Search results for: P450

Authors: Sara Arafeh, Kovriguine Evguine

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Cytochromes P450 are enzymes that require a supply of electrons to catalyze the synthesis of steroid hormones, fatty acids, and prostaglandin hormone. Cytochrome P450 Oxidoreductase (CYPOR), a membrane bound enzyme, provides these electrons in its open conformation. CYPOR has two cytosolic domains (FAD domain and FMN domain) and an N-terminal in the membrane. In its open conformation, electrons flow from NADPH, FAD, and finally to FMN where cytochrome P450 picks up these electrons. In the closed conformation, cytochrome P450 does not bind to the FMN domain to take the electrons. It was found that when the cytosolic domains are isolated, CYPOR could not bind to cytochrome P450. This suggested that the membrane environment is important for CYPOR function. This project takes the initiative to better understand the dynamics of CYPOR in its full length. Here, we determine the distance between specific sites in the FAD and FMN binding domains in CYPOR by Forster Resonance Energy Transfer (FRET) and Ultrafast TA spectroscopy with and without NADPH. The approach to determine these distances will rely on labeling these sites with red and infrared fluorophores. Mimic membrane attachment is done by inserting CYPOR in lipid nanodiscs. By determining the distances between the donor-acceptor sites in these domains, we can observe the open/closed conformations upon reducing CYPOR in the presence and absence of cytochrome P450. Such study is important to better understand CYPOR mechanism of action in various endosomal membranes including hepatic CYPOR which is vital in plasma cholesterol homeostasis. By investigating the conformational cycles of CYPOR, we can synthesize drugs that would be more efficient in affecting the steroid hormonal levels and metabolism of toxins catalyzed by Cytochrome P450.

Keywords: conformational cycle of CYPOR, cytochrome P450, cytochrome P450 oxidoreductase, FAD domain, FMN domain, FRET, Ultrafast TA Spectroscopy

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Authors: Leticia Leandro Rade, Amanda Silva de Sousa, Suman Das, Wesley Generoso, Mayara Chagas Ávila, Plinio Salmazo Vieira, Antonio Bonomi, Gabriela Persinoti, Mario Tyago Murakami, Thomas Michael Makris, Leticia Maria Zanphorlin

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Alkenes have an economic appeal, especially in the biofuels field, since they are precursors for drop-in biofuels production, which have similar chemical and physical properties to the conventional fossil fuels, with no oxygen in their composition. After the discovery of the first P450 CYP152 OleTJE in 2011, reported with its unique property of decarboxylating fatty acids (FA), by using hydrogen peroxide as a cofactor and producing 1-alkenes as the main product, the scientific and technological interest in this family of enzymes vastly increased. In this context, the present work presents a new decarboxylase (OleTRN) with low similarity with OleTJE (32%), its biochemical characterization, and structure elucidation. As main results, OleTRN presented a high yield of expression and purity, optimum reaction conditions at 35 °C and pH from 6.5 to 8.0, and higher specificity for oleic acid. Besides that, structure-guided mutations were performed and according to the functional characterizations, it was observed that some mutations presented different specificity and chemoselectivity by varying the chain-length of FA substrates from 12 to 20 carbons. These results are extremely interesting from a biotechnological perspective as those characteristics could diversify the applications and contribute to designing better cytochrome P450 decarboxylases. Considering that peroxygenases have the potential activity of decarboxylating and hydroxylating fatty acids and that the elucidation of the intriguing mechanistic involved in the decarboxylation preferential from OleTJE is still a challenge, the elucidation of OleTRN structure and the functional characterizations of OleTRN and its mutants contribute to new information about CYP152. Besides that, the work also contributed to the discovery of a new decarboxylase with a different selectivity profile from OleTJE, which allows a wide range of applications.

Keywords: P450, decarboxylases, alkenes, biofuels

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Authors: Mai. M. Toughan, Ahmed A. A. Sallam, Ashraf O. Abd El-Latif

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Termites are eusocial insects that are found on all continents except Antarctica. Termites have serious destructive impact, damaging local huts and crops of poor subsistence. The annual cost of termite damage and its control is determined in the billions globally. In Egypt, most of these damages are due to the subterranean termite species especially the sand termite, P. hypostoma. Pyrethroids became the primary weapon for subterranean termite control, after the use of chlorpyrifos as a soil termiticide was banned. Despite the important role of pyrethroids in termite control, its extensive use in pest control led to the eventual rise of insecticide resistance which may make many of the pyrethroids ineffective. The ability to diagnose the precise mechanism of pyrethroid resistance in any insect species would be the key component of its management at specified location for a specific population. In the present study, detailed toxicological and biochemical studies was conducted on the mechanism of pyrethroid resistance in P. hypostoma. The susceptibility of field populations of P. hypostoma against deltamethrin, α-cypermethrin and ƛ-cyhalothrin was evaluated. The obtained results revealed that the workers of P. hypostoma have developed high resistance level against the tested pyrethroids. Studies carried out through estimation of detoxification enzyme activity indicated that enhanced esterase and cytochrome P450 activities were probably important mechanisms for pyrethroid resistance in field populations. Elevated esterase activity and also additional esterase isozyme were observed in the pyrethroid-resistant populations compared to the susceptible populations. Strong positive correlation between cytochrome P450 activity and pyrethroid resistance was also reported. |Deltamethrin could be recommended as a resistance-breaking pyrethroid that is active against resistant populations of P. hypostoma.

Keywords: Psammotermes hypostoma, pyrethroid resistance, esterase, cytochrome P450

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Authors: Rachel F. Ajayi, Unathi Sidwaba, Usisipho Feleni, Samantha F. Douman, Ezo Nxusani, Lindsay Wilson, Candice Rassie, Oluwakemi Tovide, Priscilla G.L. Baker, Sibulelo L. Vilakazi, Robert Tshikhudo, Emmanuel I. Iwuoha

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Pyrazinamide (PZA) is among the first-line pro-drugs in the tuberculosis (TB) combination chemotherapy used to treat Mycobacterium tuberculosis. Numerous reports have suggested that hepatotoxicity due to pyrazinamide in patients is due to inappropriate dosing. It is therefore necessary to develop sensitive and reliable techniques for determining the PZA metabolic profile of diagnosed patients promptly and at point-of-care. This study reports the determination of PZA based on nanobiosensor systems developed from disuccinimidyl octanedioate modified Cytochrome P450-2E1 (CYP2E1) electrodeposited on gold substrates derivatised with (poly(8-anilino-1-napthalene sulphonic acid) PANSA/PVP-AgNPs nanocomposites. The rapid and sensitive amperometric PZA detection gave a dynamic linear range of 2 µM to 16 µM revealing a limit of detection of 0.044 µM and a sensitivity of 1.38 µA/µM. The Michaelis-Menten parameters; KM, KMapp and IMAX were also calculated and found to be 6.0 µM, 1.41 µM and 1.51 µA respectively indicating a nanobiosensor suitable for use in serum.

