Current Enzyme Inhibition (v.13, #1)

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In vitro and In silico: Selectivities of Seychellene Compound as Candidate Cyclooxygenase Isoenzyme Inhibitor on Pre-Osteoblast Cells by Sentot J. Raharjo, Chanif Mahdi, Nurdiana Nurdiana, Takheshi Kikuchi, Fatchiyah Fatchiyah (2-10).
Objective: To determine selectivity's single and cluster of the seychellene (CID519743) compound such as cyclooxygenase (COX-1/ COX-2) inhibitor by invitro and In silico analysis.

Methods: Fraction of seychellene obtained fractional-vacuum distillation patchouli oil by Pilodist-104. The fraction was determined through IC50 COX-1/ COX-2 value by colorimetric test COX-ovine 760700 and the effect decreases the expression of COX-1/ COX-2 on pre-osteoblast cell to induce LPS. Molecular interaction studies single/ cluster of seychellene and other compounds with COX-1 and COX-2 used the molecular docking tools by Hex 8.0 and the interactions were further visualized and the binding energy was calculated (with Generalized Born Molecular Volume (GBMV) model solvent) using Discovery Studio Client 3.5 software.

Result: Fraction of seychellene obtained from patchouli oil in fraction-5 (25.05%). The IC50 values of fraction-5 were: COX-1 (73.47μM) and COX-2 (73.31μM). The fraction of seychellene decreased the expression of COX-1/ COX-2 isoenzym. In silico analyses of scoring binding energy calculation (with GBMV model solvent) of single and cluster seychellene compounds affected the selectivity of COX-1 and COX-2 inhibitor.

Conclusion: Collectively, selectivity's of the seychellene compound as non-selective inhibitor cyclooxygenase.

An in silico Approach Towards Crop Improvement by ACC Synthase Inhibition Declining Ethylene Production by Jasarat Ali, Rupesh K. Mishra, Chhedi L. Gupta, Dinesh C. Sharma, Preeti Bajpai, Neelam Pathak (11-19).
Introduction: The increased level of ethylene inhibits root elongation and causes physiological damage, thereby reduces ethylene level imparts a positive support against various biotic and abiotic stresses viz. phytopathogens, extreme temperatures, hyper salinity, flooding, drought, metal/organic contaminants and insect predation. The metabolic pathways showed the involvement of ACC synthase inhibition for ethylene suppression in plants.

Objective: The primary objective of this study focused towards the use of In-silico approach to assess the inhibitory effect of S-adenosyl methionine (SAM) analogue on ACC synthase activity.

Methods: The 3D structure of ACC synthase of Pisum sativum was constructed using modeler 9v11 software. The reliability of developed model was evaluated by PROCHECK, ERRAT and ProSA web servers. Furthermore the molecular interactions between substrate SAM and inhibitors were performed.

Results: The docking study demonstrated that the binding energy of the substrate SAM is -5.37 Kcal/mol. The SAM analogue (Inhibitors) considered in this study were 3-dzSAHC, SAHC, sinefungin, SIBA, 7-dz-SIBA, 1-dz-SIBA,3-dz-SIBA and S-n-Butyladenosine. Among these analogues, 7-dz-SIBA was found to be most effective on ACC synthase as inhibitor due to lowest binding energy (- 5.51Kcal/mol) and strong Ki value (91.74 M). The LYS276 amino acid residue of ACC synthase was observed in the interaction with both substrates SAM and 7-dz-SIBA (S-isobutyl-7-deazaadenosine) demonstrates as most crucial catalytic residue for molecular interaction.

Conclusion: This study successfully screened most potent inhibitor for ACC synthase which have indicated the compounds 7-dz- SIBA as effective inhibitor with lowest binding energy, better hydrogen bond interaction and strong inhibition constant compared to others compounds studied. Thus 7-dz-SIBA can be projected to use as a growth enhancer for overall crop improvement. It may help in plant growth promotion, prevents the plants from various environmental stress and phytopathogenic infections etc.

