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An update of aurones: food resource, health benefit, biosynthesis and application

Yang J, Gao Z, Yu Z, Hou Y, Tang D, Yan H, et al.

Crit Rev Food Sci Nutr, 2023 Aug 21.
PMID: 37599623 DOI: 10.1080/10408398.2023.2248244

Aurones are a subclass of active flavonoids characterized with a scaffold of 2-benzylidene-3(2H)-benzofuranone. This type of chemicals are widely distributed in fruit, vegetable and flower, and contribute to human health. In this review, we summarize the natural aurones isolated from dietary plants. Their positive effects on immunomodulation, antioxidation, cancer prevention as well as maintaining the health status of cardiovascular, nervous system and liver organs are highlighted. The biosynthesis strategies of plant-derived aurones are elaborated to provide solutions for their limited natural abundance. The potential application of natural aurones in food coloration are also discussed. This paper combines the up-to-date information and gives a full image of dietary aurones.
Ancient DNA reveals genetic admixture in China during tiger evolution

Sun X, Liu YC, Tiunov MP, Gimranov DO, Zhuang Y, Han Y, et al.

Nat Ecol Evol, 2023 Nov;7(11):1914-1929.
PMID: 37652999 DOI: 10.1038/s41559-023-02185-8

The tiger (Panthera tigris) is a charismatic megafauna species that originated and diversified in Asia and probably experienced population contraction and expansion during the Pleistocene, resulting in low genetic diversity of modern tigers. However, little is known about patterns of genomic diversity in ancient populations. Here we generated whole-genome sequences from ancient or historical (100-10,000 yr old) specimens collected across mainland Asia, including a 10,600-yr-old Russian Far East specimen (RUSA21, 8× coverage) plus six ancient mitogenomes, 14 South China tigers (0.1-12×) and three Caspian tigers (4-8×). Admixture analysis showed that RUSA21 clustered within modern Northeast Asian phylogroups and partially derived from an extinct Late Pleistocene lineage. While some of the 8,000-10,000-yr-old Russian Far East mitogenomes are basal to all tigers, one 2,000-yr-old specimen resembles present Amur tigers. Phylogenomic analyses suggested that the Caspian tiger probably dispersed from an ancestral Northeast Asian population and experienced gene flow from southern Bengal tigers. Lastly, genome-wide monophyly supported the South China tiger as a distinct subspecies, albeit with mitochondrial paraphyly, hence resolving its longstanding taxonomic controversy. The distribution of mitochondrial haplogroups corroborated by biogeographical modelling suggested that Southwest China was a Late Pleistocene refugium for a relic basal lineage. As suitable habitat returned, admixture between divergent lineages of South China tigers took place in Eastern China, promoting the evolution of other northern subspecies. Altogether, our analysis of ancient genomes sheds light on the evolutionary history of tigers and supports the existence of nine modern subspecies.
Assessment of the level of knowledge of colorectal cancer among public at outpatient clinics in Serdang Hospital: a survey based study

Pan Y, Chieng CY, Haris AAH, Ang SY

Med J Malaysia, 2017 12;72(6):338-344.
PMID: 29308770 MyJurnal

OBJECTIVES: Colorectal cancer (CRC) is one of the most common gastrointestinal cancers in the world. In the Asia- Pacific region, it is the fastest emerging gastrointestinal cancer. Level of awareness on CRC warning signs and risk factors in the rural population of Malaysia is reported of very low. The aim of this study was to assess the level of knowledge of CRC among the public at medical outpatient clinics in Serdang Hospital. The association between sociodemographic factors with level of knowledge among the respondents was further studied.
STUDY DESIGN: A study was conducted among the non-CRC patients' relatives accompanying their relatives to the medical outpatient clinics in Serdang Hospital from 1st April to 31st August 2016. The study was carried out with cluster sampling method.
METHODS: The respondents were assessed using validated and modified Cancer Awareness Measures (CAM) questionnaire consists of three parts which are knowledge on warning signs, knowledge on risk factors and sociodemographic factors. All data were analysed using IBM SPSS Statistics 21.0.
RESULTS: Altogether 308 subjects completed the questionnaires. It was shown high percentage of good knowledge for warning signs and risk factors of CRC among the respondents. A significant association between age groups and level of income with level of knowledge on warning signs was observed.
CONCLUSIONS: The level of knowledge of CRC among the general public in Serdang Hospital was sufficient. The respondents with higher income or younger age had higher level of knowledge regarding CRC.
Fulltext Author Correction: Native diversity buffers against severity of non-native tree invasions

Delavaux CS, Crowther TW, Zohner CM, Robmann NM, Lauber T, van den Hoogen J, et al.

