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  1. Ong, Chin-Eng, Yan, Pan, Tiong, Kai-Hung, Yiap, Beow-Chin, Tan, Eng-Lai, Pook, Peter, et al.
    MyJurnal
    Pharmacogenomics (or pharmacogenetics), the study of the effects of genetic differences on a person’s response to drugs, can help in optimizing drug efficacy and minimizing adverse drug reactions. Interperson difference in drug metabolism is one of the important consequences of such genetic variation. This variation is determined in part by mutations in cytochrome P450 enzymes (CYPs). IMU is part of a major collaborative research project in the area of phamacogenetics and drug metabolism. Working together with USM and UiTM, our group has, since 2000, generated useful population database on genetic polymorphism of various CYP isoforms. We have successfully genotyped three major ethnic groups, Malay, Indian and Chinese for their allelic frequency of important isoforms. These include CYP2D6, CYP2C9, CYP2C8 and CYP2A6. Data generated so far collectively have contributed to our effort in mapping and constructing genomic database for Malaysian population.
    Since early 2002, our research has been focusing on developing in vitro methods in studying the functional consequences of genetic polymorphism of CYP enzymes. Using site-directed mutagenesis, CYP mutants, carrying nucleotide changes as reported in known alleles in human populations, were generated and expressed in E. coli system, and the expressed recombinant proteins were characterized using enzyme assays to determine the functional consequences of mutations. We have established a series of HPLC (high performance liquid chromatography)-based and fluorescence-based assays to investigate CYP activities. Assays that have been developed include tolbutamide methylhydroxylase, paclitaxel 6α-hydroxylase, dextromethorphan O-demethylation, testosterone 6β-hydroxylation and coumarin 7-hydroxylase assays. These assays serve as activity markers allowing comparison of catalytic activities of mutant proteins generated. Another focus of our work is to use the developed assays as a screening tool to investigate drug-herb interactions. This was achieved by co-incubation of herbal extracts and active constituents with the probe substrates in the assays followed by characterization of the kinetic behaviors of the enzymes involved using various pharmacokinetic parameters such as Km, Vmax, IC50 and Ki. This work is currently carried out with collaboration from the Institute for Medical Research (IMR) and is supported by MOSTI’s eScienceFund under RM9. It is envisaged that this screening work will give us insights on the potential of the commonly used herbs to cause pharmacokinetic interactions with other drug substrates, and allow us to elucidate the mechanisms involved in the interactions.
  2. Tan SC, Yiap BC
    J Biomed Biotechnol, 2009;2009:574398.
    PMID: 20011662 DOI: 10.1155/2009/574398
    Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput. Currently, there are many specialized methods that can be used to extract pure biomolecules, such as solution-based and column-based protocols. Manual method has certainly come a long way over time with various commercial offerings which included complete kits containing most of the components needed to isolate nucleic acid, but most of them require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment. Automated systems designed for medium-to-large laboratories have grown in demand over recent years. It is an alternative to labor-intensive manual methods. The technology should allow a high throughput of samples; the yield, purity, reproducibility, and scalability of the biomolecules as well as the speed, accuracy, and reliability of the assay should be maximal, while minimizing the risk of cross-contamination.
  3. Yap PS, Yiap BC, Ping HC, Lim SH
    Open Microbiol J, 2014;8:6-14.
    PMID: 24627729 DOI: 10.2174/1874285801408010006
    For many years, the battle between humans and the multitudes of infection and disease causing pathogens continues. Emerging at the battlefield as some of the most significant challenges to human health are bacterial resistance and its rapid rise. These have become a major concern in global public health invigorating the need for new antimicrobial compounds. A rational approach to deal with antibiotic resistance problems requires detailed knowledge of the different biological and non-biological factors that affect the rate and extent of resistance development. Combination therapy combining conventional antibiotics and essential oils is currently blooming and represents a potential area for future investigations. This new generation of phytopharmaceuticals may shed light on the development of new pharmacological regimes in combating antibiotic resistance. This review consolidated and described the observed synergistic outcome between essential oils and antibiotics, and highlighted the possibilities of essential oils as the potential resistance modifying agent.
  4. Yap PS, Lim SH, Hu CP, Yiap BC
    Phytomedicine, 2013 Jun 15;20(8-9):710-3.
    PMID: 23537749 DOI: 10.1016/j.phymed.2013.02.013
