Current Enzyme Inhibition (v.12, #2)

Meet Our Editorial Board Member by Simone Carradori (109-109).

Protein Engineering Studies for C-C Chemokine Receptor Type 2 (CCR2) by Ramin E. Salmas, Mine Yurtsever, Serdar Durdagi (110-114).
C-C chemokine receptor type 2 (CCR2) belongs to large GPCR family and it plays a critical role in cognitive function. Inhibition of CCR2 is important for autoimmune diseases including atherosclerosis, pain, and metabolic diseases. 3D structure of this receptor was not solved yet. In the current study, 3D structure of the CCR2 is predicted using recently solved high resolution crystal structure of C-C chemokine receptor type 5 (CCR5) which shares a high amino acid sequence homology with CCR2. Derived model firstly refined with molecular dynamics simulations and then validated with Ramachandran plot as well as available validation tools such as PROCHECK (a program to check the sterochemical quality of protein structures). Correctness of the topology of the binding cavity of the target structure is externally tested with known CCR2 inhibitors using molecular docking simulations. In addition, in order to discover novel CCR2 inhibitors through approved drugs, high throughput virtual screening of marketed drugs against derived CCR2 model was performed.

Monoamine Oxidase Inhibitors: Perspective Design for the Treatment of Depression and Neurological Disorders by Bijo Mathew, Githa E. Mathew, Jerad Suresh, Gülberk Ucar, Rani Sasidharan, Sockalingam Anbazhagan, Jobin K. Vilapurathu, Venkatesan Jayaprakash (115-122).
Monoamine oxidase-A and B have been studied over a long period as one of the promising drug targets for the treatment of depression and neurodegenerative disorders. Commonly, MAO-A is associated with depression because of its relation with the control of serotonin levels. On the other hand, MAO-B has been associated with Alzheimer's and Parkinson's diseases because this enzyme modulates dopamine levels in the CNS. The major objective of the research in this field is devoted to identify and isolate selective ligands of MAO-A/MAO-B so that the undesirable side effects due to non-selective inhibition of monoamine catabolism by the isoforms can be avoided. This review will give an overview of the inhibition mechanism of MOA and its biochemistry, along with the history and development of MAO inhibitors including the significance of molecular modeling studies for the identification of novel class of MAO inhibitors.

Virtual Screening on Analogs of 2 Methyl Heptyl Isonicotinate as GlmU Inhibitors of Mycobacterium tuberculosis by Salam P. Singh, Chitta R. Deb, Sharif Uddin Ahmed, Yenisetti Saratchandra, Bolin K. Konwar (123-133).
As we are aware of the alarming rate of the tuberculosis multi drug resistant and extensive drug resistance, it necessitates for a novel drug target. In this investigation, we performed pharmacophore assisted virtual screening on analogs of 2-methyl heptyl isonicotinate against GlmU enzyme of Mycobacterium tuberculosis. Recent reports suggested the GlmU enzyme as a key protein in UDPGlcNAc biosynthesis. Inhibition of the enzyme often results in cell lysis and cell death, and hence GlmU is a potential drug target. Besides, 2-methylheptyl isonicotinate has a strong antibacterial activity against Mycobacterium tuberculosis. In the present investigation, pharmacophore modeling was performed for GlmU enzyme and molecular docking was carried out at the predicted site. The molecular docking simulation results revealed the analogs exhibiting favourable docking score inhibiting the enzyme. The analogs used in the present investigation are 90% similar with 2- methyl heptyl isonicotinate. Hence the analogs could also possess strong anti bacterial property against Mycobacterium tuberculosis. Additionally, the ADME-Toxicity parameters for the top docking hits showed two analogs viz CID55259454, CID4147645, CID20033037 and CID531736 possessed enhanced pharmacological parameters than market approved drug isoniazid. And hence these candidates may serve as a novel inhibitor against Mtb GlmU enzyme.

Extracellular Phytase in Candida albicans from Different Sources by Ali Z. Mahmoudabadi, Shahrzad Heidarinia, Ali Rezaei-Matehkolaei (134-136).
Background: The activity and role of different extracellular secreted enzymes has been characterized in the pathogenesis of different fungal species. Phytase, a special class of histidine acid phosphatases has recently been specified in some fungal species but not in Candida albicans. The objective of the present study was to elucidate the ability of C. albicans strains from different sources to secrete phytate and its correlation to pathogenicity.
Materials and Methods: A total of 100 C. albicans strains of which 25 isolated from patients with Vulvovaginal Candidiasis (VVC), 25 from normal vagina, 25 from candiduria and 25 from buccal cavity (healthy people) were included in the study. All strains were cultured on phytase specific agar medium. Phytase activity was evaluated as phytase index (PI) according to visual method presented by Tsang using.
Results: Overall, 63 strains (63%) including all of the oral isolates, 88% of VVC and 64% of normal vagina isolates had a high level of phytase activity (Phytase Index ≤ 0.50) while the remaining (37%) included all urinary tracts strains that were unable to secrete phytase (0.50 Conclusions: It is concluded that, most of the phytase activity was detected in oral and vaginal isolates however to provide further explanation on the phytase activity of Candida strains, a broader sampling from other clinical and environmental sources is recommended.