Nature, 2023 Oct;622(7982):E2.
PMID: 37752352 DOI: 10.1038/s41586-023-06654-9
CYP35 family in Caenorhabditis elegans biological processes: fatty acid synthesis, xenobiotic metabolism, and stress responses

Lim SYM, Alshagga M, Kong C, Alshawsh MA, Alshehade SA, Pan Y

Arch Toxicol, 2022 12;96(12):3163-3174.
PMID: 36175686 DOI: 10.1007/s00204-022-03382-3

With more than 80 cytochrome P450 (CYP) encoding genes found in the nematode Caenorhabditis elegans (C. elegans), the cyp35 genes are one of the important genes involved in many biological processes such as fatty acid synthesis and storage, xenobiotic stress response, dauer and eggshell formation, and xenobiotic metabolism. The C. elegans CYP35 subfamily consisted of A, B, C, and D, which have the closest homolog to human CYP2 family. C. elegans homologs could answer part of the hunt for human disease genes. This review aims to provide an overview of CYP35 in C. elegans and their human homologs, to explore the roles of CYP35 in various C. elegans biological processes, and how the genes of cyp35 upregulation or downregulation are influenced by biological processes, upon exposure to xenobiotics or changes in diet and environment. The C. elegans CYP35 gene expression could be upregulated by heavy metals, pesticides, anti-parasitic and anti-chemotherapeutic agents, polycyclic aromatic hydrocarbons (PAHs), nanoparticles, drugs, and organic chemical compounds. Among the cyp35 genes, cyp-35A2 is involved in most of the C. elegans biological processes regulation. Further venture of cyp35 genes, the closest homolog of CYP2 which is the largest family of human CYPs, may have the power to locate cyps gene targets, discovery of novel therapeutic strategies, and possibly a successful medical regime to combat obesity, cancers, and cyps gene-related diseases.
Chemotherapy impairs ovarian function through excessive ROS-induced ferroptosis

Zhang S, Liu Q, Chang M, Pan Y, Yahaya BH, Liu Y, et al.

Cell Death Dis, 2023 May 24;14(5):340.
PMID: 37225709 DOI: 10.1038/s41419-023-05859-0

Chemotherapy was conventionally applied to kill cancer cells, but regrettably, they also induce damage to normal cells with high-proliferative capacity resulting in cardiotoxicity, nephrotoxicity, peripheral nerve toxicity, and ovarian toxicity. Of these, chemotherapy-induced ovarian damages mainly include but are not limited to decreased ovarian reserve, infertility, and ovarian atrophy. Therefore, exploring the underlying mechanism of chemotherapeutic drug-induced ovarian damage will pave the way to develop fertility-protective adjuvants for female patients during conventional cancer treatment. Herein, we firstly confirmed the abnormal gonadal hormone levels in patients who received chemotherapy and further found that conventional chemotherapeutic drugs (cyclophosphamide, CTX; paclitaxel, Tax; doxorubicin, Dox and cisplatin, Cis) treatment significantly decreased both the ovarian volume of mice and the number of primordial and antral follicles and accompanied with the ovarian fibrosis and reduced ovarian reserve in animal models. Subsequently, Tax, Dox, and Cis treatment can induce the apoptosis of ovarian granulosa cells (GCs), likely resulting from excessive reactive oxygen species (ROS) production-induced oxidative damage and impaired cellular anti-oxidative capacity. Thirdly, the following experiments demonstrated that Cis treatment could induce mitochondrial dysfunction through overproducing superoxide in GCs and trigger lipid peroxidation leading to ferroptosis, first reported in chemotherapy-induced ovarian damage. In addition, N-acetylcysteine (NAC) treatment could alleviate the Cis-induced toxicity in GCs by downregulating cellular ROS levels and enhancing the anti-oxidative capacity (promoting the expression of glutathione peroxidase, GPX4; nuclear factor erythroid 2-related factor 2, Nrf2 and heme oxygenase-1, HO-1). Our study confirmed the chemotherapy-induced chaotic hormonal state and ovarian damage in preclinical and clinical examination and indicated that chemotherapeutic drugs initiated ferroptosis in ovarian cells through excessive ROS-induced lipid peroxidation and mitochondrial dysfunction, leading to ovarian cell death. Consequently, developing fertility protectants from the chemotherapy-induced oxidative stress and ferroptosis perspective will ameliorate ovarian damage and further improve the life quality of cancer patients.
Cis-9, Trans-11 Conjugated Linoleic Acid Reduces Phosphoenolpyruvate Carboxykinase Expression and Hepatic Glucose Production in HepG2 Cells

