Search results for: angiotensin converting enzyme inhibition

Authors: Drago Bratkovic, Curtis Gravance, David Ketteridge, Ravi Krishnan, Michael Imperiale

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The mucopolysaccharidoses (MPSs) are a group of lysosomal storage diseases that have a common defect in the catabolism of glycosaminoglycans (GAGs). MPS I is the most common of the MPS diseases. Manifestations of MPS I include coarsening of facial features, corneal clouding, developmental delay, short stature, skeletal manifestations, hearing loss, cardiac valve disease, hepatosplenomegaly, and umbilical and inguinal hernias. Treatments for MPS I restore or activate the missing or deficient enzyme in the case of enzyme replacement therapy (ERT) and haematopoietic stem cell transplantation (HSCT). Pentosan polysulfate sodium (PPS) is a potential treatment to improve bone and joint manifestations of MPS I. The mechanisms of action of PPS that are relevant to the treatment of MPS I are the ability to: (i) Reduce systemic and accumulated GAG, (ii) Reduce inflammatory effects via the inhibition of NF-kB, resulting in the reduction in pro-inflammatory mediators. (iii) Reduce the expression of the pain mediator nerve growth factor in osteocytes from degenerating joints. (iv) Inhibit the cartilage degrading enzymes related to joint dysfunction in MPS I. PPS is being evaluated as an adjunctive therapy to ERT and/or HSCT in an open-label, single-centre, phase 2 study. Patients are ≥ 5 years of age with a diagnosis of MPS I and previously received HSCT and/or ERT. Three white, female, patients with MPS I-Hurler, ages 14, 15, and 19 years, and one, white male patient aged 15 years are enrolled. All were diagnosed at ≤2 years of age. All patients received HSCT ≤ 6 months after diagnosis. Two of the patients were treated with ERT prior to HSCT, and 1 patient received ERT commencing 3 months prior to HSCT. Two patients received 0.75mg/kg and 2 patients received 1.5mg/kg of PPS. PPS was well tolerated at doses of 0.75 and 1.5 mg/kg to 47 weeks of continuous dosing. Of the 19 adverse events (AEs), 2 were related to PPS. One AE was moderate (pre-syncope) and 1 was mild (injection site bruising), experienced in the same patient. All AEs were reported as mild or moderate. There have been no SAEs. One subject experienced a COVID-19 infection and PPS was interrupted. The MPS I signature GAG fragments, sulfated disaccharide and UA-HNAc S, tended to decrease in 3 patients from baseline through Week 25. Week 25 GAG data are pending for the 4th patient. Overall, most biomarkers (inflammatory, cartilage degeneration, and bone turnover) evaluated in the 3 patients with 25-week assessments have indicated either no change or a reduction in levels compared to baseline. In 3 patients, there was a trend toward improvement in the 2MWT from baseline to Week 48 with > 100% increase in 1 patient (01-201). In the 3 patients that had Week 48 assessments, patients and proxies reported improvement in PGIC, including “worthwhile difference” (n=1), or “made all the difference” (n=2).

Keywords: MPS I, pentosan polysulfate sodium, clinical study, 2MWT, QoL

Procedia PDF Downloads 111

Authors: M. Naimi, M. B. Khaled

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The aim of the present work was to test in vitro inhibition of food pathogens and spoilage bacteria by crude bacteriocins from autochthonous lactic acid bacteria. Thirty autochthonous lactic acid bacteria isolated previously, belonging to the genera: Lactobacillus, Carnobacterium, Lactococcus, Vagococcus, Streptococcus, and Pediococcus, have been screened by an agar spot test and a well diffusion assay against Gram-positive and Gram-negative harmful bacteria: Bacillus cereus, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 8739, Salmonella typhimurium ATCC 14028, Staphylococcus aureus ATCC 6538, and Pseudomonas aeruginosa under conditions means to reduce lactic acid and hydrogen peroxide effect to select bacteria with high bacteriocinogenic potential. Furthermore, crude bacteriocins semiquantification and heat sensitivity to different temperatures (80, 95, 110°C, and 121°C) were performed. Another exploratory test concerning the response of St. aureus ATCC 6538 to the presence of crude bacteriocins was realized. It has been observed by the agar spot test that fifteen candidates were active toward Gram-positive targets strains. The secondary screening demonstrated an antagonistic activity oriented only against St. aureus ATCC 6538, leading to the selection of five isolates: Lm14, Lm21, Lm23, Lm24, and Lm25 with a larger inhibition zone compared to the others. The ANOVA statistical analysis reveals a small variation of repeatability: Lm21: 0.56%, Lm23: 0%, Lm25: 1.67%, Lm14: 1.88%, Lm24: 2.14%. Conversely, slight variation was reported in terms of inhibition diameters: 9.58± 0.40, 9.83± 0.46, and 10.16± 0.24 8.5 ± 0.40 10 mm for, Lm21, Lm23, Lm25, Lm14and Lm24, indicating that the observed potential showed a heterogeneous distribution (BMS = 0.383, WMS = 0.117). The repeatability coefficient calculated displayed 7.35%. As for the bacteriocins semiquantification, the five samples exhibited production amounts about 4.16 for Lm21, Lm23, Lm25 and 2.08 AU/ml for Lm14, Lm24. Concerning the sensitivity the crude bacteriocins were fully insensitive to heat inactivation, until 121°C, they preserved the same inhibition diameter. As to, kinetic of growth , the µmax showed reductions in pathogens load for Lm21, Lm23, Lm25, Lm14, Lm24 of about 42.92%, 84.12%, 88.55%, 54.95%, 29.97% in the second trails. Inversely, this pathogen growth after five hours displayed differences of 79.45%, 12.64%, 11.82%, 87.88%, 85.66% in the second trails, compared to the control. This study showed potential inhibition to the growth of this food pathogen, suggesting the possibility to improve the hygienic food quality.

Keywords: exploratory test, lactic acid bacteria, crude bacteriocins, spoilage, pathogens

Procedia PDF Downloads 213

Authors: Fadilah Fadilah, Rochmadi Rochmadi, Siti Syamsiah, Djagal W. Marseno

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Glucomannan can be found in the tuber of porang together with starch and proteinaceous components which were regarded as impurities. An enzymatic process for obtaining higher glucomannan content from Porang flour have been conducted. Papain was used for hydrolysing proteinaceous components in Porang flour which was conducted after a simultaneous extraction of glucomannan and enzymatic starch hydrolysis. Three variables affecting the rate were studied, i.e. temperature, the amount of enzyme and the stirring speed. The ninhydrin method was used to determine degree of protein hydrolysis. Results showed that the rising of degree of hydrolysis were fast in the first ten minutes of the reaction and then proceeded slowly afterward. The optimum temperature for hydrolysis was 60 ^oC. Increasing the amount of enzyme showed a remarkable effect to degree of hydrolysis, but the stirring speed had no significant effect. This indicated that the reaction controlled the rate of hydrolysis.