Keywords: tuberculosis, cytochrome P450-2E1, disuccinimidyl octanedioate, pyrazinamide

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Authors: Soodabeh Shahid Sales, Azam Rastgar Moghadam, Mehrane Mehramiz, Malihe Entezari, Kazem Anvari, Mohammad Sadegh Khorrami, Saeideh Ahmadi Simab, Ali Moradi, Seyed Mahdi Hassanian, Majid Ghayour-Mobarhan, Gordon A. Ferns, Amir Avan

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Background Esophageal cancer has been reported as the eighth most common cancer universal and the seventh cause of cancer-related death in men .recent studies have revealed that cytochrome P450, family 1, subfamily B, polypeptide 1, which plays a role in metabolizing xenobiotics, is associated with different cancers. Therefore in the present study, we investigated the impact of CYP1B1-rs1056836 on esophagus squamous cell carcinoma (ESCC) patients. Method: 317 subjects, with and without ESCC were recruited. DNA was extracted and genotyped via Real-time PCR-Based Taq Man. Kaplan Meier curves were utilized to assess overall and progression-free survival. To evaluate the relationship between patients clinicopathological data, genotypic frequencies, disease prognosis, and patients survival, Pearson chi-square and t-test were used. Logistic regression was utilized to assess the association between the risk of ESCC and genotypes. Results: the genotypic frequency for GG, GC, and CC are respectively 58.6% , 29.8%, 11.5% in the healthy group and 51.8%, 36.14% and 12% in ESCC group. With respect to the recessive genetic inheritance model, an association between the GG genotype and stage of ESCC were found. Also, statistically significant results were not found for this variation and risk of ESCC. Patients with GG genotype had a decreased risk of nodal metastasis in comparison with patients with CC/CG genotype, although this link was not statistically significant. Conclusion: Our findings illustrated the correlation of CYP1B1-rs1056836 as a potential biomarker for ESCC patients, supporting further studies in larger populations in different ethnic groups. Moreover, further investigations are warranted to evaluate the association of emerging marker with dietary intake and lifestyle.

Keywords: Cytochrome P450, esophagus squamous cell carcinoma, dietary intake, lifestyle

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Authors: Manohar Salla, Mark S. Butler, Ruby Pelingon, Geraldine Kaeslin, Daniel E. Croker, Janet C. Reid, Jong Min Baek, Paul V. Bernhardt, Elizabeth M. J. Gillam, Matthew A. Cooper, Avril A. B. Robertson

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MCC950 is a potent and selective inhibitor of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome that shows early promise for treatment of inflammatory diseases. The identification of major metabolites of lead molecule is an important step during drug development process. It provides an information about the metabolically labile sites in the molecule and thereby helping medicinal chemists to design metabolically stable molecules. To identify major metabolites of MCC950, the compound was incubated with human liver microsomes and subsequent analysis by (+)- and (−)-QTOF-ESI-MS/MS revealed a major metabolite formed due to hydroxylation on 1,2,3,5,6,7-hexahydro-s-indacene moiety of MCC950. This major metabolite can lose two water molecules and three possible regioisomers were synthesized. Co-elution of major metabolite with each of the synthesized compounds using HPLC-ESI-SRM-MS/MS revealed the structure of the metabolite (±) N-((1-hydroxy-1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide. Subsequent synthesis of individual enantiomers and coelution in HPLC-ESI-SRM-MS/MS using a chiral column revealed the metabolite was R-(+)- N-((1-hydroxy-1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide. To study the possible cytochrome P450 enzyme(s) responsible for the formation of major metabolite, MCC950 was incubated with a panel of cytochrome P450 enzymes. The result indicated that CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C18, CYP2C19, CYP2J2 and CYP3A4 are most likely responsible for the formation of the major metabolite. The biological activity of the major metabolite and the other synthesized regioisomers was also investigated by screening for for NLRP3 inflammasome inhibitory activity and cytotoxicity. The major metabolite had 170-fold less inhibitory activity (IC50-1238 nM) than MCC950 (IC50-7.5 nM). Interestingly, one regioisomer had shown nanomolar inhibitory activity (IC50-232 nM). However, no evidence of cytotoxicity was observed with any of these synthesized compounds when tested in human embryonic kidney 293 cells (HEK293) and human liver hepatocellular carcinoma G2 cells (HepG2). These key findings give an insight into the SAR of the hexahydroindacene moiety of MCC950 and reveal a metabolic soft spot which could be blocked by chemical modification.

Keywords: Cytochrome P450, inflammasome, MCC950, metabolite, microsome, NLRP3

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Authors: Gesti Prastiti, Maylina Adani, Yuyun darma A. N., M. Khilmi F., Yunita Wahyu Pratiwi

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Combination therapy may allow interaction on two drugs or more that can give adverse effects on patients. Simvastatin is a drug of antihyperlipidemia it can interact with drugs which work on cytochrome P450 CYP3A4 because it can interfere the performance of simvastatin. Flavonoid found in plants can inhibit the cytochrome P450 CYP3A4 if taken with simvastatin and can increase simvastatin levels in the body and increases the potential side effects of simvastatin such as myopati and rhabdomyolysis. Green tea leaves and mint are herbal medicine which has the effect of antihiperlipidemia. This study aims to determine the potential interaction of simvastatin with herbal drinks (green tea leaves and mint). This research method are experimental post-test only control design. Test subjects were divided into 5 groups: normal group, negative control group, simvastatin group, a combination of green tea group and the combination group mint leaves. The study was conducted over 32 days and total cholesterol levels were analyzed by enzymatic colorimetric test method. Results of this study is the obtainment of average value of total cholesterol in each group, the normal group (65.92 mg/dL), the negative control group the average total cholesterol test in the normal group was (69.86 mg/dL), simvastatin group (58.96 mg/dL), the combination of green tea group (58.96 mg/dL), and the combination of mint leaves (63.68 mg/dL). The conclusion is between simvastatin combination therapy with herbal drinks have the potential for pharmacodynamic interactions with a synergistic effect, antagonist, and a powerful additive, so the combination therapy are no more effective than a single administration of simvastatin therapy.