Chai BK, Al-Shagga M, Pan Y, Then SM, Ting KN, Loh HS, et al.

Lipids, 2019 06;54(6-7):369-379.
PMID: 31124166 DOI: 10.1002/lipd.12154

Dysregulated hepatic gluconeogenesis is a hallmark of insulin resistance and type 2 diabetes mellitus (T2DM). Although existing drugs have been proven to improve gluconeogenesis, achieving this objective with functional food is of interest, especially using conjugated linoleic acid (CLA) found in dairy products. Both cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12) isomers of CLA were tested in human (HepG2) and rat (H4IIE) hepatocytes for their potential effects on gluconeogenesis. The hepatocytes exposed for 24 h with 20 μM of c9,t11-CLA had attenuated the gluconeogenesis in both HepG2 and H4IIE by 62.5% and 80.1%, respectively. In contrast, t10,c12-CLA had no effect. Of note, in HepG2 cells, the exposure of c9,t11-CLA decreased the transcription of gluconeogenic enzymes, cytosolic phosphoenolpyruvate carboxykinase (PCK1) by 87.7%, and glucose-6-phosphatase catalytic subunit (G6PC) by 38.0%, while t10,c12-CLA increased the expression of G6PC, suggesting the isomer-specific effects of CLA on hepatic glucose production. In HepG2, the peroxisome proliferator-activated receptor (PPAR) agonist, rosiglitazone, reduced the glucose production by 72.9%. However, co-administration of c9,t11-CLA and rosiglitazone neither exacerbated nor attenuated the efficacy of rosiglitazone to inhibit glucose production; meanwhile, t10,c12-CLA abrogated the efficacy of rosiglitazone. Paradoxically, PPARγ antagonist GW 9662 also led to 70.2% reduction of glucose production and near undetectable PCK1 expression by abrogating CLA actions. Together, while the precise mechanisms by which CLA isomers modulate hepatic gluconeogenesis directly or via PPAR warrant further investigation, our findings establish that c9,t11-CLA suppresses gluconeogenesis by decreasing PEPCK on hepatocytes.
Fulltext Colorectal cancer screening using immunochemical fecal occult blood test

Tan MC, Ng OC, Yap RYP, Pan Y, Chieng JY

Malaysian Journal of Public Health Medicine, 2017;17(1):33-37.

Fecal occult blood test (FOBT) screening has been shown to decrease the incidence and mortality of colorectal cancer (CRC). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the immunochemical fecal occult blood test (i-FOBT) in diagnosing CRC were assessed among the patients in a tertiary referral hospital in Malaysia. A total sample of 814 patients aged 16 to 85 years old who performed i-FOBT and endoscopic screenings was obtained. The patients were recruited for a retrospective investigation. Sensitivity, specificity, PPV, and NPV were derived for the CRC screenees. Out of the 814 patients screened using i-FOBT, half of them were above 59 years old (49.6%), and 36% had positive i-FOBT. Gender distribution was almost equal, where 53.4% of the patients were female, and 46.6% were male. Majority of the patients were Malays (56.6%), followed by Chinese (24.0%), Indians (16.5%), and others (2.9%). Among the 71 patients referred for colonoscopy, 57.7% and 42.3% corresponded to positive and negative i-FOBT cases, respectively. Polyps were found to be most common among the patients (25.6%), 7.0% were found positive for invasive CRC, and 35.2% had normal colonoscopic findings. There was a significant association between colonoscopic finding and positive i-FOBT (p=0.001). The sensitivity, specificity, PPV, and NPV for CRC detection were 66.7%, 43.0%, 9.8%, and 93.3%, respectively. The results indicate that i-FOBT is a useful tool in the detection of abnormalities in the lower gastrointestinal tract and therefore serves as a cornerstone for potential large-scale screening programmes.
Cytochrome P450 2C9-natural antiarthritic interactions: Evaluation of inhibition magnitude and prediction from in vitro data

Tan BH, Ahemad N, Pan Y, Palanisamy UD, Othman I, Yiap BC, et al.