Introduction: HIV viral envelope proteins are targets for small inhibitor molecules aimed at disrupting the cellular entry process. Potential peptide-class inhibitor molecules (rDNA drugs) have been previously identified, with mixed results, through biomimicry and phage display experimental methods. Here we describe a new approach based on computational fragment discovery. The method has the potential to not only optimize peptide binding affinity but also to rapidly produce alternative inhibitors against mutated strains.

Methods: A comprehensive, all-atom implicit solvent method is used to bombard the C-heptad repeat unit of HIV-1 target envelope protein GP41 with single Damino acid residues as they exist in their native state. A nascent peptide computational search process then identifies potential favorable sequences of attached ligands based on four peptide bond criteria. Finally, dynamic simulations of nascent peptides attached to host targets help refine potential peptide inhibitors for experimental HIV-1 challenge assays and testing.

Results and Discussion: Initial testing of the method was done using 64,000 total ligands of D-amino acid residues at a total computational time of 0.05 microseconds per ligand, which resulted in several thousand attached ligands. Peptide bond criteria search employing three of the four bond constraints with a tolerance of 20 percent, resulted in four potential peptide inhibitors of 5 to 6 residues in length. Only one of the four peptides demonstrated IC50 values and partial viral inhibition based on cell challenge assays using CEM-SS host cells. That peptide inhibitor also computationally demonstrated longtime attachment and stability to a helical groove in its C-heptad target. This initial testing of peptide fragment discovery against HIV-1 has helped us refine the protocols and identify key areas of improvement.

Conclusion: Our methods demonstrate the potential to design efficient peptide inhibitors to viral target proteins based on an all-atom dynamic simulation and using a ligand library as fragments of potential nascent peptides. Our methods can be greatly improved through the use of higher numbers of ligands, increased time of bombardment, and tighter constraints on the peptide bond search step. Our method may be important in the need to rapidly respond to target mutations and to advance multiple targeting methods based multiple peptide inhibitors.

Acethylcholinesterase Inhibition, Antibacterial and Antioxidant Properties of Diaryl Oxalates by Gunay K. Kantar, Nimet Baltas, Fatih S. Beris, Selami Sasmaz (27-33).
Objective: The main study was to synthesize the diaryl oxalates containing pharmacophore groups like eugenol and guaiacol moieties, investigate antioxidant, antibacterial and acethylcholinesterase inhibitory properties and compare with each other.

Method: The compounds were screened for their antioxidant properties by employing three in vitro assays like CUPric Reducing Antioxidant Capacity (CUPRAC) and 2,2-Diphenyl-1-Picrylhydrazyl (DPPH.) radical scavenging assay, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS.+) assay and they exhibited very promising results. Also, The compounds were evaluated for their antiacetylcholinesterase activity (anti-AChE) and anti-microbial effects against Bacillus subtilis ATCC 6633, S. pyogenes ATCC 19615, Y. pseudotuberculosis ATCC 911, E. cloaceae ATCC 25923, and E. coli ATCC 25922.

Results: The compounds were found to possess good antioxidant (TEAC values:7.834-9.353 mM TEAC/mg compound) and inhibitiory activity against AChE (IC50=11.95±0.27 for compound 2). Antimicrobial activity results show that compound 1 has the same antimicrobial effect on the bacteria in this study and is more effective than compound 2.

Conclusion: The results indicate that these compounds have not only good antioxidant and antimicrobial activity, but is also a powerful AChE inhibitors.

Inhibitory Effects of Some Bryophytes on Glutathione-S -Transferase by Ebru K. Kocazorbaz, Rabia N. Un, Adnan Erdag, Figen Zihnioglu (34-40).
Background: Nine extracts from selected Bryophytes, A. californica, B. pomiformis B. stricta, C. conicum, F. antipyretica var. gracilis, G. lisae, L. sciuroides, M. polymorpha, T. barbuloides were investigated for their total phenolic content and glutathione S-transferase (GST) inhibitory activity.