Keywords: hyperlipidemia, simvastatin, herbal drinks, green tea leaves, mint leaves, drug interactions

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Authors: Naso Isaiah Thanavisuth

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Introduction: Medical cannabis can be used for treatment, including anorexia, pain, inflammation, multiple sclerosis, Parkinson's disease, epilepsy, cancer, and metabolic syndrome-related disorders. However, medical cannabis leads to adverse effects (AEs), which is delta-9-tetrahydrocannabinol (THC). In previous studies, the major of THC metabolism enzymes are CYP2C9. Especially, the variation of CYP2C9 gene consist of CYP2C9*2 on exon 3 (C430T) (Arg144Cys) and CYP2C9*3 on exon 7 (A1075C) (Ile359Leu) to decrease enzyme activity. Notwithstanding, there is no data describing whether the variant of CYP2C9 genes are a pharmacogenetics marker for prediction of THC-induced AEs in Thai patients. Objective: We want to investigate the association between CYP2C9 gene and THC-induced AEs in Thai patients. Method: We enrolled 39 Thai patients with medical cannabis treatment consisting of men and women who were classified by clinical data. The quality of DNA extraction was assessed by using NanoDrop ND-1000. The CYP2C9*2 and *3 genotyping were conducted using the TaqMan real time PCR assay (ABI, Foster City, CA, USA). Results: All Thai patients who received the medical cannabis consist of twenty four (61.54%) patients who were female and fifteen (38.46%) were male, with age range 27- 87 years. Moreover, the most AEs in Thai patients who were treated with medical cannabis between cases and controls were tachycardia, arrhythmia, dry mouth, and nausea. Particularly, thirteen (72.22%) medical cannabis-induced AEs were female and age range 33 – 69 years. In this study, none of the medical cannabis groups carried CYP2C9*2 variants in Thai patients. The CYP2C9*3 variants (*1/*3, intermediate metabolizer, IM) and (*3/*3, poor metabolizer, PM) were found, three of thirty nine (7.69%) and one of thirty nine (2.56%) , respectively. Conclusion: This is the first study to confirm the genetic polymorphism of CYP2C9 and medical cannabis-induced AEs in the Thai population. Although, our results indicates that there is no found the CYP2C9*2. However, the variation of CYP2C9 allele might serve as a pharmacogenetics marker for screening before initiating the therapy with medical cannabis for prevention of medical cannabis-induced AEs.

Keywords: CYP2C9, medical cannabis, adverse effects, THC, P450

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Authors: Sara Franco Ortega, Alina Goldberg Cavalleri, Nawaporn Onkokesung, Richard Dale, Melissa Brazier-Hicks, Robert Edwards

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Safeners are agrochemicals that enhance the selective chemical control of wild grasses by increasing the ability of the crop to metabolise the herbicide. Although these compounds are widely used, their mode of action is not well understood. It is known that safeners enhance the metabolism of herbicides, by up-regulating the associated detoxification system we have termed the xenome. The xenome proteins involved in herbicide metabolism have been previously divided into four different phases, with cytochrome P450s (CYPs) playing a key role in phase I metabolism by catalysing hydroxylation and dealkylation reactions. Subsequently, glutathione S-transferases (GSTs) and UDP-glucosyltransferases lead to the formation of Phase II conjugates prior to their transport into the vacuole by ABCs transporters (Phase III). Maize (Zea mays), was been treated with different safeners to explore the selective induction of xenome proteins, with a special interest in the regulation of the CYP superfamily. Transcriptome analysis enabled the identification of key safener-inducible CYPs that were then functionally assessed to determine their role in herbicide detoxification. In order to do that, CYP’s were codon optimised, synthesised and inserted into the yeast expression vector pYES3 using in-fusion cloning. CYP’s expressed as recombinant proteins in a strain of yeast engineered to contain the P450 co-enzyme (cytochrome P450 reductase) from Arabidopsis. Microsomes were extracted and treated with herbicides of different chemical classes in the presence of the cofactor NADPH. The reaction products were then analysed by LCMS to identify any herbicide metabolites. The results of these studies will be presented with the key CYPs identified in maize used as the starting point to find orthologs in other crops and weeds to better understand their roles in herbicide selectivity and safening.

Keywords: CYPs, herbicide detoxification, LCMS, RNA-Seq, safeners

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Authors: Viktoriia Burkina, Vladimir Zlabek, Galia Zamaratskaia

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Cytochromes P450 (CYP) are important family of enzymes in Phase I metabolism. Environmental pollutants often act as inducers of the gene expression and activities CYP1A1 and CYP3A-like isoforms in fish. The activities are generally measured in the fish liver or gills, and less is known about tissue distribution of expression. In present study, the CYP1A1 and CYP3A-like activities were measured in rainbow trout liver, gill, intestine, heart, brain and gonads. The activities of CYP1A1 and CYP3A-like proteins were estimated as the rates of 7-ethoxyresorufin-O-deethylation (EROD) and benzyloxy-4-trifluoromethylcoumarin-O-debenzyloxylation (BFCOD), respectively. The CYP1A1 and CYP3A-like activities were detectable in all investigated fish tissues, with the highest activity in hepatic tissue followed by heart > brain > gill > intestine > gonads. To confirm the presence of CYP1A1 in different tissues, EROD activity was measured in presence of the selective inhibitors ellipticine (CYP1A1), ketoconazole (CYP3A), 8-methoxypsoralen (human CYP2A) and diallyl sulphide (CYP2E1). It was found that ellipticine, ketoconazole and 8-methoxypsoralen inhibited hepatic EROD activity by 88-98%. Ellipticine inhibited gill, intestine, and gonad EROD activity by 50%. In conclusion, EROD and BFCOD activities were detected in rainbow trout liver, gill, intestine, heart, brain and gonads. Further studies are needed to fully identify all CYP450 isoforms responsible for these activities. Acknowledgement: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic - projects „CENAKVA “(No. CZ.1.05/2.1.00/01.0024), “CENAKVA Center Development “(No. CZ.1.05/2.1.00/19.0380), “CENAKVA II “(No. LO1205 under the NPU I program), and "Development of USB - International mobility (No. CZ.02.2.69/0.0/0.0/16_027/0008364).

