Displaying publications 21 - 33 of 33 in total

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  1. Physicochemical Characterization of Mitragyna speciosa Alkaloid Extract and Mitragynine using In Vitro High Throughput Assays
    Kong WM, Chik Z, Mohamed Z, Alshawsh MA
    Comb Chem High Throughput Screen, 2017;20(9):796-803.
    PMID: 29076424 DOI: 10.2174/1386207320666171026121820
    AIM AND OBJECTIVE: Mitragynine, a major active alkaloid of Mitragyna speciosa, acts as an agonist on µ-opioid receptors, producing effects similar to morphine and other opioids. It has been traditionally utilized to alleviate opiate withdrawal symptoms. Besides consideration about potency and selectivity, a good drug must possess a suitable pharmacokinetic profile, with suitable absorption, distribution, metabolism, excretion and toxicity (ADME-Tox) profile, in order to have a high chance of success in clinical trials.

    MATERIAL AND METHOD: The purity of mitragynine in a Mitragyna speciosa alkaloid extract (MSAE) was determined using Ultra-Fast Liquid Chromatography (UFLC). In vitro high throughput ADMETox studies such as aqueous solubility, plasma protein binding, metabolic stability, permeability and cytotoxicity tests were carried out to analyze the physicochemical properties of MSAE and mitragynine. The UFLC quantification revealed that the purity of mitragynine in the MSAE was 40.9%.

    RESULTS: MSAE and mitragynine are highly soluble in aqueous solution at pH 4.0 but less soluble at pH 7.4. A parallel artificial membrane permeability assay demonstrated that it is extensively absorbed through the semi-permeable membrane at pH 7.4 but very poorly at pH 4.0. Both are relatively highly bound to plasma proteins (> 85 % bound) and are metabolically stable to liver microsomes (> 84 % remained unchanged). In comparison to MSAE, mitragynine showed higher cytotoxicity against WRL 68, HepG2 and Clone 9 hepatocytes after 72 h treatment.

    CONCLUSION: The obtained ADME and cytotoxicity data demonstrated that both MSAE and mitragynine have poor bioavailability and have the potential to be significantly cytotoxic.

    Matched MeSH terms: High-Throughput Screening Assays*
  2. Fulltext Endogenous HIF2A reporter systems for high-throughput functional screening
    Zaini MN, Patel SA, Syafruddin SE, Rodrigues P, Vanharanta S
    Sci Rep, 2018 08 13;8(1):12063.
    PMID: 30104738 DOI: 10.1038/s41598-018-30499-2
    Tissue-specific transcriptional programs control most biological phenotypes, including disease states such as cancer. However, the molecular details underlying transcriptional specificity is largely unknown, hindering the development of therapeutic approaches. Here, we describe novel experimental reporter systems that allow interrogation of the endogenous expression of HIF2A, a critical driver of renal oncogenesis. Using a focused CRISPR-Cas9 library targeting chromatin regulators, we provide evidence that these reporter systems are compatible with high-throughput screening. Our data also suggests redundancy in the control of cancer type-specific transcriptional traits. Reporter systems such as those described here could facilitate large-scale mechanistic dissection of transcriptional programmes underlying cancer phenotypes, thus paving the way for novel therapeutic approaches.
    Matched MeSH terms: High-Throughput Screening Assays/methods*
  3. High-Throughput Screening Assays for Dengue Antiviral Drug Development
    Jabanathan SG, Xuan LZ, Ramanathan B
    Methods Mol Biol, 2021;2296:279-302.
    PMID: 33977455 DOI: 10.1007/978-1-0716-1358-0_17
    Dengue is an arthropod-borne viral disease that has become endemic and a global threat in over 100 countries. The increase in prevalence would require a long-term measure to control outbreaks. Sanofi Pasteur has licensed the tetravalent dengue vaccine (Dengvaxia) in certain dengue endemic countries. However, the efficacy of the vaccine is limited against certain dengue serotypes and can only be used for individuals from the age from 9 to 45 years old. Over the years, there has been intense research conducted on the development of antivirals against dengue virus (DENV) through either inhibiting the virus replication or targeting the host cell mechanism to block the virus entry. However, no approved antiviral drug against dengue is yet available. In this chapter, we describe the dengue antiviral development workflow including (i) prophylactic, (ii) virucidal, and (iii) postinfection assays that are employed in the antiviral drug screening process against DENV. Further, we demonstrate different methods that can be used to enumerate the reduction in virus foci number including foci-forming unit reduction assay (FFURA), estimation of viral RNA copy number through quantitative real-time PCR, and a high-throughput enzyme linked immunosorbent assay (ELISA)-based quantification of virus particles.