2-(2-Arylidenehydrazinyl) thiazol-4(5H)-ones having epidermal growth factor receptor (EGFR) inhibitory activity were subjected to quantitative structure activity relationship (QSAR) (2 and 3 dimensional) analysis and pharmacophore study in the present work. The best 2 dimensional (2D) QSAR model had r2 (squared correlation coefficient), q2 (cross validated correlation coefficient) and pred_r2 (predictive correlation coefficient) values of 0.8630, 0.7652 and 0.9533 respectively with Partial least square regression (PLSR) analysis. This model showed that count of number of nitrogen atoms separated from oxygen atom by four bonds (T_N_O_4) and RadiusOfGyration (size descriptor) descriptors contribute positively and count of number of any atoms separated from carbon atom by four bonds (T_T_C_4) contributes inversely to the biological activity. The k-nearest neighbor (kNN) method produced a significant 3 dimensional (3D) QSAR model exhibiting q2 and pred_r2 values of 0.6615 and 0.8833 respectively. PLSR gave a significant 3D QSAR model having r2, q2 and pred_r2 values of 0.8449, 0.7816 and 0.7834 respectively. Both 3D QSAR models depict the need of less bulky substituents at R2 position while PLSR 3D QSAR model also portrays the need of more bulky substituent at R4 position. The identified common pharmacophore features are one aromatic (AroC) and three hydrogen bond acceptors (HAc) obtained from Molsign and Pharmagist approaches. The present work may be useful for further lead optimization and designing of potent anticancer agents.

New Dehydroepiandrosterone-triazole Derivatives Identified as Inhibitors of 17β-Hydroxysteroid Dehydrogenase Enzyme in the Prostate by Araceli Sánchez-Márquez, Aylin Silva-Ortíz, Eugene Bratoeff, Yvonne Heuze, Juan Soriano, Yesica Medina, Marisa Cabeza (145-154).
Introduction: Synthesis and biological activity of three different dehydroepiandrosteronederivatives which possess a triazole group at C-17 and various substituents at C-3 of the steroidal skeleton are presented. These compounds were designed to inhibit the activity of type 5 17-β hydroxysteroid dehydrogenase (17β-HSD 5) which is present in human prostate. These steroids have been proposed as an alternative for treatment of prostate cancer patients showing resistance to anti-androgen therapy.
Materials and Methods: The role of these derivatives as inhibitors of 17β-HSD activity was determined both in in vivo and in vitro conditions. In in vitro studies both human and hamster prostate were used as the source of this enzyme membrane fraction. The pharmacological effect on the weight of the prostate and seminal vesicles of castrated hamsters treated with androstenedione (4-dione) was determined for each of the derivatives tested.
Results: The results showed that 17β-HSD 5 activity is present in the prostate membrane of both species. However, it could be inhibited by the three derivatives studied. Pharmacological studies have demonstrated that these three compounds were able to decrease prostate weight.
Conclusion: In conclusion, these dehydroepiandrosterone-triazole derivatives inhibited the activity of 17β-HSD 5 and also displayed pharmacological activity, so they could have therapeutic potential for cancer patients who do not respond to treatment with anti-androgens.

Extracellular Enzymes Production and Biofilm Formation in Rhodotorula Species by Zahra Seifi, Ali Z. Mahmoudabadi, Sharzad Hydrinia, Marzieh Halvaeezadeh (155-160).
Background: The different species of Rhodotorula are living in the environment and considered as normal inhabitants in the human body. In addition, they are found as common contaminants of skin, urine, respiratory and gastrointestinal systems among normal individuals. Out of several species of Rhodotorula, Rhodotorula mucilaginosa, R. glutinis and R. minuta were considered as human pathogens. They are causing invasive Rhodotorulosis among immunocompromised patients. Several studies show that extracellular enzymes are among the most important pathogenic factors in yeasts. In the present study we aimed to the detection of extracellular enzymes and the formation of biofilm by different species of Rhodotorula.
Materials and Methods: In the present study 68 isolates of Rhodotorula including, R. glutinis (58 isolates) R. mucilaginosa (5 isolates), R. minuta (3 isolates) and Rhodotorula species (2 isolates) were examined for extracellular enzymes excretion. Standard methods were used for the evaluation of phospholipase, proteinase, esterase and hemolytic activity. In addition biofilm formation was also evaluated using polystyrene microplates.
Results: Our results show that 82.4% of tested Rhodotorula isolates had phospholipase activity with higher Pz values (<0.70) in 69.1% of isolates. Esterase activity was detected among 29.4% of R. glutinis, R. minuta and Rhodotorula species, whereas 5 isolates of R. mucilaginosa were negative for this enzyme. The best biofilm formation results were obtained from incubation at 25°C after 72h (23.5%).
Conclusions: According to this study there is a remarkable difference in the extracellular enzymatic activity among tested Rhodotorula species. In addition, 82.4% of isolates were positive for phospholipase activity, in comparison to 29.4% for esterase and 69.1% for hemolytic activity. Biofilm formation was very weak among tested isolates.