Keywords: BFCOD, EROD, fish, phase I metabolism, selective inhibitors

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Authors: Shanna Swart, Caryn Fenner, Athanasios Kotsiopoulos, Susan Harrison

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Linear alkanes are produced as by-products from the increasing use of gas-to-liquid fuel technologies for synthetic fuel production and offer great potential for value addition. Their current use as low-value fuels and solvents do not maximize this potential. Therefore, attention has been drawn towards direct activation of these aliphatic alkanes to more useful products such as alcohols, aldehydes, carboxylic acids and derivatives. Cytochrome P450 monooxygenases (P450s) can be used for activation of these aliphatic alkanes using whole-cells or cell-free systems. Some limitations of whole-cell systems include reduced mass transfer, stability and possible side reactions. Since the P450 systems are little studied as cell-free systems, they form the focus of this study. Challenges of a cell-free system include co-factor regeneration, substrate availability and enzyme stability. Enzyme immobilization offers a positive outlook on this dilemma, as it may enhance stability of the enzyme. In the present study, 2 different P450s (CYP153A6 and CYP102A1) as well as the relevant accessory enzymes required for electron transfer (ferredoxin and ferredoxin reductase) and co-factor regeneration (glucose dehydrogenase) have been expressed in E. coli and purified by metal affinity chromatography. Glucose dehydrogenase (GDH), was used as a model enzyme to assess the potential of various enzyme immobilization strategies including; surface attachment on MagReSyn® microspheres with various functionalities and on electrospun nanofibers, using self-assembly based methods forming Cross Linked Enzymes (CLE), Cross Linked Enzyme Aggregates (CLEAs) and spherezymes as well as in a sol gel. The nanofibers were synthesized by electrospinning, which required the building of an electrospinning machine. The nanofiber morphology has been analyzed by SEM and binding will be further verified by FT-IR. Covalent attachment based methods showed limitations where only ferredoxin reductase and GDH retained activity after immobilization which were largely attributed to insufficient electron transfer and inactivation caused by the crosslinkers (60% and 90% relative activity loss for the free enzyme when using 0.5% glutaraldehyde and glutaraldehyde/ethylenediamine (1:1 v/v), respectively). So far, initial experiments with GDH have shown the most potential when immobilized via their His-tag onto the surface of MagReSyn® microspheres functionalized with Ni-NTA. It was found that Crude GDH could be simultaneously purified and immobilized with sufficient activity retention. Immobilized pure and crude GDH could be recycled 9 and 10 times, respectively, with approximately 10% activity remaining. The immobilized GDH was also more stable than the free enzyme after storage for 14 days at 4˚C. This immobilization strategy will also be applied to the P450s and optimized with regards to enzyme loading and immobilization time, as well as characterized and compared with the free enzymes. It is anticipated that the proposed immobilization set-up will offer enhanced enzyme stability (as well as reusability and easy recovery), minimal mass transfer limitation, with continuous co-factor regeneration and minimal enzyme leaching. All of which provide a positive outlook on this robust multi-enzyme system for efficient activation of linear alkanes as well as the potential for immobilization of various multiple enzymes, including multimeric enzymes for different bio-catalytic applications beyond alkane activation.

Keywords: alkane activation, cytochrome P450 monooxygenase, enzyme catalysis, enzyme immobilization

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Authors: Kuladip Jana, Bhaswati Banerjee, Parimal C. Sen

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Benzo(a)pyrene [B(a)P] is an environmental toxicant present mostly in cigarette smoke and car exhaust, is an aryl hydrocarbon receptor (AhR) ligand that exerts its toxic effects on both male and female reproductive systems along with carcinogenesis in skin, prostate, ovary, lung and mammary glands. Our study was focused on elucidating the molecular mechanism of B(a)P induced male reproductive toxicity and its prevention with phytochemical like resveratrol. In this study, the effect of B(a)P at different doses (0.1, 0.25, 0.5, 1 and 5 mg /kg body weight) was studied on male reproductive system of Wistar rat. A significant decrease in cauda epididymal sperm count and motility along with the presence of sperm head abnormalities and altered epididymal and testicular histology were documented following B(a)P treatment. B(a)P treatment resulted apoptotic sperm cells as observed by TUNEL and Annexin V-PI assay with increased Reactive Oxygen Species (ROS), altered sperm mitochondrial membrane potential (ΔΨm) with a simultaneous decrease in the activity of antioxidant enzymes and GSH status. TUNEL positive apoptotic cells also observed in testis as well as isolated germ and Leydig cells following B(a)P exposure. Western Blot analysis revealed the activation of p38 mitogen activated protein kinase (p38MAPK), cytosolic translocation of cytochrome-c, upregulation of Bax and inducible nitric oxide synthase (iNOS) with cleavage of poly ADP ribose polymerase (PARP) and down regulation of BCl2 in testis upon B(a)P treatment. The protein and mRNA levels of testicular key steroidogenesis regulatory proteins like steroidogenic acute regulatory protein (StAR), cytochrome P450 IIA1 (CYPIIA1), 3β hydroxy steroid dehydrogenase (3β HSD), 17β hydroxy steroid dehydrogenase (17β HSD) showed a significant decrease in a dose dependent manner while an increase in the expression of cytochrome P450 1A1 (CYP1A1), Aryl hydrocarbon Receptor (AhR), active caspase- 9 and caspase- 3 following B(a)P exposure. We conclude that exposure of benzo(a)pyrene caused testicular gamatogenic and steroidogenic disorders by induction of oxidative stress, inhibition of StAR and other steroidogenic enzymes along with activation of p38MAPK and initiated caspase-3 mediated germ and Leydig cell apoptosis. Next we investigated the role of resveratrol on B(a)P induced male reproductive toxicity. Our study highlighted that resveratrol co-treatment with B(a)P maintained testicular redox potential, increased serum testosterone level and prevented steroidogenic dysfunction with enhanced expression of major testicular steroidogenic proteins (CYPIIA1, StAR, 3β HSD,17β HSD) relative to treatment with B(a)P only. Resveratrol suppressed B(a)P-induced testicular activation of p38 MAPK, ATF2, iNOS and ROS production; cytosolic translocation of Cytochome c and Caspase 3 activation thereby prevented oxidative stress of testis and inhibited apoptosis. Resveratrol co-treatment also decreased B(a)P-induced AhR protein level, its nuclear translocation and subsequent CYP1A1 promoter activation, thereby decreased protein and mRNA levels of testicular cytochrome P4501A1 (CYP1A1) and prevented BPDE-DNA adduct formation. Our findings cumulatively suggest that resveratrol prevents activation of B(a)P by modulating the transcriptional regulation of CYP1A1 and acting as an antioxidant thus prevents B(a)P-induced oxidative stress and testicular apoptosis.