Keywords: degree of hydrolysis, ninhydrin, papain, porang flour, proteinaceous components

Procedia PDF Downloads 250

Authors: Zohreh Soleimani, Sally Salome Shahzad, Stamatis Zoras

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As a result of current energy and environmental issues, the human body is known as one of the promising candidate for converting wasted heat to electricity (Seebeck effect). Thermoelectric generator (TEG) is one of the most prevalent means of harvesting body heat and converting that to eco-friendly electrical power. However, the uneven distribution of the body heat and its curvature geometry restrict harvesting adequate amount of energy. To perfectly transform the heat radiated by the body into power, the most direct solution is conforming the thermoelectric generators (TEG) with the arbitrary surface of the body and increase the temperature difference across the thermoelectric legs. Due to this, a computational survey through COMSOL Multiphysics is presented in this paper with the main focus on the impact of integrating a flexible wearable TEG with a corrugated shaped heat sink on the module power output. To eliminate external parameters (temperature, air flow, humidity), the simulations are conducted within indoor thermal level and when the wearer is stationary. The full thermoelectric characterization of the proposed TEG fabricated by a wavy shape heat sink has been computed leading to a maximum power output of 25µW/cm2 at a temperature gradient nearly 13°C. It is noteworthy that for the flexibility of the proposed TEG and heat sink, the applicability and efficiency of the module stay high even on the curved surfaces of the body. As a consequence, the results demonstrate the superiority of such a TEG to the most state of the art counterparts fabricated with no heat sink and offer a new train of thought for the development of self-sustained and unobtrusive wearable power suppliers which generate energy from low grade dissipated heat from the body.

Keywords: device simulation, flexible thermoelectric module, heat sink, human body heat

Procedia PDF Downloads 151

Authors: Akimitsu Kugimiya, Kouhei Iwato, Toru Saito, Jiro Kohda, Yasuhisa Nakano, Yu Takano

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The measurement of amino acid content is reported to be useful for the diagnosis of several types of diseases, including lung cancer, gastric cancer, colorectal cancer, breast cancer, prostate cancer, and diabetes. The conventional detection methods for amino acid are high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS), but they have several drawbacks as the equipment is cumbersome and the techniques are costly in terms of time and costs. In contrast, biosensors and biosensing methods provide more rapid and facile detection strategies that use simple equipment. The authors have reported a novel approach for the detection of each amino acid that involved the use of aminoacyl-tRNA synthetase (aaRS) as a molecular recognition element because aaRS is expected to a selective binding ability for corresponding amino acid. The consecutive enzymatic reactions used in this study are as follows: aaRS binds to its cognate amino acid and releases inorganic pyrophosphate. Hydrogen peroxide (H₂O₂) was produced by the enzyme reactions of inorganic pyrophosphatase and pyruvate oxidase. The Trinder’s reagent was added into the reaction mixture, and the absorbance change at 556 nm was measured using a microplate reader. In this study, an amino acid-sensing method using histidyl-tRNA synthetase (HisRS; histidine-specific aaRS) as molecular recognition element in combination with the Trinder’s reagent spectrophotometric method was developed. The quantitative performance and selectivity of the method were evaluated, and the optimal enzyme reaction and detection conditions were determined. The authors developed a simple and rapid method for detecting histidine with a combination of enzymatic reaction and spectrophotometric detection. In this study, HisRS was used to detect histidine, and the reaction and detection conditions were optimized for quantitation of these amino acids in the ranges of 1–100 µM histidine. The detection limits are sufficient to analyze these amino acids in biological fluids. This work was partly supported by Hiroshima City University Grant for Special Academic Research (General Studies).

Keywords: amino acid, aminoacyl-tRNA synthetase, biosensing, enzyme reaction

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Authors: Kwang Sok Jong

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Apatite is separated from carbonate minerals via direct flotation by using lignosulphonate as a depressant, but its dosage is high, and its inhibition ability is insufficient. Therefore a combination of depressant calcium lignosulphonate and depressant tannin was considered to improve flotation selectivity and decrease the dosage of depressant. In the present work, the effects of several reagents- pH regulators (sodium carbonate and sodium hydroxide), combined depressant (calcium lignosulphonate and tannin) and collector (fatty acid amide soap) on the flotation performance of apatite ore were investigated using Design Expert software. Flotation results showed that the combined depressant had not only more excellent inhibition ability compared with the individual depressant respectively, but also lower dosage. In the raw ore containing 6.65% P₂O₅, a concentrate containing 32.93% P₂O₅ with 93.24% recovery was obtained using 3.5kg/t sodium carbonate, 0.75kg/t sodium hydroxide, 1kg/t calcium lignosulphonate, 50g/t tannin and 100g/t fatty acid amide soap in the rougher flotation.

Keywords: apatite flotation, combined depressant, calcium lignosulphonate, tannin

Procedia PDF Downloads 37

Authors: Nikolai Nosik, Vladislav Podmasterjev, Nina Kondrashina, Marina Chataeva, Olga Lobach, Dmitry Noosik, Sergei Razumovskii

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Virucidal activity of ozone is well known: dissolved in water it kill viruses very fast. The virucidal capacity of ozone in ozone-air mixture is less known. The goal of the study was to investigate the virucidal potentials of the ozone–air mixture and kinetics of virus inactivation. Materials and methods. Ozone (O3 ) was generated from oxygen with ozonizer ( 1.0 – 75.0 mg\l). The ozone concentration was determined by the spectrophotometric methods. Virus contaminated samples were placed into the flowing reactor. Viruses: poliovirus type 1, vaccine strain (Sabin) and adenovirus, type 5, were obtained from the State virus collection. Titrations of viruses were carried out in appropriate cell cultures. CxT value ( mg\l x min) was calculated. Results. Metallic, polycarbonic and fiber “Kevlar” samples were contaminated with virus, dried and treated with ozone-air mixture in the flowing reactor. Kinetics of poliovirus inactivation: in 15 min at 5.0 mg\l -2.0 lg TCID50 inhibition , in 15 min at 10 mg\l – 2.5 lg TCID50 , 4.0 lg TCID50 inactivation of poliovirus was achieved after 75min at ozone concentration 20.0mg\l (99.99%). ( CxT = 75, 150 and 1500 mg\l x min on all three types of surfaces). It was found that the inactivation of poliovirus was more effective when the virus contaminated samples were wet (in 15 min at 20mg\l inhibition of virus in dry samples was 2.0 TCID50 , in wet samples – 4.0 TCID50). Adenovirus was less resistant to ozone treatment then poliovirus: 4.0 lg TCID50 inhibition was observed after 30 min of the treatment with ozone at 20mg\l ( CxT mg\l x min = 300 for adenovirus as for poliovirus it was 1500). Conclusion. It was found that ozone-air mixture inactivates viruses at rather high concentrations (compared to the reported effect of ozone dissolved in water). Despite of that there is a difference in the resistance to ozone action between viruses – poliovirus is more resistant then adenovirus-ozone-air mixture can be used for disinfection of large rooms. The maintaining of the virus-contaminated surfaces in wet condition allow to decrease the ozone load for virus inactivation.