    In this study we investigated the relationship between several selected commercially available essential oils and beta-lactam antibiotics on their antibacterial effect against multidrug resistant bacteria. The antibacterial activity of essential oils and antibiotics was assessed using broth microdilution. The combined effects between essential oils of cinnamon bark, lavender, marjoram, tea tree, peppermint and ampicillin, piperacillin, cefazolin, cefuroxime, carbenicillin, ceftazidime, meropenem, were evaluated by means of the checkerboard method against beta-lactamase-producing Escherichia coli. In the latter assays, fractional inhibitory concentration (FIC) values were calculated to characterize interaction between the combinations. Substantial susceptibility of the bacteria toward natural antibiotics and a considerable reduction in the minimum inhibitory concentrations (MIC) of the antibiotics were noted in some paired combinations of antibiotics and essential oils. Out of 35 antibiotic-essential oil pairs tested, four of them showed synergistic effect (FIC≤0.5) and 31 pairs showed no interaction (FIC>0.5-4.0). The preliminary results obtained highlighted the occurrence of a pronounced synergistic relationship between piperacillin/cinnamon bark oil, piperacillin/lavender oil, piperacillin/peppermint oil as well as meropenem/peppermint oil against two of the three bacteria under study with a FIC index in the range 0.26-0.5. The finding highlighted the potential of peppermint, cinnamon bark and lavender essential oils being as antibiotic resistance modifying agent. Reduced usage of antibiotics could be employed as a treatment strategy to decrease the adverse effects and possibly to reverse the beta-lactam antibiotic resistance.
  5. Tiong KH, Mohammed Yunus NA, Yiap BC, Tan EL, Ismail R, Ong CE
    PLoS One, 2014;9(1):e86230.
    PMID: 24475091 DOI: 10.1371/journal.pone.0086230
    Human cytochrome P450 2A6 (CYP2A6) is a highly polymorphic isoform of CYP2A subfamily. Our previous kinetic study on four CYP2A6 allelic variants (CYP2A6 15, CYP2A6 16, CYP2A6 21 and CYP2A6 22) have unveiled the functional significance of sequence mutations in these variants on coumarin 7-hydroxylation activity. In the present study, we further explored the ability of a typical CYP2A6 inhibitor, 8-methoxypsoralen (8-MOP), in inhibition of these alleles and we hypothesized that translational mutations in these variants are likely to give impact on 8-MOP inhibitory potency. The CYP2A6 variant and the wild type proteins were subjected to 8-MOP inhibition to yield IC50 values. In general, a similar trend of change in the IC50 and Km values was noted among the four mutants towards coumarin oxidation. With the exception of CYP2A6 16, differences in IC50 values were highly significant which implied compromised interaction of the mutants with 8-MOP. Molecular models of CYP2A6 were subsequently constructed and ligand-docking experiments were performed to rationalize experimental data. Our docking study has shown that mutations have induced enlargement of the active site volume in all mutants with the exception of CYP2A6 16. Furthermore, loss of hydrogen bond between 8-MOP and active site residue Asn297 was evidenced in all mutants. Our data indicate that the structural changes elicited by the sequence mutations could affect 8-MOP binding to yield differential enzymatic activities in the mutant CYP2A6 proteins.
  6. Tiong KH, Yiap BC, Tan EL, Ismail R, Ong CE
    Drug Metab. Dispos., 2010 May;38(5):745-51.
    PMID: 20139165 DOI: 10.1124/dmd.109.031054
    Variation in CYP2A6 levels and activity can be attributed to genetic polymorphism and, thus, functional characterization of allelic variants is necessary to define the importance of CYP2A6 polymorphism in humans. The aim of the present study was to investigate the reported alleles CYP2A6*15, CYP2A6*16, CYP2A6*21, and CYP2A6*22, in terms of the functional consequences of their mutations on the enzyme catalytic activity. With use of the wild-type CYP2A6 cDNA as template, site-directed mutagenesis was performed to introduce nucleotide changes encoding K194E substitution in CYP2A6*15, R203S substitution in CYP2A6*16, K476R substitution in CYP2A6*21, and concurrent D158E and L160I substitutions in CYP2A6*22. Upon sequence verification, the CYP2A6 wild-type and mutant constructs were individually coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. A kinetic study using a coumarin 7-hydroxylase assay indicated that CYP2A6*15 exhibited higher V(max) than the wild type, whereas all mutant constructs, except for variant CYP2A6*16, exhibited higher K(m) values. Analysis of the V(max)/K(m) ratio revealed that all mutants demonstrated 0.85- to 1.05-fold differences from the wild type, with the exception of variant CYP2A6*22, which only portrayed 39% of the wild-type intrinsic clearance. These data suggested that individuals carrying the CYP2A6*22 allele are likely to have lower metabolism of CYP2A6 substrate than individuals expressing CYP2A6*15, CYP2A6*16, CYP2A6*21, and the wild type.
  7. 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.
  8. Dong AN, Ahemad N, Pan Y, Palanisamy UD, Yiap BC, Ong CE
    Curr Mol Pharmacol, 2020;13(3):233-244.
    PMID: 31713493 DOI: 10.2174/1874467212666191111110429
    BACKGROUND: There is a large inter-individual variation in cytochrome P450 2C19 (CYP2C19) activity. The variability can be caused by the genetic polymorphism of CYP2C19 gene. This study aimed to investigate the molecular and kinetics basis for activity changes in three alleles including CYP2C19*23, CYP2C19*24 and CYP2C19*25found in the Chinese population.