Methods: The flavonoid and phenolic content of nine extracts from Bryophytes were estimated by aluminium chloride and Folin-Ciocalteu method. The sheep liver fraction incubated with or without plant extracts and the activity GST was assayed using reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) secondary substrate.

Results: These results indicate the potential use of A. californica, C. conicum, G. lisae, M. polymorpha, and T. barbuloides can be regarded as promising candidates for natural plant sources of GST inhibitory effects.

Conclusion: These plants can be examined in order to isolate and identify the active ingredients, and this may serve as a foundation to find safer and more effective agents for therapeutic use.

Screening of Potential Plant Compounds as Survivin Inhibitors and its Anti-Cancer Efficacy by Molecular Docking by Safreen S.D. Amanulla, Sneeha Veerakumar, Kumaresan Ramanathan (41-48).
Background: Survivin, smallest Inhibitor of Apoptosis Protein (IAP) mediates apoptosis pathway and cell cycle is tumor specific and therapeutic target for cancer research. Breast cancer is a dreadful disease with greater mortality rate, commonly in women globally.

Aim: This study was undertaken to investigate the effect of indigenous Plant drugs by in-silico determination of antagonist for Survivin protein that was up regulated in breast cancer.

Materials and Methods: The 3D structure of Survivin protein was retrieved from PDB (Protein Data Bank). Plant compounds were retrieved from NPACT (Naturally occurring Plant based Anti-Cancerous Target) database and converted into 3D- PDB format using Open babel software. Based on the active sites we have screened the essential plant compound as an effective binder against modelled protein using iGEMDock 2.1.

Results and conclusion: The lead compound of plant as well as plant protein molecule would be scaled out on the basis of binding efficiency. Remangilones A showed greater interaction effect with Total energy (-121.465kcal mol-1). However, further in-vivo investigation is essential for the confirmation of drug efficacy and bio-compatibility.

Phytochemical and Biological Activities of five Turanecio Hamzaoglu (Asteraceae) Species from Turkey by Sevil Albayrak, Ahmet Aksoy, Abit Yasar, Lutfiye Yurtseven, Umit Budak (49-55).
Objectives: In vitro biological activities of methanolic extracts of five Turanecio species have been studied.

Materials and Methods: The phenolic compositions of the extracts were evaluated by the Folin- Ciocalteu assay and by HPLC analysis. Antioxidant activities were determined with two in vitro assays namely, DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging assay and phosphomolybdenum assay. The antimicrobial activities of the extracts were evaluated against 13 microorganisms.

Results: T. hypochionaeus var. hypochionaeus was showed the highest DPPH inhibition with 88.84% at 100 μg·mL-1. All of the extracts were exerted high total antioxidant activities 128.00- 243.13 mg AAE g-1) and seem to be a promising source of natural antioxidants. The phenolic contents in the extracts varied from 26.17 to 60.99 mg·g-1 as gallic acid equivalent. Chlorogenic acid, caffeic acid and p- coumaric acid were the predominant constituents. The methanolic extracts revealed promising antibacterial activities against most bacteria. No activity was recorded against yeasts tested.

Conclusion: The polyphenolic constituents appear to be responsible, at least in part, for the extract's activities. The present study confirms that tested Turanecio species contains significant source of phenolics have antioxidant and antimicrobial activities and may have therapeutic potential.

Objective: A carboxylesterase (CbE) or carboxylic-ester hydrolase (EC is an enzyme that catalyzes the hydrolysis of carboxylesters to alcohol and carboxylate. The objective of this study is to obtain this enzyme from liver and lung of rabbit with a comparison between the effects of two specific substrates, - naphthyl acetate (-NA) and p-nitrophenyl acetate (p-NPA) on the enzymes activity. The kinetic characteristics of the enzymes have been also investigated in details.