    Matched MeSH terms: High-Throughput Screening Assays/methods*
  4. Fluorescence polarisation for high-throughput screening of adulterated food products via phosphodiesterase 5 inhibition assay
    Mohd Yusop AY, Xiao L, Fu S
    Drug Test Anal, 2021 May;13(5):953-964.
    PMID: 32959983 DOI: 10.1002/dta.2926
    The surge in the consumption of food products containing herbal aphrodisiacs has driven their widespread adulteration. A rapid screening strategy is, therefore, warranted to curb this problem. This study established an enzyme inhibition assay to screen phosphodiesterase 5 (PDE5) inhibitors as adulterants in selected food products. Fluorescein-labelled cyclic-3',5'-guanosine monophosphate was utilised as substrates for the PDE5A1 enzyme, aided by the presence of nanoparticle phosphate-binding beads on their fluorescence polarisation. The sample preparation was optimised to improve the enzyme inhibition efficiency and applied to calculate the threshold values of six blank food matrices. The assay was validated using sildenafil, producing an IC50 of 4.2 nM. The applicability of the assay procedure was demonstrated by screening 55 distinct food samples. The results were subsequently verified using confirmatory liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis. Altogether, 49 samples inhibited the PDE5 enzyme above the threshold values (75.7%-105.5%) and were registered as potentially adulterated samples. The remaining six samples were marked as nonadulterated with percentage inhibition below the threshold values (-3.3%-18.2%). The LC-HRMS analysis agreed with the assay results for all food products except for the instant coffee premix (ICP) samples. False-positive results were obtained for the ICP samples at 32% (8/25), due to possible PDE5 inhibition by caffeine. Contrarily, all other food samples were found to produce 0% (0/30) false-positive or false-negative results. The broad-based assay, established via a simple mix-incubate-read format, exhibited promising potential for high-throughput screening of PDE5 inhibitors in various food products, except those with naturally occurring phosphodiesterase inhibitors such as caffeine.
    Matched MeSH terms: High-Throughput Screening Assays
  5. A facile three-component [3+2]-cycloaddition for the regioselective synthesis of highly functionalised dispiropyrrolidines acting as antimycobacterial agents
    Wei AC, Ali MA, Yoon YK, Ismail R, Choon TS, Kumar RS
    Bioorg Med Chem Lett, 2013 Mar 1;23(5):1383-6.
    PMID: 23352268 DOI: 10.1016/j.bmcl.2012.12.069
    A series of fourteen dispiropyrrolidines were synthesized using [3+2]-cycloaddition reactions and were screened for their antimycobacterial activity against Mycobacterium tuberculosis H(37)Rv in HTS (High Throughput Screen). Most of the compounds showed moderate to good activity with MIC of less than 20 μM. Compound 4'-(4-bromophenyl)-1'-methyldispiro[acenaphthylene-1,2'-pyrrolidine-3',2″-indane]-2,1″(1H)-dione (4c) was found to be the most active with MIC of 12.50 μM.
    Matched MeSH terms: High-Throughput Screening Assays
  6. Fulltext Evaluation of the effects of Mitragyna speciosa alkaloid extract on cytochrome P450 enzymes using a high throughput assay
    Kong WM, Chik Z, Ramachandra M, Subramaniam U, Aziddin RE, Mohamed Z
    Molecules, 2011 Aug 29;16(9):7344-56.
    PMID: 21876481 DOI: 10.3390/molecules16097344
    The extract from Mitragyna speciosa has been widely used as an opium substitute, mainly due to its morphine-like pharmacological effects. This study investigated the effects of M. speciosa alkaloid extract (MSE) on human recombinant cytochrome P450 (CYP) enzyme activities using a modified Crespi method. As compared with the liquid chromatography-mass spectrometry method, this method has shown to be a fast and cost-effective way to perform CYP inhibition studies. The results indicated that MSE has the most potent inhibitory effect on CYP3A4 and CYP2D6, with apparent half-maximal inhibitory concentration (IC(50)) values of 0.78 µg/mL and 0.636 µg/mL, respectively. In addition, moderate inhibition was observed for CYP1A2, with an IC(50) of 39 µg/mL, and weak inhibition was detected for CYP2C19. The IC(50) of CYP2C19 could not be determined, however, because inhibition was <50%. Competitive inhibition was found for the MSE-treated CYP2D6 inhibition assay, whereas non-competitive inhibition was shown in inhibition assays using CYP3A4, CYP1A2 and CYP2C19. Quinidine (CYP2D6), ketoconazole (CYP3A4), tranylcypromine (CYP2C19) and furafylline (CYP1A2) were ACCESSused as positive controls throughout the experiments. This study shows that MSE may contribute to an herb-drug interaction if administered concomitantly with drugs that are substrates for CYP3A4, CYP2D6 and CYP1A2.