    METHODS: The three variants expressed by bacteria were investigated using substrate (omeprazole and 3- cyano-7-ethoxycoumarin[CEC]) and inhibitor (ketoconazole, fluoxetine, sertraline and loratadine) probes in enzyme assays along with molecular docking.

    RESULTS: All alleles exhibited very low enzyme activity and affinity towards omeprazole and CEC (6.1% or less in intrinsic clearance). The inhibition studies with the four inhibitors, however, suggested that mutations in different variants have a tendency to cause enhanced binding (reduced IC50 values). The enhanced binding could partially be explained by the lower polar solvent accessible surface area of the inhibitors relative to the substrates. Molecular docking indicated that G91R, R335Q and F448L, the unique mutations in the alleles, have caused slight alteration in the substrate access channel morphology and a more compact active site cavity hence affecting ligand access and binding. It is likely that these structural alterations in CYP2C19 proteins have caused ligand-specific alteration in catalytic and inhibitory specificities as observed in the in vitro assays.

    CONCLUSION: This study indicates that CYP2C19 variant selectivity for ligands was not solely governed by mutation-induced modifications in the active site architecture, but the intrinsic properties of the probe compounds also played a vital role.

  9. 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.
  10. Dong AN, Pan Y, Palanisamy UD, Yiap BC, Ahemad N, Ong CE
    Appl Biochem Biotechnol, 2018 Sep;186(1):132-144.
    PMID: 29524040 DOI: 10.1007/s12010-018-2728-0
    Genetic polymorphism of the cytochrome P450 (CYP) genes particularly affects CYP2D6 and CYP2C19 to a functionally relevant extent, and it is therefore crucial to elucidate the enzyme kinetic and molecular basis for altered catalytic activity of these allelic variants. This study explored the expression and function of the reported alleles CYP2D6*2, CYP2D6*10, CYP2D6*17, CYP2C19*23, CYP2C19*24, and CYP2C19*25 with respect to gene polymorphisms. Site-directed mutagenesis (SDM) was carried out to generate these six alleles. After DNA sequencing, the CYP2D6 and CYP2C19 wild types alongside with their alleles were each independently co-expressed with NADPH-CYP oxidoreductase (OxR) in Escherichia coli. The expressed proteins were analyzed using Western blotting, reduced carbon monoxide (CO) difference spectral scanning, and cytochrome c reductase assay. Results from Western blot revealed the presence of all CYP wild-type and allelic proteins in E. coli membrane fractions. The reduced CO difference spectra scanning presented the distinct peak of absorbance at 450 nm, and the cytochrome c reductase assay has confirmed that spectrally active OxR was expressed in each protein preparation. As a conclusion, the results obtained from this study have proven the CYP variants to be immunoreactive and spectrally active and are suitable for use to examine biotransformation and interaction mechanism of the enzymes.
  11. Dong AN, Ahemad N, Pan Y, Palanisamy UD, Yiap BC, Ong CE
    Drug Metab Bioanal Lett, 2022;15(1):51-63.
    PMID: 35049443 DOI: 10.2174/1872312815666220113125232
    BACKGROUND: Genetic polymorphism of cytochrome P450 (CYP) contributes to variability in drug metabolism, clearance, and response. This study aimed to investigate the functional and molecular basis for altered ligand binding and catalysis in CYP2D6*14A and CYP2D6*14B, two unique alleles common in the Asian population.

    METHODS: CYP proteins expressed in Escherichia coli were studied using the substrate 3-cyano-7- ethoxycoumarin (CEC) and inhibitor probes (quinidine, fluoxetine, paroxetine, terbinafine) in the enzyme assay. Computer modelling was additionally used to create three-dimensional structures of the CYP2D6*14 variants.

    RESULTS: Kinetics data indicated significantly reduced intrinsic clearance in CYP2D6*14 variants, suggesting that P34S, G169R, R296C, and S486T substitutions worked cooperatively to alter the conformation of the active site that negatively impacted the deethylase activity of CYP2D6. For the inhibition studies, IC50 values decreased in quinidine, paroxetine, and terbinafine but increased in fluoxetine, suggesting a varied ligand-specific susceptibility to inhibition. Molecular docking further demonstrated the role of P34S and R296C in altering access channel dimensions, thereby affecting ligand access and binding and subsequently resulting in varied inhibition potencies.

    CONCLUSION: In summary, the differential selectivity of CYP2D6*14 variants for the ligands (substrate and inhibitor) was governed by the alteration of the active site and access channel architecture induced by the natural mutations found in the alleles.

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