Keywords: adenovirus, disinfection, ozone, poliovirus

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Authors: Aoxue Yang, Weili Tian, Haikun Liu

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Brain tumor stem cells (BTSCs) are resistant to therapy and give rise to recurrent tumors. These rare and elusive cells are likely to disseminate during cancer progression, and some may enter dormancy, remaining viable but not increasing. The identification of dormant BTSCs is thus necessary to design effective therapies for glioblastoma (GBM) patients. Glucocorticoids (GCs) are used to treat GBM-associated edema. However, glucocorticoids participate in the physiological response to psychosocial stress, linked to poor cancer prognosis. This raises concern that glucocorticoids affect the tumor and BTSCs. Identifying markers specifically expressed by brain tumor stem cells (BTSCs) may enable specific therapies that spare their regular tissue-resident counterparts. By ribosome profiling analysis, we have identified that glycerol-3-phosphate dehydrogenase 1 (GPD1) is expressed by dormant BTSCs but not by NSCs. Through different stress-induced experiments in vitro, we found that only dexamethasone (DEXA) can significantly increase the expression of GPD1 in NSCs. Adversely, mifepristone (MIFE) which is classified as glucocorticoid receptors antagonists, could decrease GPD1 protein level and weaken the proliferation and stemness in BTSCs. Furthermore, DEXA can induce GPD1 expression in tumor-bearing mice brains and shorten animal survival, whereas MIFE has a distinct adverse effect that prolonged mice lifespan. Knocking out GR in NSC can block the upregulation of GPD1 inducing by DEXA, and we find the specific sequences on GPD1 promotor combined with GR, thus improving the efficiency of GPD1 transcription from CHIP-Seq. Moreover, GR and GPD1 are highly co-stained on GBM sections obtained from patients and mice. All these findings confirmed that GR could regulate GPD1 and loss of GPD1 Impairs Multiple Pathways Important for BTSCs Maintenance GPD1 is also a critical enzyme regulating glycolysis and lipid synthesis. We observed that DEXA and MIFE could change the metabolic profiles of BTSCs by regulating GPD1 to shift the transition of cell dormancy. Our transcriptome and lipidomics analysis demonstrated that cell cycle signaling and phosphoglycerides synthesis pathways contributed a lot to the inhibition of GPD1 caused by MIFE. In conclusion, our findings raise concern that treatment of GBM with GCs may compromise the efficacy of chemotherapy and contribute to BTSC dormancy. Inhibition of GR can dramatically reduce GPD1 and extend the survival duration of GBM-bearing mice. The molecular link between GPD1 and GR may give us an attractive therapeutic target for glioblastoma.

Keywords: cancer stem cell, dormancy, glioblastoma, glycerol-3-phosphate dehydrogenase 1, glucocorticoid receptor, dexamethasone, RNA-sequencing, phosphoglycerides

Procedia PDF Downloads 132

Authors: Debajyoti Bose

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Chitosan has received much attention as a functional biopolymer for diverse applications, especially in pharmaceutics and medicine. Chitosan is a positively charged natural biodegradable and biocompatible polymer. It is a linear polysaccharide consisting of β-1,4 linked monomers of glucosamine and N-acetylglucosamine. Chitosan can be mainly obtained from fungal sources during large fermentation process. In this study,three different fungal strains Aspergillus niger NCIM 1045, Aspergillus oryzae NCIM 645 and Mucor indicus MTCC 3318 were used for the production of chitosan. The growth mediums were optimized for maximum fungal production. The produced chitosan was characterized by determining degree of deacetylation. Chitosan possesses one reactive amino at the C-2 position of the glucosamine residue, and these amines confer important functional properties to chitosan which can be exploited for biofabrication to generate various chemically modified derivatives and explore their potential for pharmaceutical field. Chitosan nanoparticles were prepared by ionic cross-linking with tripolyphosphate (TPP). The major effect on encapsulation and release of protein (e.g. enzyme diastase) in chitosan-TPP nanoparticles was investigated in order to control the loading and release efficiency. It was noted that the chitosan loading and releasing efficiency as a nanocapsule, obtained from different fungal sources was almost near to initial enzyme activity(12026 U/ml) with a negligible loss. This signify, chitosan can be used as a polymeric drug as well as active component or protein carrier material in dosage by design due to its appealing properties such as biocompatibility, biodegradability, low toxicity and relatively low production cost from abundant natural sources. Based upon these initial experiments, studies were also carried out on modification of chitosan based nanocapsules incorporated with physiologically important enzymes and nutraceuticals for target delivery.

Keywords: fungi, chitosan, enzyme, nanocapsule

Procedia PDF Downloads 502

Authors: Sema Şenoğlu, Sevgi Karakuş

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Hydrazone scaffold is important to design new drug groups and is found to possess numerous uses in pharmaceutical chemistry. Besides, hydrazone derivatives are also known for biological activities such as anticancer, antimicrobial, antiviral, and antifungal. Hydrazone derivatives are promising anticancer agents because they inhibit cancer proliferation and induce apoptosis. Human carbonic anhydrase IX has a high potential to be an antiproliferative drug target, and targeting this protein is also important for obtaining potential anticancer inhibitors. The protein construct was retrieved as a PDB file from the RCSB protein database. This binding interaction of proteins and ligands was performed using Discovery Studio Visualizer. In vitro inhibitory activity of hydrazone derivatives was tested against enzyme carbonic anhydrase IX on the PyRx programme. Most of these molecules showed remarkable human carbonic anhydrase IX inhibitory activity compared to the acetazolamide. As a result, these compounds appear to be a potential target in drug design against human carbonic anhydrase IX.