Methods: CbE was obtained from rabbit liver and lung with Tris/HCl buffer (pH 7.4) extraction, ammonium sulfate precipitation, dialysis, and lyophilized.

Results: The lyophilized enzyme appeared to be homogeneous on native polyacrylamide gel electrophoresis and its molecular mass was estimated to be 30 kDa suggesting a monomeric structure for the obtained enzyme. The enzyme exhibited a broad activity at different pH and temperature. The pH optimum was 7.5 and optimum temperature was 35°C. The enzyme showed specific activities of 1.36 and 1.04 mol -naphthol hydrolyzed/mg protein/min for liver and lung CbEs, respectively using -naphthyl acetate (-NA). However, the specific activities of the liver and lung CbE were 0.44 and 0.23 mol p-nitrophenol hydrolyzed/mg protein/min, respectively using p-nitrophenyl acetate (p- NPA). The relationships between estimates of Km and Vmax calculated from the Michaelis-Menten equation have been explored. The Km values of liver and lung CbEs were 59.28 and 28.10 M with -NA, respectively however, the values were 86.39 and 90.05 M with p-NPA. The Vmax values of liver and lung CbEs were 2.32 and 1.05 with -NA, respectively however, the values were 0.55 and 0.19 with p- NPA. The enzyme showed residual activity when incubated at wide range of pesticides chlorpyrifos and methomyl however it was totally inhibited by concentrations higher than 200 M. The extent of the inhibition was different, as estimated by the values of the inhibition constant Ki that were found to be 2.14 and 5.14 for chlorpyrifos and methomyl, respectively.

Conclusion: This enzyme may play an important role in the detoxification of organophosphorous and carbamate pesticides at low levels.

α-Glucosidase Inhibitory Effect and Antioxidant Activity of the Extracts of Eighteen Plant Traditionally Used in Algeria for Diabetes by Ihcen Khacheba, Hadjer Boussoussa, Amar Djeridane, Abir Bekhaoua, Noussiba Bensayah, Mohamed Yousfi (67-78).
Background: This paper focuses on one of the most recurrent hypotheses for the antidiabetic effect of medicinal plants by inhibiting the alpha -glucosidase

Objective: In the present study aqueous and hydro -alcoholic extracts of eighteen medicinal plants, collected in the town of Laghouat in the steppe region of Algeria, and which are used in the treatment of diabetes, were examined for α-glucosidase inhibition using an in vitro model. Also total phenol content and antioxidant activity of the extracts was investigated.

Methods: The extracts were prepared using distilled water and hydro -alcoholic solvent (methanol/water). The amount of total phenolics and flavonoids in the samples was determined with the Folin-Ciocalteu reagent and with aluminum chloride respectively. To identify plants with inhibitory capacities, we have studied the effects of ours extracts on the kinetics catalyzed of α-glucosidase an enzymes belonging to the class of hydrolase responsible for the digestion and we have subjected our extracts to inhibition assay to determine the inhibition percentage for each extract. Finally, we assessed the potential antioxydant of the best inhibitory plants with the DPPH test.

Results: The major plants were found to inhibit enzymatic activity of α-glucosidase and the values of the IC50 constants have been determined for the best inhibitory plants common in both extraction methods (Cistus SP, Salvia officinalis and Zygophylum album), with values ranged from 0.38 to 28.95 mg/ml. The evaluation of antioxidant activity of this plants using DPPH test, shows that the phenolic extract of Salvia officinalis exhibit good antioxidant capacity (8.46 μg/ml) comparatively to the antioxidants taken as reference (BHA, Gallic acid, Rutin, Trolox and Vitamin C).

Conclusion: This study is the first report on potential inhibition of these plants extracts on the digestive enzyme, α-glucosidase. The obtained results support the traditionally use of a number of the analyzed species in the in the management of diabetes.