Biopharm Drug Dispos, 2018 Apr;39(4):205-217.
PMID: 29488228 DOI: 10.1002/bdd.2127

Many dietary supplements are promoted to patients with osteoarthritis (OA) including the three naturally derived compounds, glucosamine, chondroitin and diacerein. Despite their wide spread use, research on interaction of these antiarthritic compounds with human hepatic cytochrome P450 (CYP) enzymes is limited. This study aimed to examine the modulatory effects of these compounds on CYP2C9, a major CYP isoform, using in vitro biochemical assay and in silico models. Utilizing valsartan hydroxylase assay as probe, all forms of glucosamine and chondroitin exhibited IC50 values beyond 1000 μM, indicating very weak potential in inhibiting CYP2C9. In silico docking postulated no interaction with CYP2C9 for chondroitin and weak bonding for glucosamine. On the other hand, diacerein exhibited mixed-type inhibition with IC50 value of 32.23 μM and Ki value of 30.80 μM, indicating moderately weak inhibition. Diacerein's main metabolite, rhein, demonstrated the same mode of inhibition as diacerein but stronger potency, with IC50 of 6.08 μM and Ki of 1.16 μM. The docking of both compounds acquired lower CDOCKER interaction energy values, with interactions dominated by hydrogen and hydrophobic bondings. The ranking with respect to inhibition potency for the investigated compounds was generally the same in both in vitro enzyme assay and in silico modeling with order of potency being diacerein/rhein > various glucosamine/chondroitin forms. In vitro-in vivo extrapolation of inhibition kinetics (using 1 + [I]/Ki ratio) demonstrated negligible potential of diacerein to cause interaction in vivo, whereas rhein was predicted to cause in vivo interaction, suggesting potential interaction risk with the CYP2C9 drug substrates.
Cytochrome P450 2W1 (CYP2W1) - ready for use as the biomarker and drug target for cancer?

Pan Y, Ong EC

Xenobiotica, 2017 Oct;47(10):923-932.
PMID: 27690753 DOI: 10.1080/00498254.2016.1244370

1. This article aims to evaluate the potentials of using cytochrome P450 2W1 (CYP2W1) as a biomarker and a drug target of cancer because of its characteristic cancer-specific expression. 2. Discrepant findings comparing the expression levels of CYP2W1 in cancer and non-cancer samples were reported. In general, the expression followed a developmental pattern. The demethylation status of CpG island and the expression levels of CYP2W1 genes was positively correlated. 3. CYP2W1 was able to activate several procarcinogens, anticancer pro-drugs and to metabolise many endogenous substances including fatty acids and lysophospholipids. 4. CYP2W1 expression level was suggested to serve as an independent prognostic biomarker in colorectal cancer and hepatocellular carcinoma. The correlation of genetic polymorphisms of CYP2W1 and cancer risk was uncertain. 5. Further characterisation of CYP2W1 structure is suggested to link to its functions. More studies are warranted to reveal the true status and the regulation of CYP2W1 expression across normal and cancer tissues. Catalytic activity of CYP2W1 should be tested on a wider spectrum of endogenous and exogenous substances before its use as the drug target. Larger size of clinical samples can be included to verify the potential of CYP2W1 as the prognostic or cancer risk biomarker.
Cytochrome P450 4B1 (CYP4B1) as a target in cancer treatment

Lim S, Alshagga M, Ong CE, Chieng JY, Pan Y

Hum Exp Toxicol, 2020 Jun;39(6):785-796.
PMID: 32054340 DOI: 10.1177/0960327120905959