Keywords: benzo(a)pyrene, resveratrol, testis, apoptosis, cytochrome P450 1A1 (CYP1A1), aryl hydrocarbon receptor (AhR), p38 MAPK/ATF2/iNOS

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Authors: M. Ouassaf, S. Belaidi

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Aromatase is an estrogen biosynthetic enzyme belonging to the cytochrome P450 family, which catalyzes the limiting step in the conversion of androgens to estrogens. As it is relevant for the promotion of tumor cell growth. A set of thirty 1,2,3-triazole derivatives was used in the quantitative structure activity relationship (QSAR) study using regression multiple linear (MLR), We divided the data into two training and testing groups. The results showed a good predictive ability of the MLR model, the models were statistically robust internally (R² = 0.982) and the predictability of the model was tested by several parameters. including external criteria (R²pred = 0.851, CCC = 0.946). The knowledge gained in this study should provide relevant information that contributes to the origins of aromatase inhibitory activity and, therefore, facilitates our ongoing quest for aromatase inhibitors with robust properties.

Keywords: aromatase inhibitors, QSAR, MLR, 1, 2, 3-triazole

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Authors: Kuladip Jana

Abstract:

Benzo(a)pyrene [B(a)P] is an environmental toxicant present mostly in cigarette smoke and car exhaust, is an aryl hydrocarbon receptor (AhR) ligand that exerts its toxic effects on both male and female reproductive systems. In this study, the effect of B(a)P at different doses (0.1, 0.25, 0.5, 1 and 5 mg /kg body weight) was studied on male reproductive system of rat. A significant decrease in cauda epididymal sperm count and motility along with the presence of sperm head abnormalities and altered epididymal and testicular histology were documented following B(a)P treatment. B(a)P treatment resulted apoptotic sperm cells as observed by TUNEL and Annexin V-PI assay with increased ROS, altered sperm mitochondrial membrane potential (ΔΨm) with a simultaneous decrease in the activity of antioxidant enzymes and GSH status. TUNEL positive apoptotic cells also observed in testis as well as isolated germ and Leydig cells following B(a)P exposure. Western Blot analysis revealed the activation of p38MAPK, cytosolic translocation of cytochrome-c, up-regulation of Bax and inducible nitric oxide synthase (iNOS) with cleavage of PARP and down-regulation of BCl2 in testis upon B(a)P treatment. The protein and mRNA levels of testicular key steroidogenesis regulatory proteins like StAR, cytochrome P450 IIA1 (CYPIIA1), 3β HSD, 17β HSD showed a significant decrease in a dose dependent manner while an increase in the expression of cytochrome P450 1A1 (CYP1A1), Aryl hydrocarbon Receptor (AhR), active caspase- 9 and caspase- 3 following B(a)P exposure. We conclude that exposure of benzo(a)pyrene caused testicular gamatogenic and steroidogenic disorders by induction of oxidative stress, inhibition of StAR and other steroidogenic enzymes along with activation of p38MAPK and initiated caspase-3 mediated germ and Leydig cell apoptosis.The possible protective role of naturally occurring phytochemicals against B(a)P induced testicular toxicity needs immediate consideration. Curcumin and resveratrol separately were found to protect against B(a)P induced germ cell apoptosis, and their combinatorial effect was more significant. Our present study in isolated testicular germ cell population from adult male Wistar rats, highlighted their synergistic protective effect against B(a)P induced germ cell apoptosis. Curcumin-resveratrol co-treatment decreased the expression of pro-apoptotic proteins like cleaved caspase 3,8,9, cleaved PARP, Apaf1, FasL, tBid. Curcumin-resveratrol co-treatment decreased Bax/Bcl2 ratio, mitochondria to cytosolic translocation of cytochrome c and activated the survival protein Akt. Curcumin-resveratrol decreased the expression of p53 dependent apoptotic genes like Fas, FasL, Bax, Bcl2, Apaf1.Curcumin-resveratrol co-treatment thus prevented B(a)P induced germ cell apoptosis. B(a)P induced testicular ROS generation and oxidative stress were significantly ameliorated with curcumin and resveratrol. Curcumin-resveratrol co-treatment prevented B(a)P induced nuclear translocation of AhR and CYP1A1 production. The combinatorial treatment significantly inhibited B(a)P induced ERK 1/2, p38 MAPK and JNK 1/2 activation. B(a)P treatment increased the expression of p53 and its phosphorylation (p53 ser 15). Curcumin-resveratrol co-treatment significantly decreased p53 level and its phosphorylation (p53 ser 15). The study concludes that curcumin-resveratrol synergistically modulated MAPKs and p53, prevented oxidative stress, regulated the expression of pro and anti-apoptotic proteins as well as the proteins involved in B(a)P metabolism thus protected germ cells from B(a)P induced apoptosis.

Keywords: benzo(a)pyrene, germ cell, apoptosis, oxidative stress, resveratrol, curcumin

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Authors: Nadeem Bashir, Ghulam Mustafa Peerzada

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The present study pertains to the nonlinear behavior of acetaminophen based uncatalyzed Belousov-Zhabotinsky (BZ) oscillator and its dynamics in the presence of Ferroin as the catalyst. The role of free metal ions as catalysts was examined and the results compared with corresponding complexed catalysts. Free metal ions were found to be sluggish with respect to the evolution of the oscillatory regime as compared to complexed ones. Effect of change of the ligand moiety of the catalyst complex on the oscillatory parameters was monitored. Since ethanol potentiates the hepatotoxicity caused by acetaminophen in-vivo, it is thought to understand this interaction by virtue of causing perturbation of the acetaminophen based oscillator with different concentrations of the ethanol with and without ferroin as the catalyst. Another dimension to the ethanol effect was added by perturbation of the system with ethanol at different stages of the reaction so as to get an idea whether it is acetaminophen or some reactive intermediate generated in the reaction system which reacts with ethanol. Further, the ferroin-catalyzed oscillator is taken as a prototype inorganic model of the acetaminophen-ethanol syndrome, as ferroin and HOBr were inorganic replacements to Cyt P450 and NADPH in the alcohol metabolism.