    Matched MeSH terms: High-Throughput Screening Assays
  7. Fulltext High-throughput screening of ancient forest plant extracts shows cytotoxicity towards triple-negative breast cancer
    Li Y, Ling Ma N, Chen H, Zhong J, Zhang D, Peng W, et al.
    Environ Int, 2023 Nov;181:108279.
    PMID: 37924601 DOI: 10.1016/j.envint.2023.108279
    According to the World Health Organization, women's breast cancer is among the most common cancers with 7.8 million diagnosed cases during 2016-2020 and encompasses 15 % of all female cancer-related mortalities. These mortality events from triple-negative breast cancer are a significant health issue worldwide calling for a continuous search of bioactive compounds for better cancer treatments. Historically, plants are important sources for identifying such new bioactive chemicals for treatments. Here we use high-throughput screening and mass spectrometry analyses of extracts from 100 plant species collected in Chinese ancient forests to detect novel bioactive breast cancer phytochemicals. First, to study the effects on viability of the plant extracts, we used a MTT and CCK-8 cytotoxicity assay employing triple-negative breast cancer (TNBC) MDA-MB-231 and normal epithelial MCF-10A cell lines and cell cycle arrest to estimate apoptosis using flow cytometry for the most potent three speices. Based on these analyses, the final most potent extracts were from the Amur honeysuckle (Lonicera maackii) wood/root bark and Nigaki (Picrasma quassioides) wood/root bark. Then, 5 × 106 MDA-MB-231 cells were injected subcutaneously into the right hind leg of nude mice and a tumour was allowed to grow before treatment for seven days. Subsequently, the four exposed groups received gavage extracts from Amur honeysuckle and Nigaki (Amur honeysuckle wood distilled water, Amur honeysuckle root bark ethanol, Nigaki wood ethanol or Nigaki root bark distilled water/ethanol (1:1) extracts) in phosphate-buffered saline (PBS), while the control group received only PBS. The tumour weight of treated nude mice was reduced significantly by 60.5 % within 2 weeks, while on average killing 70 % of the MDA-MB-231 breast cancer cells after 48 h treatment (MTT test). In addition, screening of target genes using the Swiss Target Prediction, STITCH, STRING and NCBI-gene database showed that the four plant extracts possess desirable activity towards several known breast cancer genes. This reflects that the extracts may kill MBD-MB-231 breast cancer cells. This is the first screening of plant extracts with high efficiency in 2 decades, showing promising results for future development of novel cancer treatments.
    Matched MeSH terms: High-Throughput Screening Assays
  8. Fulltext A high-throughput oxidative stress biosensor based on Escherichia coli roGFP2 cells immobilized in a k-carrageenan matrix
    Ooi L, Heng LY, Mori IC
    Sensors (Basel), 2015;15(2):2354-68.
    PMID: 25621608 DOI: 10.3390/s150202354
    Biosensors fabricated with whole-cell bacteria appear to be suitable for detecting bioavailability and toxicity effects of the chemical(s) of concern, but they are usually reported to have drawbacks like long response times (ranging from hours to days), narrow dynamic range and instability during long term storage. Our aim is to fabricate a sensitive whole-cell oxidative stress biosensor which has improved properties that address the mentioned weaknesses. In this paper, we report a novel high-throughput whole-cell biosensor fabricated by immobilizing roGFP2 expressing Escherichia coli cells in a k-carrageenan matrix, for the detection of oxidative stress challenged by metalloid compounds. The E. coli roGFP2 oxidative stress biosensor shows high sensitivity towards arsenite and selenite, with wide linear range and low detection limit (arsenite: 1.0 × 10(-3)-1.0 × 10(1) mg·L(-1), LOD: 2.0 × 10(-4) mg·L(-1); selenite: 1.0 × 10(-5)-1.0 × 10(2) mg·L(-1), LOD: 5.8 × 10(-6) mg·L(-1)), short response times (0-9 min), high stability and reproducibility. This research is expected to provide a new direction in performing high-throughput environmental toxicity screening with living bacterial cells which is capable of measuring the bioavailability and toxicity of environmental stressors in a friction of a second.