Cytochrome P450 4B1 (CYP4B1) plays crucial roles in biotransforming of xenobiotics. Its predominant extrahepatic expression has been associated with certain tissue-specific toxicities. However, the expressions of CYP4B1 in various cancers and hence their potential roles in cancer development were inclusive. In this work, existing knowledge on expression and regulation of CYP4B1 gene and protein, catalysis of CYP4B1, association of CYP4B1 with cancers, contradicting findings about human CYP4B1 activities as well as the employing CYP4B1 in suicide gene approach for cancer treatment were reviewed. To date, it appears that there is a wide spectrum of tissue distribution of CYP4B1 with lungs as the predominant sites. Several nuclear receptors are possibly responsible for regulating its gene expression. The involvement of CYP4B1 in cancer was considered via activation of procarcinogens and neovascularization. However, human CYP4B1 was found to be inactive due to a substitution of proline with serine at position 427. Suicide gene approach combining reengineered CYP4B1 and prodrug 4-ipomeanol (4-IPO) has shown a promising potential for targeted cancer therapy. Further studies should focus on the verification of human CYP4B1 catalytic activities. More compounds with similar structure as 4-IPO should be tested to identify more alternative agents for the suicide gene approach in cancer treatment.
Cytochrome P450 genotype-guided drug therapies: An update on current states

Dong AN, Tan BH, Pan Y, Ong CE

Clin Exp Pharmacol Physiol, 2018 10;45(10):991-1001.
PMID: 29858511 DOI: 10.1111/1440-1681.12978

Over the past 2 decades, knowledge of the role and clinical value of pharmacogenetic markers has expanded so that individualized pre-emptive therapy based on genetic background of patients could be within reach for clinical implementation. This is evidenced from the frequent updating of drug labels that incorporates pharmacogenetic information (where compelling data become available) by the regulatory agencies (such as the US FDA), and the periodical publication of guidelines of specific therapeutic recommendations based on the results of pharmacogenetic tests by the pharmacogenetics working groups or consortiums of professional bodies. Clinical relevance of the cytochrome P450 (CYP) polymorphism related to dose, effectiveness and/or toxicity of key drugs are presented in this review, including that of warfarin, clopidogrel, tricyclic antidepressants, and proton pump inhibitors. Prospect for routine clinical application of CYP genotyping before prescribing drugs is still currently unclear due to challenges and barriers associated with availability of well-defined and validated pharmacogenetic studies, the interpretation, result reporting and potential error of genotype testing, involvement of non-genetic factors, and other patient's demographic and disease conditions. Further studies to provide additional supporting clinical data and acceleration of pharmacogenetic testing standards and techniques should help improve the evidence base needed for clinical utility and hence move the implementation of genotype-guided therapy in clinical practice a step closer to reality.
Effect of 95% Ethanol Khat Extract and Cathinone on in vitro Human Recombinant Cytochrome P450 (CYP) 2C9, CYP2D6, and CYP3A4 Activity

Lim SYM, Binti Azidin AR, Ung YT, Al-Shagga M, Alshawsh MA, Mohamed Z, et al.

Eur J Drug Metab Pharmacokinet, 2019 Jun;44(3):423-431.
PMID: 30306496 DOI: 10.1007/s13318-018-0518-2

BACKGROUND AND OBJECTIVE: A significant number of people worldwide consume khat on daily basis. Long term of khat chewing has shown negative impact on several organ systems. It is likely that these people are co-administered khat preparations and conventional medication, which may lead to khat-drug interactions. This study aimed to reveal the inhibitory potencies of khat ethanol extract (KEE) and its major active ingredient (cathinone) on human cytochrome P450 (CYP) 2C9, CYP2D6, and CYP3A4 enzymes activities, which are collectively responsible for metabolizing 70-80% clinically used drugs.
METHODS: In vitro fluorescence-based enzyme assays were developed and the CYP enzyme activities were quantified in the presence and absence of KEE and cathinone employing Vivid® CYP450 Screening Kits.
RESULTS: KEE inhibited human CYP2C9, CYP2D6, and CYP3A4 enzyme activities with IC50 of 42, 62, and 18 μg/ml. On the other hand, cathinone showed negligible inhibitory effect on these CYPs. Further experiments with KEE revealed that KEE inhibited CYP2C9 via non-competitive or mixed mode with Ki of 14.7 μg/ml, CYP2D6 through competitive or mixed mode with Ki of 17.6 μg/ml, CYP3A4 by mixed inhibition mode with Ki of 12.1 μg/ml.
CONCLUSION: Khat-drug interactions are possible due to administration of clinical drugs metabolized by CYP2C9/CYP2D6/CYP3A4 together with khat chewing. Further in vivo studies are required to confirm our findings and identify the causative constituents of these inhibitory effects.
Effect of eurycomanone on cytochrome P450 isoforms CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 in vitro

Pan Y, Tiong KH, Abd-Rashid BA, Ismail Z, Ismail R, Mak JW, et al.