Keywords: cancer, carbonic anhydrase IX enzyme, docking, hydrazone

Procedia PDF Downloads 82

Authors: Shaohua Li, Aihua Zhang, Kelly Zatopek, Saba Parvez, Andrew F. Gardner, Ivan R. Corrêa Jr., Christopher J. Noren, Ming-Qun Xu

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Covalent immobilization of enzymes on solid supports is an alternative approach to biocatalysis with the added benefits of simple enzyme removal, improved stability, and adaptability to automation and high-throughput applications. Nevertheless, immobilized enzymes generally suffer from reduced activities compared to their soluble counterparts. One major factor leading to activity loss is the intrinsic hydrophobic property of the supporting material surface, which could result in the conformational change/confinement of enzymes. We report a strategy of utilizing flexible poly (ethylene oxide) (PEO) moieties as to improve the surface hydrophilicity of solid supports used for enzyme immobilization. DNA modifying enzymes were covalently conjugated to PEO-coated magnetic-beads. Kinetics studies proved that the activities of the covalently-immobilized DNA modifying enzymes were greatly enhanced by the PEO modification on the bead surface.

Keywords: immobilized enzymes, biocatalysis, poly(ethylene oxide), surface modification

Procedia PDF Downloads 308

Authors: Mohamed H. Khalifa, Fikry I. El-Shahawi, Nabil A. Mansour

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The cotton leafworm, Spodoptera littoralis (Boisduval) is a major insect pest of vegetables and cotton crops in Egypt, and exhibits different levels of tolerance to certain insecticides. Chlorantraniliprole has been registered recently in Egypt for control this insect. The susceptibilities of three S. littoralis populations collected from El Behaira governorate, north Egypt to chlorantraniliprole were determined by leaf-dipping technique on 4^th instar larvae. Obvious variation of toxicity was observed among the laboratory susceptible, and three field populations with LC₅₀ values ranged between 1.53 µg/ml and 6.22 µg/ml. However, all the three field populations were less susceptible to chlorantraniliprole than a laboratory susceptible population. The most tolerant populations were sampled from El Delengat (ED) Province where S. littoralis had been frequently challenged by insecticides. Certain enzyme activity assays were carried out to be correlated with the mechanism of the observed field population tolerance. All field populations showed significantly enhanced activities of detoxification enzymes compared with the susceptible strain. The regression analysis between chlorantraniliprole toxicities and enzyme activities revealed that the highest correlation is between α-esterase or β-esterase (α-β-EST) activity and collected field strains susceptibility, otherwise this correlation is not significant (P > 0.05). Synergism assays showed the ED and susceptible strains could be synergized by known detoxification inhibitors such as piperonyl butoxide (PBO), triphenyl phosphate (TPP) and diethyl-maleate (DEM) at different levels (1.01-8.76-fold and 1.09-2.94 fold, respectively), TPP showed the maximum synergism in both strains. The results show that there is a correlation between the enzyme activity and tolerance, and carboxylic-esterase (Car-EST) is likely the main detoxification mechanism responsible for tolerance of S. littoralis to chlorantraniliprole.

Keywords: chlorantraniliprole, detoxification enzymes, Egypt, Spodoptera littoralis

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Authors: Zerrin Erginkaya, Gözde Konuray

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In this study, antimicrobial effect of Greek clover was determined with usage of MIC (minimum inhibition concentration) and agar diffusion method. Moreover, pH, water activity and microbial change were determined during storage of fenugreek paste. At first part of our study, microbial load of spices was evaluated. Two different fenugreek pastes were produced with mixing of Greek clover, spices, garlic and water. Fenugreek pastes were stored at 4 °C. At the second part, antimicrobial effect of Greek clover was determined on Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Debaryomyces hansenii, Aspergillus parasiticus, Candida rugosa, Mucor spp., when the concentrations of Greek clover were 8%, 12% and 16%. According to the results obtained, mould growth was determined at 15^th and 30^th days of storage in first and second fenugreek samples, respectively. Greek clover showed only antifungal effect on Aspergillus parasiticus at previously mentioned concentrations.

Keywords: antimicrobial, fenugreek, Greek clover, minimum inhibition concentration

Procedia PDF Downloads 254

Authors: Dharshika Rajalingam, Jeffery W. Peng

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Acinetobacter baumannii is a challenging threat to global health, recognized as a multidrug-resistant pathogen. -lactamase is one of the principal resistant mechanisms developed by A. baumannii to survive against -lactam antibiotics. OXA24/40 is one of the types of -lactamases, a well-documented carbapenem hydrolyzing class D -lactamases (CHDL). It was revealed that OXA24/40 showed resistivity against doripenem, one of the carbapenems, by two different mechanisms as hydrolysis and -lactonization. Furthermore, it undergoes genetic mutations to broaden the -lactamase activity to survive against antibiotic environments. One of the crucial characterizations of prokaryotes to develop adaptation is post-translational modification (PTM), mainly phosphorylation. However, the PTM of OXA24/40 is an unknown feature, and the impact of PTM on antibiotic resistivity is yet to be explored. We approached these hypotheses using NMR and MS techniques and found that the OXA24/40 could be phosphorylated in vitro. The Ser81 at the active STFK motif of OXA24/40 of catalytic pocket was identified as the site of phosphorylation using 1D 31P NMR experiment, whereas S81 is required to form an acyl-enzyme complex between enzyme and -lactam antibiotics. The activity of completely phosphorylated OXA24/40 wild type against doripenem revealed that the phosphorylation of active Ser inactivates the -lactamases activity of OXA24/40. The 1D 1H CPMG NMR-based activity assay of phosphorylated OXA24/40 against doripenem confirmed that both deactivating mechanisms are inhibited by phosphorylation. Carbamylated Lysine at the active STFK motif is one of the critical features of CHDL required for the acylation and deacylation reactions of the enzyme. The 1D 13C NMR experiment confirmed that the K84 of phosphorylated OXA24/40 is de-carbamylated. Phosphorylation of OXA24/40 affects both active S81 and carbamylated K84 of OXA24 that are required for the resistivity of -lactamase. So, phosphorylation could be one of the reasons for the genetic mutation of OXA24/40 for the development of antibiotic resistivity. Further research can lead to an understanding of the effect of phosphorylation on the clinical mutants of the OXA24-like -lactamase family on the broadening of -lactamase activity.

Keywords: OXA24/40, phosphorylation, clinical mutants, resistivity

Procedia PDF Downloads 79

Authors: A. N. Ukwuani, M. G. Abubakar, S. W. Hassan

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Grewia crenata is used locally in the treatment of fractured bones, wound healing and inflammatory conditions. The anti-inflammatory activity of hydromethanolic extract of G. crenata leaves was investigated using egg albumin induced-hind paw oedema model in albino rat. The extract produced a time-dependent inhibition of egg albumin induced-hind paw oedema at 30th minutes up to 150th minutes in all the groups compared to the control. Significant reduction (p<0.05) of hind paw oedema was observed 150 minutes after egg albumin injection. The percentage inhibition produced by the extract at 200 mg/kg (22.1%) was comparable to that produced by 10 mg/kg indomethacin (24.9%) at the 150th minutes of post-egg albumin injection. Preliminary qualitative phytochemical analysis revealed the presence of saponins, steroids, flavonoids, anthraquinones and glycosides. The results obtained in this study suggest that Grewia crenata can be a potential source of anti-inflammatory agent and validates its use in the treatment of inflammatory conditions.