CoMFA, CoMSIA and Docking Studies of Saquinavir Based Peptidomimetic Inhibitors of HIV-1 Protease by Vandana Saini, Sakshi Piplani, Amita S. Dang, Ajit Kumar (161-169).
HIV protease has been one of the most considered target sites to combat HIV infection and Saquinavir is the forerunner of all therapeutic agents targeting the same. There has always been a quest for new HIV-protease inhibitor for AIDS-treatment. The current study deals with in-silico attempt to develop 3D-QSAR models based on CoMFA and CoMSIA studies, to design and evaluate new saquinavir based chemical entities for their anti-HIV Protease activity. Optimal CoMFA and CoMSIA models were generated using a set of saquinavir based 23 molecules (18 training and 05 test set). The leave-one out cross validation correlation coefficients were q2CoMFA= 0.681 and q2CoMSIA= 0.684. The correlation between the experimental activities and cross validated/predicted activities of the test set molecules was high and reflected robustness of the models (r2CoMFA= 0.967 and r2CoMSIA= 0.988). The CoMFA model suggested 76.4% steric and 23.6% electrostatic field contribution while the optimal CoMSIA model revealed that electrostatic, hydrophobic and hydrogen bonding interactions were significantly required for HIV-protease inhibition. The models were subjected to molecular docking studies for in-silico validation using different set of molecules derived from ZINC database with ≥ 95% similarity with saquinavir. 07 molecules having activity greater than saquinavir, as predicted, in common, by CoMFA and CoMSIA models, were docked against HIV-1 protease. The dock score and the predicted activity were observed to be significantly correlated with r2= -0.7142 (CoMFA); -0.6219 (CoMSIA) while the binding patterns were observed to be comparable to that of Saquinavir.

Synthesis and In Vitro Activity of Pyrrolo[3,4-d]pyrimidine-2,5-diones as Potential Non-nucleoside HCV Inhibitors by Amany S. Mostafa, Serry A. El Bialy, Waleed A. Bayoumi, Youki Ueda, Masanori Ikeda, Nobuyuki Kato, Ali M. Abdelal (170-176).
A new series of 1-methyl-4-phenyl-6-substituted-3,4,6,7-tetrahydro-1H-pyrrolo[3,4- d]pyrimidine-2,5-diones (9a-h) were investigated as potential non-nucleoside anti-HCV, through Renilla luciferase (RL) assay using HuH-7-derived OR6 assay system. The target derivatives 9a-h were synthesized in moderate to good yields through nucleophilic substitution, followed by subsequent cyclocondensation of the 6-bromomethyl dihydropyrimidine derivative 8 with the appropriate primary amine. Biological screening revealed that compound 9a (EC50 of 28 M) showed moderate anti-HCV activity; while compounds 9c, 9e and 9h showed weak activity.

Enzyme Inhibitory Effect and Antioxidant Properties of Astragalus lagurus Extracts by Gokhan Zengin, Ramazan Ceylan, Gokalp O. Guler, Simone Carradori, Sengul Uysal, Abdurrahman Aktumsek (177-182).
Antioxidant and enzyme inhibitory effects (against cholinesterase, tyrosinase, -amylase and -glucosidase) of ethyl acetate, methanol, and aqueous extracts from Astragalus lagurus (Fabaceae) were investigated with spectrophotometric methods. Antioxidant capacity were evaluated by using different assay including free radical scavenging (DPPH and ABTS), reducing power (FRAP and CUPRAC), phosphomolybdenum, and metal chelating experiments. Total phenolic and flavonoid content was also determined as 20.34-20.72 mg GAEs/g and 19.58-31.10 mg REs/g, respectively. Generally, A. lagurus ethyl acetate and aqueous extracts were observed to possess higher antioxidant and enzyme inhibitory effects compared to methanol extract. The aqueous extract exhibited the strongest free radical scavenging activity on DPPH (20.65 mg TEs/g) and ABTS (67.43 mg TEs/g) as well as on reducing power tests (73.98 mg TEs/g for CUPRAC and 53.49 mg TEs/g for FRAP). -Amylase and -glucosidase inhibition were found to be 0.12-0.56 and 1.45-1.82 mmol ACAEs/g. These findings suggest that A. lagurus could provide natural biologically active agents to be used in food and pharmaceutical industry.

