid derivatives as potential metalloprotease inhibitors has recently increased.Particular plants growing in Sicily are an excellent yielder of the flavonoids luteolin, apigenin, and their respective glycoside derivatives (7-O-rutinoside, 7-O-glucoside, and 7-O-glucuronide).The inhibitory activity of luteolin, apigenin, and their respective glycoside derivatives on the metalloproteases MMP-1, MMP-3, MMP-13, MMP-8, and MMP-9 was assessed and rationalized correlating in vitro target-oriented screening and in silico docking.The flavones apigenin, luteolin, and their respective glucosides have good ability to interact with metalloproteases and can also be lead compounds for further development. Glycones are more active on MMP-1, -3, -8, and -13 than MMP-9. Collagenases MMP-1, MMP-8, and MMP-13 are inhibited by compounds having rutinoside glycones. Apigenin and luteolin are inactive on MMP-1, -3, and -8, which can be interpreted as a better selectivity for both -9 and -13 peptidases. The more active compounds are apigenin-7-O-rutinoside on MMP-1 and luteolin-7-O-rutinoside on MMP-3. The lowest IC50 values were also found for apigenin-7-O-glucuronide, apigenin-7-O-rutinoside, and luteolin-7-O-glucuronide. The glycoside moiety might allow for a better anchoring to the active site of MMP-1, -3, -8, -9, and -13. Overall, the in silico data are substantially in agreement with the in vitro ones (fluorimetric assay).

scholarly journals Self-acetylation at the active site of phosphoenolpyruvate carboxykinase (PCK1) controls enzyme activity

2020 ◽  
pp. jbc.RA120.015103
Author(s):  
Pedro Latorre-Muro ◽  
Josue Baeza ◽  
Ramon Hurtado-Guerrero ◽  
Thomas Hicks ◽  
Ignacio Delso ◽  
...  
Keyword(s):  
Active Site ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Direct Evidence ◽  
Protein Crystallization ◽  
Metabolic Stress ◽  
Titration Calorimetry ◽  
Acetyl Coa ◽  
Key Enzyme ◽  
Chemically Reacting

Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to oxaloacetate). It is also known that the histone acetyltransferase p300 can induce PCK1 acetylation in cells, but whether that is a direct or indirect function was not known. Here we initially set out to determine whether p300 can acetylate directly PCK1 in vitro. We report that p300 weakly acetylates PCK1, but surprisingly, using several techniques including protein crystallization, mass spectrometry, isothermal titration calorimetry (ITC), saturation-transfer difference nuclear magnetic resonance (STD-NMR) and molecular docking, we found that PCK1 is also able to acetylate itself using acetyl-CoA independently of p300. This reaction yielded an acetylated recombinant PCK1 with a 3-fold decrease in kcat without changes in Km for all substrates. Acetylation stoichiometry was determined for 14 residues, including residues lining the active site. Structural and kinetic analyses determined that site-directed acetylation of K244, located inside the active site, altered this site and rendered the enzyme inactive. Additionally, we found that acetyl-CoA binding to the active site is specific and metal dependent. Our findings provide direct evidence for acetyl-CoA binding and chemically reacting with the active site of PCK1 and suggest a newly discovered regulatory mechanism of PCK1 during metabolic stress.

scholarly journals The P3 O-Tert-Butyl-Threonine is Key to High Cellular and Antiviral Potency for Aldehyde-Based SARS-CoV-2 Main Protease Inhibitors

2021 ◽  
Author(s):  
Yuying Ma ◽  
Kai Yang ◽  
Zhi Zachary Geng ◽  
Yugendar R. Alugubellia ◽  
Namir Shaabani ◽  
...  
Keyword(s):  
Active Site ◽  
Unnatural Amino Acids ◽  
Chemical Compositions ◽  
Structural Variations ◽  
Covalent Interaction ◽  
Tert Butyl ◽  
Main Protease ◽  
Ic50 Values ◽  
Inhibition Potency

As an essential enzyme to SARS-CoV-2, main protease (MPro) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 sites and the N-terminal protection group, we synthesized a series of MPro inhibitors that contain 𝛽-(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 site. These inhibitors have a large variation of determined IC50 values that range from 4.8 to 650 nM. The determined IC50 values reveal that relatively small side chains at both P2 and P3 sites are favorable for achieving high in vitro MPro inhibition potency, the P3 site is tolerable toward unnatural amino acids with two alkyl substituents on the 𝛼-carbon, and the inhibition potency is sensitive toward the N-terminal protection group. X-ray crystal structures of MPro bound with 16 inhibitors were determined. All structures show similar binding patterns of inhibitors at the MPro active site. A covalent interaction between the active site cysteine and a bound inhibitor was observed in all structures. In MPro, large structural variations were observed on residues N142 and Q189. All inhibitors were also characterized on their inhibition of MPro in 293T cells, which revealed their in cellulo potency that is drastically different from their in vitro enzyme inhibition potency. Inhibitors that showed high in cellulo potency all contain O-tert-butyl-threonine at the P3 site. Based on the current and a previous study, we conclude that O-tert-butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for peptidyl aldehyde inhibitors of MPro. This finding will be critical to the development of novel antivirals to address the current global emergency of concerning the COVID-19 pandemic.

scholarly journals Overexpression of restructured pyruvate dehydrogenase complexes and site-directed mutagenesis of a potential active-site histidine residue

1990 ◽  
Vol 269 (2) ◽  
pp. 443-450 ◽  
Author(s):  
G C Russell ◽  
J R Guest
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Expression System ◽  
Site Directed Mutagenesis ◽  
Histidine Residue ◽  
Complex Activity ◽  
Putative Active Site ◽  
Active Site Histidine

The aceEF-lpd operon of Escherichia coli encodes the pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase multienzyme complex (PDH complex). A thermoinducible expression system was developed to amplify a variety of genetically restructured PDH complexes, including those containing three, two, one and no lipoyl domains per E2p chain. Although large quantities of the corresponding complexes were produced, they had only 20-50% of the predicted specific activities. The activities of the E1p components were diminished to the same extent, and this could account for the shortfall in overall complex activity. Thermoinduction was used to express a mutant PDH complex in which the putative active-site histidine residue of the E2p component (His-602) was replaced by cysteine in the H602C E2p component. This substitution abolished dihydrolipoamide acetyltransferase activity of the complex without affecting other E2p functions. The results support the view that His-602 is an active-site residue. The inactivation could mean that the histidine residue performs an essential role in the acetyltransferase reaction mechanism, or that the reaction is blocked by an irreversible modification of the cysteine substituent. Complementation was observed between the H602C PDH complex and a complex that is totally deficient in lipoyl domains, both in vitro, by the restoration of overall complex activity in mixed extracts, and in vivo, from the nutritional independence of strains that co-express the two complexes from different plasmids.

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