Keywords: Grewia crenata, anti-inflammatory, hind paw, oedema

Procedia PDF Downloads 310

Authors: S. Mat Radzi, N. J. Abd Rahman, H. Mohd Noor, N. Ariffin

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Ferulic acid has widespread industrial potential by virtue of its antioxidant properties. However, it is partially soluble in aqueous media, limiting their usefulness in oil-based processes in food, cosmetic, pharmaceutical, and material industry. Therefore, modification of ferulic acid should be made by producing of more lipophilic derivatives. In this study, a preliminary investigation of lipase-catalyzed trans-esterification reaction of ethyl ferulate and olive oil was investigated. The reaction was catalyzed by immobilized lipase from Candida antarctica (Novozym 435), to produce ferulate ester, a sunscreen agent. A statistical approach of Response surface methodology (RSM) was used to evaluate the interactive effects of reaction temperature (40-80°C), reaction time (4-12 hours), and amount of enzyme (0.1-0.5 g). The optimum conditions derived via RSM were reaction temperature 60°C, reaction time 2.34 hours, and amount of enzyme 0.3 g. The actual experimental yield was 59.6% ferulate ester under optimum condition, which compared well to the maximum predicted value of 58.0%.

Keywords: ferulic acid, enzymatic synthesis, esters, RSM

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Authors: Nora Laref, Bettache Guessas

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Preservation of foods mainly depends on delaying or inhibiting the growth of spoilage microorganisms, and antifungal activity of lactic acid bacteria is one of the technological properties researched. The antifungal activity was screened with overlay method of six strains of lactic acid bacteria (Lactobacillus plantarum LB54, LB52, LB51, LB20, LB24 Lactobacillus farciminis LB53) isolated from silage, camel milk and carrot against Aspergillus sp. Lactobacillus plantarum and farciminis inhibit spore germination and mycelia growth of Aspergillus sp., the production of antifungal compounds by these strains was detectable after 4h of incubation at 30°C and show total inhibition after 24h in liquid media, but in solid media showed a good inhibition after 96h of incubation, these compounds cause malformations in the thalle, conidiophore and conidia. These strains could be used as agents of biopreservation since have the ability to retard Aspergillus sp., growth in apricot juice with and without sugar conserved in refrigerator but not in bread.

Keywords: lactobacillus, antifungal substances, aspergillus, biopreservation

Procedia PDF Downloads 346

Authors: Istvan Szilagyi, Marko Pavlovic, Paul Rouster

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Antioxidant enzymes are the most efficient defense systems against reactive oxygen species, which cause severe damage in living organisms and industrial products. However, their supplementation is problematic due to their high sensitivity to the environmental conditions. Immobilization on carrier nanoparticles is a promising research direction towards the improvement of their functional and colloidal stability. In that way, their applications in biomedical treatments and manufacturing processes in the food, textile and cosmetic industry can be extended. The main goal of the present research was to prepare and formulate antioxidant bionanocomposites composed of superoxide dismutase (SOD) enzyme, anionic clay (layered double hydroxide, LDH) nanoparticle and heparin (HEP) polyelectrolyte. To characterize the structure and the colloidal stability of the obtained compounds in suspension and solid state, electrophoresis, dynamic light scattering, transmission electron microscopy, spectrophotometry, thermogravimetry, X-ray diffraction, infrared and fluorescence spectroscopy were used as experimental techniques. LDH-SOD composite was synthesized by enzyme immobilization on the clay particles via electrostatic and hydrophobic interactions, which resulted in a strong adsorption of the SOD on the LDH surface, i.e., no enzyme leakage was observed once the material was suspended in aqueous solutions. However, the LDH-SOD showed only limited resistance against salt-induced aggregation and large irregularly shaped clusters formed during short term interval even at lower ionic strengths. Since sufficiently high colloidal stability is a key requirement in most of the applications mentioned above, the nanocomposite was coated with HEP polyelectrolyte to develop highly stable suspensions of primary LDH-SOD-HEP particles. HEP is a natural anticoagulant with one of the highest negative line charge density among the known macromolecules. The experimental results indicated that it strongly adsorbed on the oppositely charged LDH-SOD surface leading to charge inversion and to the formation of negatively charged LDH-SOD-HEP. The obtained hybrid materials formed stable suspension even under extreme conditions, where classical colloid chemistry theories predict rapid aggregation of the particles and unstable suspensions. Such a stabilization effect originated from electrostatic repulsion between the particles of the same sign of charge as well as from steric repulsion due to the osmotic pressure raised during the overlap of the polyelectrolyte chains adsorbed on the surface. In addition, the SOD enzyme kept its structural and functional integrity during the immobilization and coating processes and hence, the LDH-SOD-HEP bionanocomposite possessed excellent activity in decomposition of superoxide radical anions, as revealed in biochemical test reactions. In conclusion, due to the improved colloidal stability and the good efficiency in scavenging superoxide radical ions, the developed enzymatic system is a promising antioxidant candidate for biomedical or other manufacturing processes, wherever the aim is to decompose reactive oxygen species in suspensions.

Keywords: clay, enzyme, polyelectrolyte, formulation

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Authors: D. Bhowmik, Y. Tian, Q. Yin, F. Zhu

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Cyclic GMP-AMP synthase (cGAS), recognises cytoplasmic double-stranded DNA (dsDNA), indicative of bacterial and viral infections, as well as the leakage of self DNA by cellular dysfunction and stresses, to elicit the host's immune responses. Viruses also have developed numerous strategies to antagonize the cGAS-STING pathway. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human DNA tumor virus that is the causative agent of Kaposi’s sarcoma and several other malignancies. To persist in the host, consequently causing diseases, KSHV must overcome the host innate immune responses, including the cGAS-STING DNA sensing pathway. We already found that ORF52 or KicGAS (KSHV inhibitor of cGAS), an abundant and basic gamma herpesvirus-conserved tegument protein, directly inhibits cGAS enzymatic activity. To better understand the mechanism, we have performed the biochemical and structural characterization of full-length KicGAS and various mutants in regarding binding to DNA. We observed that KicGAS is capable of self-association and identified the critical residues involved in the oligomerization process. We also characterized the DNA-binding of KicGAS and found that KicGAS cooperatively oligomerizes along the length of the double stranded DNA, the highly conserved basic residues at the c-terminal disordered region are crucial for DNA recognition. Deficiency in oligomerization also affects DNA binding. Thus DNA binding by KicGAS sequesters DNA and prevents it from being detected by cGAS, consequently inhibiting cGAS activation. KicGAS homologues also inhibit cGAS efficiently, suggesting inhibition of cGAS is evolutionarily conserved mechanism among gamma herpesvirus. These results highlight the important viral strategy to evade this innate immune sensor.