    Matched MeSH terms: High-Throughput Screening Assays
  9. Simple and fast LC-MS/MS method for quantification of terazosin in human plasma and application to bioequivalence study
    Loh GOK, Wong EYL, Tan YTF, Ong LM, Ng RS, Wee HC, et al.
    J Chromatogr B Analyt Technol Biomed Life Sci, 2021 Jan 15;1163:122517.
    PMID: 33429127 DOI: 10.1016/j.jchromb.2020.122517
    A simple, fast and sensitive LC-MS/MS method was developed to quantify terazosin in human plasma. The mobile phase consisted of acetonitrile-0.1% (v/v) formic acid (70:30, v/v). Prazosin was used as internal standard (IS). As deproteinization agent, acetonitrile produced a clean sample. A higher response intensity with more symmetrical peak was obtained using Agilent Poroshell 120 EC-C18 - Fast LC column (100 × 2.1mmID, 2.7 μm) compared with Kinetex XB-C18 (100 × 2.1 mm, 2.6 µm) column. The response of terazosin and IS were approximately two times in citrate phosphate dextrose (CPD) plasma compared with dipotassium ethylenediaminetetraacetic acid (K2EDTA) plasma. Plasma calibration curve was linear from 1.0 to 100.0 ng/mL, with coefficient of determination r2 ≥ 0.99. The within-run and between-run precision values (CV, %) were <5.2% and <7.8%, while accuracy values were 102.8-112.7% and 103.4-112.2%. The extended run accuracy was 98.6-102.8% and precision (CV, %) 4.3-10.4%. The recovery of analyte was >98% and IS >94%. Terazosin in plasma kept at benchtop was stable for 24 h, in autosampler tray for 48 h, in instrumentation room for 48 h, for 7 freeze-thaw cycles and in freezer for 140 days. Terazosin and IS stock standard solutions were stable for 140 days at room temperature and in the chiller. The high throughput method was successfully utilized to measure 935 samples in a bioequivalence study of terazosin.
    Matched MeSH terms: High-Throughput Screening Assays
  10. Molecular characterization of Antarctic actinobacteria and screening for antimicrobial metabolite production
    Lee LH, Cheah YK, Mohd Sidik S, Ab Mutalib NS, Tang YL, Lin HP, et al.
    World J Microbiol Biotechnol, 2012 May;28(5):2125-37.
    PMID: 22806035 DOI: 10.1007/s11274-012-1018-1
    The present study aimed to isolate actinobacteria from soil samples and characterized them using molecular tools and screened their secondary metabolites for antimicrobial activities. Thirty-nine strains from four different location of Barrientos Island, Antarctica using 12 types of isolation media was isolated. The isolates were preceded to screening of secondary metabolites for antimicrobial and antifungal activities. Using high-throughput screening methods, 38% (15/39) of isolates produced bioactive metabolites. Approximately 18% (7/39), 18% (7/39), 10% (4/39) and 2.5% (1/39) of isolates inhibited growth of Candida albicans ATCC 10231(T), Staphylococcus aurues ATCC 51650(T), methicillin-resistant Staphylococcus aurues (MRSA) ATCC BAA-44(T) and Pseudomonas aeruginosa ATCC 10145(T), respectively. Molecular characterization techniques like 16S rRNA analysis, Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), Random amplified polymorphic DNA (RAPD) and composite analyses were used to characterize the actinobacteria strains. Analysis of 16S rRNA sequences is still one of the most powerful methods to determine higher taxonomic relationships of Actinobacteria. Both RAPD and ERIC-PCR fingerprinting have shown good discriminatory capability but RAPD proved to be better in discriminatory power than ERIC-PCR. Our results demonstrated that composite analysis of both fingerprinting generally increased the discrimination ability and generated best clustering for actinobacteria strains in this study.
    Matched MeSH terms: High-Throughput Screening Assays
  11. Fulltext Structure-based and ligand-based virtual screening of novel methyltransferase inhibitors of the dengue virus
    Lim SV, Rahman MB, Tejo BA
    BMC Bioinformatics, 2011;12 Suppl 13:S24.