J Nat Med, 2014 Apr;68(2):402-6.
PMID: 23881640 DOI: 10.1007/s11418-013-0794-8

Eurycomanone, an active constituent isolated from Eurycoma longifolia Jack, was examined for modulatory effects on cytochrome P450 (CYP) isoforms CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 using in vitro assays. The IC50 value was determined to assess the potencies of modulation for each CYP isoform. Our results indicated that eurycomanone did not potently inhibit any of the CYP isoforms investigated, with IC50 values greater than 250 μg/ml. Hence there appears to be little likelihood of drug-herb interaction between eurycomanone or herbal products with high content of this compound and CYP drug substrates via CYP inhibition.
Functional and structural characterisation of common cytochrome P450 2D6 allelic variants-roles of Pro34 and Thr107 in catalysis and inhibition

Dong AN, Ahemad N, Pan Y, Palanisamy UD, Yiap BC, Ong CE

Naunyn Schmiedebergs Arch Pharmacol, 2019 08;392(8):1015-1029.
PMID: 31025144 DOI: 10.1007/s00210-019-01651-0

One major source of inter-individual variability in drug pharmacokinetics is genetic polymorphism of the cytochrome P450 (CYP) genes. This study aimed to elucidate the enzyme kinetic and molecular basis for altered activity in three major alleles of CYP2D6, namely CYP2D6*2, CYP2D6*10 and CYP2D6*17. The E. coli-expressed allelic variants were examined using substrate (venlafaxine and 3-cyano-7-ethoxycoumarin[CEC]) and inhibitor (quinidine, fluoxetine, paroxetine, terbinafine) probes in enzyme assays as well as molecular docking. The kinetics data indicated that R296C and S486T mutations in CYP2D6*2 have caused enhanced ligand binding (enhanced intrinsic clearance for venlafaxine and reduced IC50 for quinidine, paroxetine and terbinafine), suggesting morphological changes within the active site cavity that favoured ligand docking and binding. Mutations in CYP2D6*10 and CYP2D6*17 tended to cause deleterious effect on catalysis, with reduced clearance for venlafaxine and CEC. Molecular docking indicated that P34S and T107I, the unique mutations in the alleles, have negatively impacted activity by affecting ligand access and binding due to alteration of the substrate access channel and active site morphology. IC50 values however were quite variable for quinidine, fluoxetine and terbinafine, and a general decrease in IC50 was observed for paroxetine, suggesting ligand-specific altered susceptibility to inhibition in the alleles. This study indicates that CYP2D6 allele selectivity for ligands was not solely governed by changes in the active site architecture induced by the mutations, but that the intrinsic properties of the substrates and inhibitors also played vital role.
Functional role of Ile264 in CYP2C8: mutations affect haem incorporation and catalytic activity

Singh R, Ting JG, Pan Y, Teh LK, Ismail R, Ong CE

Drug Metab. Pharmacokinet., 2008;23(3):165-74.
PMID: 18574320

The work described in this study aimed to express CYP2C8 wild-type and mutant proteins in bacterial expression system and to use the expressed proteins to investigate the structural and functional consequences of a reported allele CYP2C8(*)4 (carrying Ile264Met substitution) on protein activity. Ile264 was replaced by three different amino acids resulting in three mutant constructs, 2C8I264M, 2C8I264R and 2C8I264D. The presence of isoleucine at position 264 in CYP2C8 was found to be important for proper haem insertion and protein folding; whereas bulkier or charged residues were highly disruptive resulting in inactive proteins with minimum spectral and catalytic activities. This was evidenced from the low levels of Soret peak at 450 nm and negligible levels of tolbutamide methylhydroxylase activity. Kinetic study using paclitaxel indicated that all three mutants exhibited only 9.7 to 35.4% of the activity level observed in the wild-type. In addition, the mutants were more sensitive to proteinase K digestion, indicating a possible alteration of conformation. The combined effects of protein instability and compromised catalytic activity resulted in defective CYP2C8 protein which may have clinical implications in carriers of CYP2C8*4, particularly in terms of their capacity to clear potent drugs and their susceptibility to adverse drug reactions.
Fulltext Genetic Connections and Convergent Evolution of Tropical Indigenous Peoples in Asia

Deng L, Pan Y, Wang Y, Chen H, Yuan K, Chen S, et al.