Keywords: Kaposi's sarcoma-associated herpesvirus, KSHV, cGAS, DNA binding, inhibition

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Authors: T. Büyüksırıt, H. Kuleaşan

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Natural antimicrobials are used to preserve foods that can be found in plants, animals, and microorganisms. Antimicrobial substances are natural or artificial agents that produced by microorganisms or obtained semi/total chemical synthesis are used at low concentrations to inhibit the growth of other microorganisms. Food borne pathogens and spoilage microorganisms are inactivated by the use of antagonistic microorganisms and their metabolites. Yeasts can produce toxic proteins or glycoproteins (toxins) that cause inhibition of sensitive bacteria and yeast species. Antimicrobial substance producing phenotypes belonging different yeast genus were isolated from different sources. Toxins secreted by many yeast strains inhibiting the growth of other yeast strains. These strains show antimicrobial activity, inhibiting the growth of mold and bacteria. The effect of antimicrobial agents produced by yeasts can be extremely fast, and therefore may be used in various treatment procedures. Rapid inhibition of microorganisms is possibly caused by microbial cell membrane lipopolysaccharide binding and in activation (neutralization) effect. Antimicrobial agents inhibit the target cells via different mechanisms of action.

Keywords: antimicrobial agents, yeast, toxic protein, glycoprotein

Procedia PDF Downloads 362

Authors: Boudjenah-Haroun Saliha, Isselnane Souad, Nouani Abdelwahab, Baaissa Babelhadj, Mati Abderrahmane

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Algeria is experiencing significant development of the dairy sector, where consumption of milk and milk products increased by 2.7 million tons in 2008 to 4,400,000 tons in 2013, and cheese production has reached 1640 tons in the year 2014 with average consumption of 0.7 kg/person/year. Although rennet is still the most used coagulating enzyme in cheese, its production has been growing worldwide shortage. This shortage is primarily due to a growing increase in the production and consumption of cheese, and the inability to increase in parallel the production of rennet. This shortage has caused very large fluctuations in its price). To overcome these obstacles, much research has been undertaken to find effective and competitive substitutes used industrially. For this, the selection of a local production of rennet substitute is desirable. It would allow a permanent supply with limited dependence on imports and price fluctuations. Investigations conducted by our research team showed that extracts coagulants from the stomachs of older camels are characterized by a coagulating power than those from younger camels. The objective of this work is to study the possibility of substituting commercial rennet coagulant by gastric enzymes from adult camels for coagulation bovine milk. Excerpts from the raw camel coagulants obtained are characterized through their teneures proteins and clotting and proteolytic activities. Milk clotting conditions by the action of these extracts were optimized. Milk clotting time all treated with enzyme preparations and under different conditions was calculated. Bovine rennet has been used for comparison. The results show that crude extracts from gastric adult camel can be good substituting bovine rennet.

Keywords: Algeria, camel, cheese, coagulation, gastric extracts, milk

Procedia PDF Downloads 441

Authors: Yahia Massinissa, Benhouda Afaf, Benbia Souhila, Meddour Noura, Takellalet Karima, Zeroual Amina

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Introduction: chenopodiaceae family species are known for their important biological activity, in which Atriplex halimus belongs . However, the inflammatory effect of this plant leaves has not been studied. This work aimed at assessing the anti- inflammatory and hemostatic activities of the methanolic extract AHMeOH of Atriplex halimus’s leaves. Methods: The extract was obtained using sonication of leaves powder in 80 % methanol. The analysis of phenolic compounds was carried out using thin-layer chromatography (TLC).The anti-inflammatory activity was determined by studying the plasmical membrane stabilization and albumin denaturation inhibition, the hemostatic activity was evaluated by measuring the plasma in the blood. Results: Quantitative determination of total flavonoids reveals that AHMeOH is rich in flavonoids (16 ± 0.88 μg Q / mg extract) and polyphenols (20 ± 0.20 μg AG / mg extract). about anti-inflammatory activity, the tests show that AHMeOH has a significant effect (P≤0.05) of inhibiting hypotonic-induced hemolysis with concentrations (100 and 200 μg / ml) with 77.55 and 90% respectively, and heat-induced hemolysis with percentages 81.75% and 87.44% respectively with significant difference (P ≤0.05). The obtained results with this plant reveal that the inhibition of denaturation of albumin is dose dependent. The concentration of 400 μg / ml gives denaturation inhibition of 81.00 ± 17.70% and the concentration 600 μg / ml gives an effect of 82.95 ± 17.40%. Regarding the haemostatic activity our extract with the doses 10 mg / ml, 20 mg / ml and 30 mg / ml confer a decrease of the plasma recalcification time in the tube, these concentrations could prolong the time of coagulation significantly compared to the control (P≤0.001). This result is an interesting indication in favor of haemostatic activity of AHMeOH. Conclusion: Atriplex Halimus has a strong anti-inflammatory activity and constitutes a potential source for the development of new treatments.

Keywords: albumin, atriplex halimus, hemostatic activity, methanolic extract

Procedia PDF Downloads 75

Authors: Heidarali Malmir

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The effects of acid rain (AR), smog’s cars (SC), and combined AR+SC on the antioxidants enzymes, lipid-soluble antioxidants, and water-soluble antioxidants were studied in the two cultivars of rice. The results showed that simulated AR significantly increased the total glutathione (TGSH), thiobarbituric acid (TBA), and α-tocopherol, accompanied by decreases in dry weight and leaves area in the two cultivars, and this change was more obvious in Shirudi cultivar than in Aus cultivar (p≤0.05). Under SC stress cultivar shirudi had higher H+-ATPase, glutathione peroxidase (GSH-px), and catalase (CAT) activities than cultivar Aus. The results of superoxide dismutase (SOD) activity, TGSH, and α-tocopherol levels affected by AR treatments were very different to those of SOD activity, TGSH, and α-tocopherol levels, as shown in SC treatment. It seems that SOD activity coupled with the water-soluble antioxidants and α-tocopherol levels correlated with the lipid-soluble antioxidants. It is suggested that α-tocopherol increases H+-ATPase activity.