Keywords: Belousov-Zhabotinsky reaction, ferroin, Paracetamol-Ethanol syndrome, kinetics

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Authors: Naso Isaiah Thanavisuth

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Medical cannabis can be used for treatment including pain, multiple sclerosis, Parkinson's disease, and cancer. However, medical cannabis leads to adverse effects (AEs), which is delta-9-tetrahydrocannabinol (THC). In previous studies, the major of THC metabolism enzymes are CYP2C9. Especially, the variation of CYP2C9 gene consist of CYP2C9*2 on exon 3 and CYP2C9*3 on exon 7 to decrease enzyme activity. Notwithstanding, there is no data describing whether the variant of CYP2C9 genes are apharmacogenetics marker for the prediction of THC-induced AEs in Thai patients. We want to investigate the association between CYP2C9 gene and THC-induced AEs in Thai patients. We enrolled 39 Thai patients with medical cannabis treatment who were classified by clinical data. The CYP2C9*2 and *3 genotyping were conducted using the TaqMan real time PCR assay. All Thai patients who received the medical cannabis consist of twenty-four (61.54%) patients were female, and fifteen (38.46%) were male, with age range 27- 87 years. Moreover, the most AEs in Thai patients who were treated with medical cannabis between cases and controls were tachycardia, arrhythmia, dry mouth, and nausea. Particularly, thirteen (72.22%) medical cannabis-induced AEs were female and age range 33 – 69 years. In this study, none of the medical cannabis groups carried CYP2C9*2 variants in Thai patients. The CYP2C9*3 variants (*1/*3, intermediate metabolizer, IM) and (*3/*3, poor metabolizer, PM) were found, three of thirty-nine (7.69%) and one of thirty-nine (2.56%), respectively. Although, our results indicate that there is no found the CYP2C9*2. However, the variation of CYP2C9 allele might serve as a pharmacogenetics marker for screening before initiating the therapy with medical cannabis for the prevention of medical cannabis-induced AEs.

Keywords: CYP2C9, medical cannabis, adverse effects, THC, P450

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Authors: Milica Karadzic, Lidija Jevric, Sanja Podunavac-Kuzmanovic, Strahinja Kovacevic, Sinisa Markov, Aleksandar Okljesa, Andrea Nikolic, Marija Sakac, Katarina Penov Gasi

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This study considered the microbiological activity determination and molecular docking study for selected steroid derivatives of biomedical importance. Minimal inhibitory concentration (MIC) was determined for steroid derivatives against Staphylococcus aureus using macrodilution method. Some of the investigated steroid derivatives express bacteriostatic effect against Staphylococcus aureus. Molecular docking approaches are the most widely used techniques for predicting the binding mode of a ligand. Molecular docking study was done for steroid derivatives for androgen receptor negative prostate cancer cell line (PC-3) toward Human Cytochrome P450 CYP17A1. The molecules that had the smallest experimental IC50 values confirmed their ability to dock into active place using suitable molecular docking procedure. The binding disposition of those molecules was thoroughly investigated. Microbiological analysis and molecular docking study were conducted with aim to additionally characterize selected steroid derivatives for future investigation regarding their biological activity and to estimate the binding-affinities of investigated derivatives. This article is based upon work from COST Action (TD1305), supported by COST (European Cooperation and Science and Technology).

Keywords: binding affinity, minimal inhibitory concentration, molecular docking, pc-3 cell line, staphylococcus aureus, steroids

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Authors: Irina S. Burashnikova, Dmitriy A. Sychev, Ruslan Y. Kazakov

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Сytochrome P450 CYP2D6 activity affects antipsychotic therapy safety. CYP2D6*4 polymorphism frequency varies among different ethnic groups. We studied CYP2D6*4 polymorphism frequency in Tatar and Russian schizophrenic patients and association of CYP2D6*4 polymorphism and extrapyramidal disorders (EPD) frequency in schizophrenic patients on haloperidol monotherapy in daily doses up to 20 mg. Results: Heterozygous CYP2D6*4 allele carrier frequency among Tatars was lower (23.8% vs 32.4% in Russians), but the differences did not reach statistical significance. CYP2D6*4 allele frequency among Tatars was also lower (11.9% vs 24.3% in Russians), but the difference was not quite significant (p=0.0592). Average daily haloperidol dose in the group without EPD was significantly higher than in the group with EPD (11.35±4.6 vs 13.87±3.3 mg, p=0.0252), but average daily haloperidol dose/weight ratios in the compared groups had no significant differences. Statistically significant association between EPD development and heterozygous CYP2D6*1/*4 genotype and CYP2D6*4 allele carrier frequency was revealed among all schizophrenic patients and among those of Tatar nationality. Further well designed pharmacogenetic studies in different Russian regions are needed to improve psychotropic therapy safety and to establish evidence-based indications for pharmacogenetic testing in clinical practice.

Keywords: antipsychotic, CYP2D6 polymorphism, ethnic differences of CYP2D6*4 allele frequency, extrapyramidal side effects/disorder, schizophrenia, pharmacogenetics, Russians, Tatars

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Authors: M. Marjaneh, M. Y. Narazah, H. Shahrul

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Array-based gene expression analysis is a powerful tool to profile expression of genes and to generate information on therapeutic effects of new anti-cancer compounds. Anti-apoptotic effect of thymoquinone was studied in MCF7 breast cancer cell line using gene expression profiling with cDNA micro array. The purity and yield of RNA samples were determined using RNeasyPlus Mini kit. The Agilent RNA 6000 Nano LabChip kit evaluated the quantity of the RNA samples. AffinityScript RT oligo-dT promoter primer was used to generate cDNA strands. T7 RNA polymerase was used to convert cDNA to cRNA. The cRNA samples and human universal reference RNA were labelled with Cy-3-CTP and Cy-5-CTP, respectively. Feature Extraction and GeneSpring software analysed the data. The single experiment analysis revealed involvement of 64 pathways with up-regulated genes and 78 pathways with down-regulated genes. The MAPK and p38-MAPK pathways were inhibited due to the up-regulation of PTPRR gene. The inhibition of p38-MAPK suggested up-regulation of TGF-ß pathway. Inhibition of p38 - MAPK caused up-regulation of TP53 and down-regulation of Bcl2 genes indicating involvement of intrinsic apoptotic pathway. Down-regulation of CARD16 gene as an adaptor molecule regulated CASP1 and suggested necrosis-like programmed cell death and involvement of caspase in apoptosis. Furthermore, down-regulation of GPCR, EGF-EGFR signalling pathways suggested reduction of ER. Involvement of AhR pathway which control cytochrome P450 and glucuronidation pathways showed metabolism of Thymoquinone. The findings showed differential expression of several genes in apoptosis pathways with thymoquinone treatment in estrogen receptor-positive breast cancer cells.