Mol Biol Evol, 2022 Feb 03;39(2).
PMID: 34940850 DOI: 10.1093/molbev/msab361

Tropical indigenous peoples in Asia (TIA) attract much attention for their unique appearance, whereas their genetic history and adaptive evolution remain mysteries. We conducted a comprehensive study to characterize the genetic distinction and connection of broad geographical TIAs. Despite the diverse genetic makeup and large interarea genetic differentiation between the TIA groups, we identified a basal Asian ancestry (bASN) specifically shared by these populations. The bASN ancestry was relatively enriched in ancient Asian human genomes dated as early as ∼50,000 years before the present and diminished in more recent history. Notably, the bASN ancestry is unlikely to be derived from archaic hominins. Instead, we suggest it may be better modeled as a survived lineage of the initial peopling of Asia. Shared adaptations inherited from the ancient Asian ancestry were detected among the TIA groups (e.g., LIMS1 for hair morphology, and COL24A1 for bone formation), and they are enriched in neurological functions either at an identical locus (e.g., NKAIN3), or different loci in an identical gene (e.g., TENM4). The bASN ancestry could also have formed the substrate of the genetic architecture of the dark pigmentation observed in the TIA peoples. We hypothesize that phenotypic convergence of the dark pigmentation in TIAs could have resulted from parallel (e.g., DDB1/DAK) or genetic convergence driven by admixture (e.g., MTHFD1 and RAD18), new mutations (e.g., STK11), or notably purifying selection (e.g., MC1R). Our results provide new insights into the initial peopling of Asia and an advanced understanding of the phenotypic convergence of the TIA peoples.
Heterologous expression of human cytochrome P450 (CYP) 2C19 in Escherichia coli and establishment of RP-HPLC method to serve as activity marker

Pan Y, Mak JW, Ong CE

Biomed Chromatogr, 2013 Jul;27(7):859-65.
PMID: 23386533 DOI: 10.1002/bmc.2872

In this study, a simple and reliable reverse-phase high-performance liquid chromatography (RP-HPLC) method was established and validated to analyze S-mephenytoin 4-hydroxylase activity of a recombinant CYP2C19 system. This system was obtained by co-expressing CYP2C19 and NADPH-CYP oxidoreductase (OxR) proteins in Escherichia coli (E. coli) cells. In addition to RP-HPLC, the expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. The RP-HPLC assay showed good linearity (r(2) = 1.00) with 4-hydroxymephenytoin concentration from 0.100 to 50.0 μm and the limit of detection was 5.00 × 10(-2) μm. Intraday and interday precisions determined were from 1.90 to 8.19% and from 2.20 to 14.9%, respectively. Recovery and accuracy of the assay were from 83.5 to 85.8% and from 95.0 to 105%. Enzyme kinetic parameters (Km , Vmax and Ki ) were comparable to reported values. The presence of CYP2C19 in bacterial membranes was confirmed by immunoblotting and the characteristic absorbance peak at 450 nm was determined in the reduced CO difference spectral assay. Moreover, the activity level of co-expressed OxR was found to be comparable to that of the literature. As a conclusion, the procedures described here have generated catalytically active CYP2C19 and the RP-HPLC assay developed is able to serve as CYP2C19 activity marker for pharmacokinetic drug interaction study in vitro.
Heterologous expression of human cytochromes P450 2D6 and CYP3A4 in Escherichia coli and their functional characterization