Keywords: H+-ATPase, membrane permeability, lipid soluble antioxidants, water soluble antioxidants, associated enzyme

Procedia PDF Downloads 83

Authors: Junie B. Billones, Maria Constancia O. Carrillo, Voltaire G. Organo, Stephani Joy Y. Macalino, Inno A. Emnacen, Jamie Bernadette A. Sy

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The drug discovery and development process is generally known to be a very lengthy and labor-intensive process. Therefore, in order to be able to deliver prompt and effective responses to cure certain diseases, there is an urgent need to reduce the time and resources needed to design, develop, and optimize potential drugs. Computer-aided drug design (CADD) is able to alleviate this issue by applying computational power in order to streamline the whole drug discovery process, starting from target identification to lead optimization. This drug design approach can be predominantly applied to diseases that cause major public health concerns, such as tuberculosis. Hitherto, there has been no concrete cure for this disease, especially with the continuing emergence of drug resistant strains. In this study, CADD is employed for tuberculosis by first identifying a key enzyme in the mycobacterium’s metabolic pathway that would make a good drug target. One such potential target is the lipoate protein ligase B enzyme (LipB), which is a key enzyme in the M. tuberculosis metabolic pathway involved in the biosynthesis of the lipoic acid cofactor. Its expression is considerably up-regulated in patients with multi-drug resistant tuberculosis (MDR-TB) and it has no known back-up mechanism that can take over its function when inhibited, making it an extremely attractive target. Using cutting-edge computational methods, compounds from AnalytiCon Discovery Natural Derivatives database were screened and docked against the LipB enzyme in order to rank them based on their binding affinities. Compounds which have better binding affinities than LipB’s known inhibitor, decanoic acid, were subjected to in silico toxicity evaluation using the ADMET and TOPKAT protocols. Out of the 31,692 compounds in the database, 112 of these showed better binding energies than decanoic acid. Furthermore, 12 out of the 112 compounds showed highly promising ADMET and TOPKAT properties. Future studies involving in vitro or in vivo bioassays may be done to further confirm the therapeutic efficacy of these 12 compounds, which eventually may then lead to a novel class of anti-tuberculosis drugs.

Keywords: pharmacophore, molecular docking, lipoate protein ligase B (LipB), ADMET, TOPKAT

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Authors: A. R. Diniz, P. Borrego, I. Bártolo, N. Taveira

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Cell-to-cell transmission plays a critical role in the spread of HIV-1 infection in vitro and in vivo. Inhibition of HIV-1 cell-associated infection by antiretroviral drugs and neutralizing antibodies (NAbs) is more difficult compared to cell-free infection. Limited data exists on cell-associated infection by HIV-2 and its inhibition. In this work, we determined the ability of entry inhibitors to inhibit HIV-1 and HIV-2 cell-to cell fusion as a proxy to cell-associated infection. We developed a method in which Hela-CD4-cells are first transfected with a Tat expressing plasmid (pcDNA3.1+/Tat101) and infected with recombinant vaccinia viruses expressing either the HIV-1 (vPE16: from isolate HTLV-IIIB, clone BH8, X4 tropism) or HIV-2 (vSC50: from HIV-2SBL/ISY, R5 and X4 tropism) envelope glycoproteins (M.O.I.=1 PFU/cell).These cells are added to TZM-bl cells. When cell-to-cell fusion (syncytia) occurs the Tat protein diffuses to the TZM-bl cells activating the expression of a reporter gene (luciferase). We tested several entry inhibitors including the fusion inhibitors T1249, T20 and P3, the CCR5 antagonists MVC and TAK-779, the CXCR4 antagonist AMD3100 and several HIV-2 neutralizing antibodies (Nabs). All compounds inhibited HIV-1 and HIV-2 cell fusion albeit to different levels. Maximum percentage of HIV-2 inhibition (MPI) was higher for fusion inhibitors (T1249- 99.8%; P3- 95%, T20-90%) followed by co-receptor antagonists (MVC- 63%; TAK-779- 55%; AMD3100- 45%). NAbs from HIV-2 infected patients did not prevent cell fusion up to the tested concentration of 4μg/ml. As for HIV-1, MPI reached 100% with TAK-779 and T1249. For the other antivirals, MPIs were: P3-79%; T20-75%; AMD3100-61%; MVC-65%.These results are consistent with published data. Maraviroc had the lowest IC50 both for HIV-2 and HIV-1 (IC50 HIV-2= 0.06 μM; HIV-1=0.0076μM). Highest IC50 were observed with T20 for HIV-2 (3.86μM) and with TAK-779 for HIV-1 (12.64μM). Overall, our results show that entry inhibitors in clinical use are less effective at preventing Env mediated cell-to-cell-fusion in HIV-2 than in HIV-1 which suggests that cell-associated HIV-2 infection will be more difficult to inhibit compared to HIV-1. The method described here will be useful to screen for new HIV entry inhibitors.

Keywords: cell-to-cell fusion, entry inhibitors, HIV, NAbs, vaccinia virus

Procedia PDF Downloads 309

Authors: T. Scattolin, E. Cavarzerani, F. Visentin, F. Rizzolio

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Organopalladium compounds have recently attracted attention for their high stability even under physiological conditions and, above all, for their remarkable in vitro cytotoxicity towards cisplatin-resistant cell lines. Among the organopalladium derivatives, those bearing at least one N-heterocyclic carbene ligand (NHC) and the Pd(II)-η³-allyl fragment have exhibited IC₅₀ values in the micro and sub-micromolar range towards several cancer cell lines in vitro and in some cases selectivity towards cancerous vs. non-tumorigenic cells. Herein, a selection of allyl and indenyl palladates were synthesized using a solvent-free method consisting of grinding the corresponding palladium precursors with different saturated and unsaturated azolium salts. All compounds have been fully characterized by NMR, XRD and elemental analyses. The intramolecular H, Cl interaction has been elucidated and quantified using the Voronoi Deformation Density scheme. Most of the complexes showed excellent cytotoxicity towards ovarian cancer cell lines, with I₅₀ values comparable to or even lower than cisplatin. Interestingly, the potent anticancer activity was also confirmed in a high-serous ovarian cancer (HGSOC) patient-derived tumoroid, with a clear superiority of this class of compounds over classical platinum-based agents. Finally, preliminary enzyme inhibition studies of the synthesized palladate complexes against the model TrxR show that the compounds have high activity comparable to or even higher than auranofin and classical Au(I) NHC complexes. Based on such promising data, further in vitro and in vivo experiments and in-depth mechanistic studies are ongoing in our laboratories.