Keywords: cDNA microarray, thymoquinone, CARD16, PTPRR, CASP10

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Authors: Tanakrit Doltanakarn

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Introduction: These days, cannabis is being accepted in many countries due to the fact that cannabis could be use in medical. The medical cannabis is used to treat and reduce the pain many diseases. For example, neuropathic pain, Parkinson, autism disorders, cancer pain reduce the adverse effect of chemotherapy, diabetes, and migraine. Active ingredients in cannabis that modulate patients' perceptions of their conditions include Δ9‐tetrahydrocannabinol (THC), cannabidiol (CBD), flavonoids, and terpenes. However, there is an adverse effect of cannabis, cardiovascular effects, psychosis, schizophrenia, mood disorder, and cognitive alternation. These effects are from the THC and CBD ingredients in the cannabis. The metabolize processes of delta-9 THC to 11-OH-delta 9 -THC (inactive form), THC were cause of adverse effects. Interestingly, the distributions of CYP2C9 gene (CYP2C9*2 and CYP2C9*3, poor metabolizer) that might affect incidences of adverse effects in patients who treated with medical cannabis. Objective: The aim of this study we want to investigate the association between genetic polymorphism of CYP2C9 frequency and Thai patients who treated with medical cannabis. Materials and Methods:We recruited sixty-five unrelated Thai patients from the College of Pharmacy, Rangsit University. DNA were extracted using Genomic DNA Mini Kit. Genotyping of CYP2C9*2 (430C>T, rs1799853) and CYP2C9*3 (1075A>C, rs1057910) were genotyped by the TaqMan Real-time PCR assay. Results: Among these 31 medicals cannabis-induced ADRs patients, they were diagnosed with 22 (33.85%) tachycardia and 3 (4.62%) arrhythmia. There were 34 (52.31%) medical cannabis-tolerant controls who were included in this study.40 (61.53%) Thai patients were female, and 25 (38.46%) were male, with median age of 57 (range 27 – 87) years. In this study, we found none of the medical cannabis-induced ADRs carried CYP2C9*2 variant along with medical cannabis-tolerant control group. CYP2C9*3 variant (intermediate metabolizer, IM) was found just only one of thirty-one (3.23%) in the medical cannabis-induced ADRs and two of thirty-fourth (5.88%) in the tolerant controls. Conclusions: Thus, the distribution of CYP2C9 alleles offer a comprehensive view of pharmacogenomics marker in Thai population that could be used as a reference for worldwide to investigate the pharmacogenomics application.

Keywords: medical cannabis, adverse effect, CYP2C9, thai patients

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Authors: Hamsa T. Tayeb, Mohammad A. Arafah, Dana M. Bakheet, Duaa M. Khalaf, Agnieszka Tarnoska, Nduna Dzimiri

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Background: CYP2D6 is a member of the cytochrome P450 mixed-function oxidase system. The enzyme is responsible for the metabolism and elimination of approximately 25% of clinically used drugs, especially in breast cancer and psychiatric therapy. Different phenotypes have been described displaying alleles that lead to a complete loss of enzyme activity, reduced function (poor metabolizers – PM), hyperfunctionality (ultrarapid metabolizers–UM) and therefore drug intoxication or loss of drug effect. The prevalence of these variants may vary among different ethnic groups. Furthermore, the xTAG system has been developed to categorized all patients into different groups based on their CYP2D6 substrate metabolization. Aim of the study: To determine the prevalence of the different CYP2D6 variants in our population, and to evaluate their clinical relevance in personalized medicine. Methodology: We used the Luminex xMAP genotyping system to sequence 305 Saudi individuals visiting the Blood Bank of our Institution and determine which polymorphisms of CYP2D6 gene are prevalent in our region. Results: xTAG genotyping showed that 36.72% (112 out of 305 individuals) carried the CYP2D6_*2. Out of the 112 individuals with the *2 SNP, 6.23% had multiple copies of *2 SNP (19 individuals out of 305 individuals), resulting in an UM phenotype. About 33.44% carried the CYP2D6_*41, which leads to decreased activity of the CYP2D6 enzyme. 19.67% had the wild-type alleles and thus had normal enzyme function. Furthermore, 15.74% carried the CYP2D6_*4, which is the most common nonfunctional form of the CYP2D6 enzyme worldwide. 6.56% carried the CYP2D6_*17, resulting in decreased enzyme activity. Approximately 5.73% carried the CYP2D6_*10, consequently decreasing the enzyme activity, resulting in a PM phenotype. 2.30% carried the CYP2D6_*29, leading to decreased metabolic activity of the enzyme, and 2.30% carried the CYP2D6_*35, resulting in an UM phenotype, 1.64% had a whole-gene deletion CYP2D6_*5, thus resulting in the loss of CYP2D6 enzyme production, 0.66% carried the CYP2D6_*6 variant. One individual carried the CYP2D6_*3(B), producing an inactive form of the enzyme, which leads to decrease of enzyme activity, resulting in a PM phenotype. Finally, one individual carried the CYP2D6_*9, which decreases the enzyme activity. Conclusions: Our study demonstrates that different CYP2D6 variants are highly prevalent in ethnic Saudi Arabs. This finding sets a basis for informed genotyping for these variants in personalized medicine. The study also suggests that xTAG is an appropriate procedure for genotyping the CYP2D6 variants in personalized medicine.

Keywords: CYP2D6, hormonal breast cancer, pharmacogenetics, polymorphism, psychiatric treatment, Saudi population

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Authors: Minu Bharati, Priyanka Rai, Satarupa Dutta, Dhiraj Saha

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Culex quinquefasciatus Say, is a mosquito of immense public health concern due to its role in transmission of filariasis, which is an endemic disease in 20 states and union territories of India, putting about 600 million people at the risk of infection. The main strategies to control filaria in India include anti-larval measures in urban areas, Indoor Residual Spray (IRS) in rural areas and mass diethylcarbamazine citrate (DEC) administration. Larval destruction measures and IRS are done with the use of insecticides. In this study, Susceptibility/ Resistance to insecticides were assessed in Culex quinquefasciatus mosquitoes collected from eight densely populated areas of Siliguri subdivision, which has a high rate of filarial infection. To unveil the insecticide susceptibility status of Culex quinquefasciatus, bioassays were performed on field-caught mosquitoes against two major groups of insecticides, i.e. Synthetic Pyrethroids (SPs): 0.05% deltamethrin and 0.05% lambda-cyhalothrin and Organophosphates (OPs): 5% malathion and temephos using World Health Organisation (WHO) discriminating doses. The knockdown rates and knockdown times (KDT50) were also noted against deltamethrin, lambda-cyhalothrin and malathion. Also, activities of major detoxifying enzymes, i.e. α-carboxylesterases, β-carboxylesterases and cytochrome P450 (CYP450) monooxygenases were determined to find the involvement of biochemical mechanisms in resistance phenomenon (if any). The results obtained showed that, majority of the mosquito populations were moderately to severely resistant against both the SPs and one OP, i.e. temephos. Whereas, most of the populations showed 100% susceptibility to malathion. The knockdown rates and KDT50 in response to above-mentioned insecticides showed significant variation among different populations. Variability in activities of carboxylesterases and CYP450 monooxygenases were also observed with hints of their involvement in contribution towards insecticide resistance in some of the tested populations. It may be concluded that, Culex quinquefasciatus has started developing resistance against deltamethrin, lambda-cyhalothrin and temephos in Siliguri subdivision. Malathion seems to hold the greatest potentiality for control of these mosquitoes in this area as revealed through this study. Adoption of Integrated mosquito management (IMM) strategy should be the prime objective of the concerned authorities to delimit the insecticide resistance phenomenon and filariasis infections.