Anti-Inflammatory Activities of Some Bee Products by Inhibition of Bovine Testes Hyaluronidase by Sevgi Kolayli, Hilal Ebru Cakir, Huseyin Sahin (183-187).
Background: Apitherapeutic products contain many valuable bioactive compounds which are responsible for biologically active features such as anti-oxidant, anti-viral, anti-microbial and anti-inflammatory properties.
Objective: This study investigated in vitro anti-inflammatory potentials in terms of hyaluronidase inhibition of three different bee products, propolis, pollens and royal jellies.
Methods: Water soluble extracts of the samples were prepared, and inhibitions of bovine serum hyaluronidase were used. Inhibitions were expressed as percentages of inhibition (IC50;g/mL or mg/mL) causing 50% inhibition of the enzyme.
Results: All the bee products exhibited anti-hyaluronidase activities in the order propolis, pollen, and finally royal jelly. Inhibition was also correlated with the samples' total phenolic contents.
Conclusion: The study results confirm that water-soluble propolis is the best inhibitory agent among the bee products, and that these may be used as anti-inflammatory agents. However, further in vivo studies are now needed to clarify their pharmaceutical potentials as medicines.

Objective: The main study was to evaluate the anti- inflammatory, anti-oxidant, and the gastro-protective properties of extracts of Stevia rebaudiana. In addition the purpose of the study was to investigate new, natural inhibition of urease and xanthine oxidase enzymes, as well as to find the sugar contents of extracts from the plants.
Method: The polyphenolic contents of methanol extracted samples were evaluated in three different ways: total phenolic contents, total flavonoid content and condensed tannin. The antioxidant activity was determined using ferric reducing antioxidant power and using the free radical scavenging activity of 2,2-diphenyl-1-picrylhydrazyl radicals and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) tests. Phenolic compounds were determined by high-performance liquid chromatography.
Results: Results of antioxidant activity gave a total pheneolic content of 12.303-8.477 mg/g gallic acid equivalent, total flavonoid content of 2.378-1.518 mg/g quercetin equivalent and condense tanin of 3.435-0.713 mg/g catechin equivalent in leaf and flower extracts. Ferric reducing antioxidant power values of the Stevia rebaudiana leaf and flower were discovered to be 202.499-116.826 M FeSO47H2O/g respectively. The radical scavenging activity values of the 2,2- diphenyl-1-picrylhydrazyl radical and the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) tests ranged between 0.533-0.398 and 6.637-17.382 mg/mL respectively. The inhibitory effect of the Stevia leaf extract on xanthine oxidase and urease was also investigated and the concentrations that gave 50% inhibition of maximal activity were found to be 23.51g/mL and 9.45g/mL respectively. Glucose and fructose were determined to be the sugars in the Stevia leaf.
Conclusion: The results indicate that the Stevia rebaudiana plant is not only rich in steviol glycosides, but is also a powerful natural antioxidant and anti-inflammatory with gastro-protective properties.

Objectives: To design novel 3,6-bis(3'- substituted propoxy) and 3,6-bis(5'- substituted pentyloxy)xanthone derivatives targeting Prostaglandin endoperoxide synthase-2 by molecular docking study and to evaluate their COX-2 inhibitory potential.
Materials and Method: The protein structure was downloaded from RCSB protein databank and the ligands were prepared using Chemdraw freeware. Discovery Studio version 2.5 was used for studying the binding interactions. Molinspiration Cheminformatics and OSIRIS property explorer were utilized online to predict the molecular properties and toxicities of the designed compounds.
Result: The in silico ligand binding interactions of compounds S3, S6, S14, S17 and S20 suggested approx. > 30% higher binding energy values than the standard Diclofenac (Binding energy -155.46 Kcal/mol). The binding energy was observed from -270.61 to -65.12 Kcal/mol. The rank of each designed compounds were determined on the basis of lowest energy score.
Conclusion: Molecular docking studies have shown significant anti-inflammatory activity in the synthesized 3,6-bis(3'- substituted propoxy) and 3,6-bis(5'- substituted pentyloxy)xanthone derivatives in comparision to the standard, Diclofenac. The incorporation of primary and secondary nitrogens in the chemical structure is found to favour the antiinflammatory potential of the designed compounds.