Keywords: anticancer activity, palladium complexes, organoids, indenyl and allyl ligands

Procedia PDF Downloads 94

Authors: Reim A. Almotiri, Manal M. Alkhamisi

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Ternary nanocomposites based on hydroxyapatite (HAP) and alumina (Al2O3) were embedded through graphene oxide (GO) nanosheets to be investigated for medical applications. The composition of the preparations has been confirmed by X-ray photoelectron spectroscopy, energy-dispersive X-ray analysis, and Fourier-Transform infrared spectroscopy. Scanning and transmission electron microscopy have shown the typical morphologies of the components of the nanocomposites with hydroxyapatite nanorods reaching an average diameter of 22.26±2 nm and an average length of 69.56±19.25 nm in the ternary nanocomposites. The ternary nanocomposite has a microhardness of 5.8±0.1 GPa and a higher average roughness of 6.5 nm compared to pure HAP preparation with an average roughness of 2.7 nm. All preparations have shown an acceptable cytotoxicity profile with a percent osteoblasts cell viability of 98.6±1.3% after culturing with the ternary nanocomposite. The TNC has also shown the highest antibacterial activity compared to preparations of each of its constituents and their nanocomposites, with a zone of inhibition’s diameter of 14.1±0.8 mm and 13.6±0.6 mm against Staphylococcus aureus and Escherichia coli, respectively, compared to no zone of inhibition for the pure hydroxyapatite preparation.

Keywords: hydroxypatite, cytotoxicity, nanocomposites, X-ray analysis

Procedia PDF Downloads 83

Authors: Somaye Eskandary

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Polycyclic aromatic hydrocarbons (PAHs) are present in crude oil and its derivatives contaminate soil and also increase carcinogen and mutagen contamination, which is a concern for researchers. Land farming is one of the methods that remove pollutants from the soil by native microorganisms. It seems that this technology is cost-effective, environmentally friendly and causes less debris problem to be disposed. This study aimed to refine the polycyclic aromatic hydrocarbons from oil-contaminated soil using the land farming method. In addition to examine the concentration of polycyclic aromatic hydrocarbons by GC-FID, some characteristics such as soil microbial respiration and dehydrogenase, peroxidase, urease, acid and alkaline phosphatase enzyme concentration were also measured. The results showed that after land farming process the concentrations of some polycyclic aromatic hydrocarbons dropped to 50 percent. The results showed that the enzyme concentration is reduced by reducing the concentration of hydrocarbons and microbial respiration. These results emphasize the process of land farming for removal of polycyclic aromatic hydrocarbons from soil by indigenous microorganisms.

Keywords: soil contamination, gas chromatography, native microorganisms, soil enzymes, microbial respiration, carcinogen

Procedia PDF Downloads 385

Authors: Amir Zeb, Shailima Rampogu, Minky Son, Ayoung Baek, Sang H. Yoon, Keun W. Lee

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Neurotoxic insults activate calpain, which in turn produces truncated p25 from p35. p25 forms hyperactivated Cdk5/p25 complex, and thereby induces severe neuropathological aberrations including hyperphosphorylated tau, neuroinflammation, apoptosis, and neuronal death. Inhibition of Cdk5/p25 complex alleviates aberrant phosphorylation of tau to mitigate AD pathology. PHA-793887 and Roscovitine have been investigated as selective inhibitors of Cdk5/p25 with IC50 values 5nM and 160nM, respectively, but their mechanistic studies remain unknown. Herein, computational simulations have explored the binding mode and interaction mechanism of PHA-793887 and Roscovitine with Cdk5/p25. Docking results suggested that PHA-793887 and Rsocovitine have occupied the ATP-binding site of Cdk5 and obtained highest docking (GOLD) score of 66.54 and 84.03, respectively. Furthermore, molecular dynamics (MD) simulation demonstrated that PHA-793887 and Roscovitine established stable RMSD of 1.09 Å and 1.48 Å with Cdk5/p25, respectively. Profiling of polar interactions suggested that each inhibitor formed hydrogen bonds (H-bond) with catalytic residues of Cdk5 and could remain stable throughout the molecular dynamics simulation. Additionally, binding free energy calculation by molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) suggested that PHA-793887 and Roscovitine had lowest binding free energies of -150.05 kJ/mol and -113.14 kJ/mol, respectively with Cdk5/p25. Free energy decomposition demonstrated that polar energy by H-bond between the Glu81 of Cdk5 and PHA-793887 is the essential factor to make PHA-793887 highly selective towards Cdk5/p25. Overall, this study provided substantial evidences to explore mechanistic interactions of the selective inhibitors of Cdk5/p25 and could be used as fundamental considerations in the development of structure-based selective inhibitors of Cdk5/p25.

Keywords: Cdk5/p25 inhibition, molecular modeling of Cdk5/p25, PHA-793887 and roscovitine, selective inhibition of Cdk5/p25

Procedia PDF Downloads 139

Authors: Agnieszka Krzemińska, Katarzyna Świderek, Vicente Molinier, Piotr Paneth

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In order to understand in details the interactions between ligands and the enzyme isotope effects were studied between clinically used drugs that bind in the active site of Human Immunodeficiency Virus Reverse Transcriptase, HIV-1 RT, as well as triazole-based inhibitor that binds in the allosteric pocket of this enzyme. The magnitudes and origins of the resulting binding isotope effects were analyzed. Subsequently, binding isotope effect of the same triazole-based inhibitor bound in the active site were analyzed and compared. Together, these results show differences in binding origins in two sites of the enzyme and allow to analyze binding mode and place of newly synthesized inhibitors. Typical protocol is described below on the example of triazole ligand in the allosteric pocket. Triazole was docked into allosteric cavity of HIV-1 RT with Glide using extra-precision mode as implemented in Schroedinger software. The structure of HIV-1 RT was obtained from Protein Data Bank as structure of PDB ID 2RKI. The pKa for titratable amino acids was calculated using PROPKA software, and in order to neutralize the system 15 Cl- were added using tLEaP package implemented in AMBERTools ver.1.5. Also N-terminals and C-terminals were build using tLEaP. The system was placed in 144x160x144Å3 orthorhombic box of water molecules using NAMD program. Missing parameters for triazole were obtained at the AM1 level using Antechamber software implemented in AMBERTools. The energy minimizations were carried out by means of a conjugate gradient algorithm using NAMD. Then system was heated from 0 to 300 K with temperature increment 0.001 K. Subsequently 2 ns Langevin−Verlet (NVT) MM MD simulation with AMBER force field implemented in NAMD was carried out. Periodic Boundary Conditions and cut-offs for the nonbonding interactions, range radius from 14.5 to 16 Å, are used. After 2 ns relaxation 200 ps of QM/MM MD at 300 K were simulated. The triazole was treated quantum mechanically at the AM1 level, protein was described using AMBER and water molecules were described using TIP3P, as implemented in fDynamo library. Molecules 20 Å apart from the triazole were kept frozen, with cut-offs established on range radius from 14.5 to 16 Å. In order to describe interactions between triazole and RT free energy of binding using Free Energy Perturbation method was done. The change in frequencies from ligand in solution to ligand bounded in enzyme was used to calculate binding isotope effects.

Keywords: binding isotope effects, molecular dynamics, HIV, reverse transcriptase

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