Keywords: Culex quinquefasciatus, detoxifying enzymes, insecticide resistance, knockdown rate

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Authors: Umara Nissar Rafiqi, Irum Gul, Nazima Nasrullah, Monica Saifi, Malik Z. Abdin

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Background: Stevia rebaudiana, a member of Asteraceae family is an important medicinal plant and produces a commercially used non-caloric natural sweetener, which is also an alternate herbal cure for diabetes. Steviol glycosides are the main sweetening compounds present in these plants. Secondary metabolites are crucial to the adaption of plants to the environment and its overcoming stress conditions. In agricultural procedures, the abiotic stresses like salinity, high metal toxicity and drought, in particular, are responsible for the majority of the reduction that differentiates yield potential from harvestable yield. Salt stress and heavy metal toxicity lead to increased production of reactive oxygen species (ROS). To avoid oxidative damage due to ROS and osmotic stress, plants have a system of anti-oxidant enzymes along with several stress induced enzymes. This helps in scavenging the ROS and relieve the osmotic stress in different cell compartments. However, whether stress induced toxicity modulates the activity of these enzymes in Stevia rebaudiana is poorly understood. Aim: The present study focussed on the effect of salinity, heavy metal toxicity (lead and mercury) on physiological traits and transcriptional profiling of Stevia rebaudiana. Method: Stevia rebaudiana plants were collected from the Central Institute of Medicinal and Aromatic plants (CIMAP), Patnagar, India and maintained under controlled conditions in a greenhouse at Hamdard University, Delhi, India. The plants were subjected to different concentrations of salt (0, 25, 50 and 75 mM respectively) and heavy metals, lead and mercury (0, 100, 200 and 300 µM respectively). The physiological traits such as shoot length, root numbers, leaf growth were evaluated. The samples were collected at different developmental stages and analysed for transcription profiling by RT-PCR. Transcriptional studies in stevia rebaudiana involves important antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), cytochrome P450 monooxygenase (CYP) and stress induced aquaporin (AQU), auxin repressed protein (ARP-1), Ndhc gene. The data was analysed using GraphPad Prism and expressed as mean ± SD. Result: Low salinity and lower metal toxicity did not affect the fresh weight of the plant. However, this was substantially decreased by 55% at high salinity and heavy metal treatment. With increasing salinity and heavy metal toxicity, the values of all studied physiological traits were significantly decreased. Chlorosis in treated plants was also observed which could be due to changes in Fe:Zn ratio. At low concentrations (upto 25 mM) of NaCl and heavy metals, we did not observe any significant difference in the gene expressions of treated plants compared to control plants. Interestingly, at high salt concentration and high metal toxicity, a significant increase in the expression profile of stress induced genes was observed in treated plants compared to control (p < 0.005). Conclusion: Stevia rebaudiana is tolerant to lower salt and heavy metal concentration. This study also suggests that with the increase in concentrations of salt and heavy metals, harvest yield of S. rebaudiana was hampered.

Keywords: Stevia rebaudiana, natural sweetener, salinity, heavy metal toxicity

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Authors: Marina Passero, Tianhua Zhai, Zuyi (Jacky) Huang

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Alzheimer’s disease has become a major public health issue, as indicated by the increasing populations of Americans living with Alzheimer’s disease. After decades of extensive research in Alzheimer’s disease, only seven drugs have been approved by Food and Drug Administration (FDA) to treat Alzheimer’s disease. Five of these drugs were designed to treat the dementia symptoms, and only two drugs (i.e., Aducanumab and Lecanemab) target the progression of Alzheimer’s disease, especially the accumulation of amyloid-b plaques. However, controversial comments were raised for the accelerated approvals of either Aducanumab or Lecanemab, especially with concerns on safety and side effects of these two drugs. There is still an urgent need for further drug discovery to target the biological processes involved in the progression of Alzheimer’s disease. Excessive cholesterol has been found to accumulate in the brain of those with Alzheimer’s disease. Cholesterol can be synthesized in both the blood and the brain, but the majority of biosynthesis in the adult brain takes place in astrocytes and is then transported to the neurons via ApoE. The blood brain barrier separates cholesterol metabolism in the brain from the rest of the body. Various proteins contribute to the metabolism of cholesterol in the brain, which offer potential targets for Alzheimer’s treatment. In the astrocytes, SREBP cleavage-activating protein (SCAP) binds to Sterol Regulatory Element-binding Protein 2 (SREBP2) in order to transport the complex from the endoplasmic reticulum to the Golgi apparatus. Cholesterol is secreted out of the astrocytes by ATP-Binding Cassette A1 (ABCA1) transporter. Lipoprotein receptors such as triggering receptor expressed on myeloid cells 2 (TREM2) internalize cholesterol into the microglia, while lipoprotein receptors such as Low-density lipoprotein receptor-related protein 1 (LRP1) internalize cholesterol into the neuron. Cytochrome P450 Family 46 Subfamily A Member 1 (CYP46A1) converts excess cholesterol to 24S-hydroxycholesterol (24S-OHC). Cholesterol has been approved for its direct effect on the production of amyloid-beta and tau proteins. The addition of cholesterol to the brain promotes the activity of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), secretase, and amyloid precursor protein (APP), which all aid in amyloid-beta production. The reduction of cholesterol esters in the brain have been found to reduce phosphorylated tau levels in mice. In this work, a computational pipeline was developed to identify the protein targets involved in cholesterol regulation in brain and further to identify chemical compounds as the inhibitors of a selected protein target. Since extensive evidence shows the strong correlation between brain cholesterol regulation and Alzheimer’s disease, a detailed literature review on genes or pathways related to the brain cholesterol synthesis and regulation was first conducted in this work. An interaction network was then built for those genes so that the top gene targets were identified. The involvement of these genes in Alzheimer’s disease progression was discussed, which was followed by the investigation of existing clinical trials for those targets. A ligand-protein docking program was finally developed to screen 1.5 million chemical compounds for the selected protein target. A machine learning program was developed to evaluate and predict the binding interaction between chemical compounds and the protein target. The results from this work pave the way for further drug discovery to regulate brain cholesterol to combat Alzheimer’s disease.

Keywords: Alzheimer’s disease, drug discovery, ligand-protein docking, gene-network analysis, cholesterol regulation

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