    PMID: 22373153 DOI: 10.1186/1471-2105-12-S13-S24
    The dengue virus is the most significant arthropod-borne human pathogen, and an increasing number of cases have been reported over the last few decades. Currently neither vaccines nor drugs against the dengue virus are available. NS5 methyltransferase (MTase), which is located on the surface of the dengue virus and assists in viral attachment to the host cell, is a promising antiviral target. In order to search for novel inhibitors of NS5 MTase, we performed a computer-aided virtual screening of more than 5 million commercially available chemical compounds using two approaches: i) structure-based screening using the crystal structure of NS5 MTase and ii) ligand-based screening using active ligands of NS5 MTase. Structure-based screening was performed using the LIDAEUS (LIgand Discovery At Edinburgh UniverSity) program. The ligand-based screening was carried out using the EDULISS (EDinburgh University LIgand Selection System) program.
    Matched MeSH terms: High-Throughput Screening Assays
  12. Molecular docking and dynamic simulation evaluation of Rohinitib - Cantharidin based novel HSF1 inhibitors for cancer therapy
    Agarwal T, Annamalai N, Khursheed A, Maiti TK, Arsad HB, Siddiqui MH
    J Mol Graph Model, 2015 Sep;61:141-9.
    PMID: 26245696 DOI: 10.1016/j.jmgm.2015.07.003
    Recent developments in the target based cancer therapies have identified HSF1 as a novel non oncogenic drug target. The present study delineates the design and molecular docking evaluation of Rohinitib (RHT) - Cantharidin (CLA) based novel HSF1 inhibitors for target-based cancer therapy. Here, we exploited the pharmacophoric features of both the parent ligands for the design of novel hybrid HSF1 inhibitors. The RHT-CLA ligands were designed and characterized for ADME/Tox features, interaction with HSF1 DNA binding domain and their pharmacophoric features essential for interaction. From the results, amino acid residues Ala17, Phe61, His63, Asn65, Ser68, Arg71 and Gln72 were found crucial for HSF1 interaction with the Heat shock elements (HSE). The hybrid ligands had better affinity towards the HSF1 DNA binding domain, in comparison to RHT or CLA and interacted with most of the active site residues. Additionally, the HSF1-ligand complex had a reduced affinity towards HSE in comparison to native HSF1. Based on the results, ligand RC15 and RC17 were non carcinogenic, non mutagenic, completely biodegradable under aerobic conditions, had better affinity for HSF1 (1.132 and 1.129 folds increase respectively) and diminished the interaction of HSF1 with HSE (1.203 and 1.239 folds decrease respectively). The simulation analysis also suggested that the ligands formed a stable complex with HSF1, restraining the movement of active site residues. In conclusion, RHT-CLA hybrid ligands can be used as a potential inhibitor of HSF1 for non-oncogene target based cancer therapy.
    Matched MeSH terms: High-Throughput Screening Assays
  13. Fulltext Identification of small molecule inhibitors of p27(Kip1) ubiquitination by high-throughput screening
    Ooi LC, Watanabe N, Futamura Y, Sulaiman SF, Darah I, Osada H
    Cancer Sci, 2013 Nov;104(11):1461-7.
    PMID: 23910095 DOI: 10.1111/cas.12246
    Dysregulation of p27(Kip1) due to proteolysis that involves the ubiquitin ligase (SCF) complex with S-phase kinase-associated protein 2 (Skp2) as the substrate-recognition component (SCF(Skp2)) frequently results in tumorigenesis. In this report, we developed a high-throughput screening system to identify small-molecule inhibitors of p27(Kip1) degradation. This system was established by tagging Skp2 with fluorescent monomeric Azami Green (mAG) and CDK subunit 1 (Cks1) (mAGSkp2-Cks1) to bind to p27(Kip1) phosphopeptides. We identified two compounds that inhibited the interaction between mAGSkp2-Cks1 and p27(Kip1): linichlorin A and gentian violet. Further studies have shown that the compounds inhibit the ubiquitination of p27(Kip1) in vitro as well as p27(Kip1) degradation in HeLa cells. Notably, both compounds exhibited preferential antiproliferative activity against HeLa and tsFT210 cells compared with NIH3T3 cells and delayed the G1 phase progression in tsFT210 cells. Our approach indicates a potential strategy for restoring p27(Kip1) levels in human cancers.
    Matched MeSH terms: High-Throughput Screening Assays
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