Pan Y, Abd-Rashid BA, Ismail Z, Ismail R, Mak JW, Ong CE

Protein J, 2011 Dec;30(8):581-91.
PMID: 22001938 DOI: 10.1007/s10930-011-9365-6

This study aimed to express two major drug-metabolizing human hepatic cytochromes P450 (CYPs), CYP2D6 and CYP3A4, together with NADPH-cytochrome P450 oxidoreductase (OxR) in Escherichia coli and to evaluate their catalytic activities. Full length cDNA clones of both isoforms in which the N-terminus was modified to incorporate bovine CYP17α sequence were inserted into a pCWori(+) vector. The modified CYP cDNAs were subsequently expressed individually, each together with OxR by means of separate, compatible plasmids with different antibiotic selection markers. The expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. Enzyme activities were examined using high performance liquid chromatography (HPLC) assays with probe substrates dextromethorphan and testosterone for CYP2D6 and CYP3A4, respectively. Results from immunoblotting demonstrated the presence of both CYP proteins in bacterial membranes and reduced CO difference spectra of the cell preparations exhibited the characteristic absorbance peak at 450 nm. Co-expressed OxR also demonstrated an activity level comparable to literature values. Kinetic parameters, K(m) and V(max) values determined from the HPLC assays also agreed well with literature values. As a conclusion, the procedures described in this study provide a relatively convenient and reliable means of producing catalytically active CYP isoforms suitable for drug metabolism and interaction studies.
In Vitro Functional Characterisation of Cytochrome P450 (CYP) 2C19 Allelic Variants CYP2C19*23 and CYP2C19*24

Lau PS, Leong KV, Ong CE, Dong AN, Pan Y

Biochem Genet, 2017 Feb;55(1):48-62.
PMID: 27578295 DOI: 10.1007/s10528-016-9771-8

Cytochrome P450 (CYP) 2C19 is essential for the metabolism of clinically used drugs including omeprazole, proguanil, and S-mephenytoin. This hepatic enzyme exhibits genetic polymorphism with inter-individual variability in catalytic activity. This study aimed to characterise the functional consequences of CYP2C19*23 (271 G>C, 991 A>G) and CYP2C19*24 (991 A>G, 1004 G>A) in vitro. Mutations in CYP2C19 cDNA were introduced by site-directed mutagenesis, and the CYP2C19 wild type (WT) as well as variants proteins were subsequently expressed using Escherichia coli cells. Catalytic activities of CYP2C19 WT and those of variants were determined by high performance liquid chromatography-based essay employing S-mephenytoin and omeprazole as probe substrates. Results showed that the level of S-mephenytoin 4'-hydroxylation activity of CYP2C19*23 (V max 111.5 ± 16.0 pmol/min/mg, K m 158.3 ± 88.0 μM) protein relative to CYP2C19 WT (V max 101.6 + 12.4 pmol/min/mg, K m 123.0 ± 19.2 μM) protein had no significant difference. In contrast, the K m of CYP2C19*24 (270.1 ± 57.2 μM) increased significantly as compared to CYP2C19 WT (123.0 ± 19.2 μM) and V max of CYP2C19*24 (23.6 ± 2.6 pmol/min/mg) protein was significantly lower than that of the WT protein (101.6 ± 12.4 pmol/min/mg). In vitro intrinsic clearance (CLint = V max/K m) for CYP2C19*23 protein was 85.4 % of that of CYP2C19 WT protein. The corresponding CLint value for CYP2C19*24 protein reduced to 11.0 % of that of WT protein. These findings suggested that catalytic activity of CYP2C19 was not affected by the corresponding amino acid substitutions in CYP2C19*23 protein; and the reverse was true for CYP2C19*24 protein. When omeprazole was employed as the substrate, K m of CYP2C19*23 (1911 ± 244.73 μM) was at least 100 times higher than that of CYP2C19 WT (18.37 ± 1.64 μM) and V max of CYP2C19*23 (3.87 ± 0.74 pmol/min/mg) dropped to 13.4 % of the CYP2C19 WT (28.84 ± 0.61 pmol/min/mg) level. Derived from V max/K m, the CLint value of CYP2C19 WT was 785 folds of CYP2C19*23. K m and V max values could not be determined for CYP2C19*24 due to its low catalytic activity towards omeprazole 5'-hydroxylation. Therefore, both CYP2C19*23 and CYP2C19*24 showed marked reduced activities of metabolising omeprazole to 5-hydroxyomeprazole. Hence, carriers of CYP2C19*23 and CYP2C19*24 allele are potentially poor metabolisers of CYP2C19-mediated substrates.

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