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Publication Open Access A unified proteochemometric model for prediction of inhibition of cytochrome P450 isoforms(2013-06) Maris Lapins; Apilak Worachartcheewan; Ola Spjuth; Valentin Georgiev; Virapong Prachayasittikul; Chanin Nantasenamat; Jarl E. S. Wikberg; Center of Data Mining and Biomedical Informatics; Department of Clinical Microbiology and Applied TechnologyA unified proteochemometric (PCM) model for the prediction of the ability of drug-like chemicals to inhibit five major drug metabolizing CYP isoforms (i.e. CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) was created and made publicly available under the Bioclipse Decision Support open source system at www.cyp450model.org. In regards to the proteochemometric modeling we represented the chemical compounds by molecular signature descriptors and the CYP-isoforms by alignment-independent description of composition and transition of amino acid properties of their protein primary sequences. The entire training dataset contained 63 391 interactions and the best PCM model was obtained using signature descriptors of height 1, 2 and 3 and inducing the model with a support vector machine. The model showed excellent predictive ability with internal AUC = 0.923 and an external AUC = 0.940, as evaluated on a large external dataset. The advantage of PCM models is their extensibility making it possible to extend our model for new CYP isoforms and polymorphic CYP forms. A key benefit of PCM is that all proteins are confined in one single model, which makes it generally more stable and predictive as compared with single target models. The inclusion of the model in Bioclipse Decision Support makes it possible to make virtual instantaneous predictions (∼100 ms per prediction) while interactively drawing or modifying chemical structures in the Bioclipse chemical structure editor.Publication Open Access Paper-based Acetylcholinesterase Inhibition Assay Combining a Wet System for Organophosphate and Carbamate Pesticides Detection(2015-02-26) Amara Apilux; Chartchalerm Isarankura-Na-Ayudhya; Virapong Prachayasittikul; Tanawut Tantimongcolwat; Center for Innovation Development and Technology Transfer; Department of Clinical Microbiology and Applied TechnologyA dramatic increase in pesticide usage in agriculture highlights the need for on-site monitoring for public health and safety. Here, a paper-based sensor combined with a wet system was developed for the simple and rapid screening of organophosphate (OP) and carbamate (CM) pesticides based on the inhibition of acetylcholinesterase (AChE). The paper-based sensor was designed as a foldable device consisting of a cover and detection sheets pre-prepared with indoxyl acetate and AChE, respectively. The paper-based sensor requires only the incubation of a sample on the test zone for 10 minutes, followed by closing of the foldable sheet to initiate the enzymatic reaction. Importantly, the buffer loading hole was additionally designed on the cover sheet to facilitate the interaction of the coated substrate and the immobilized enzyme. This subsequently facilitates the mixing of indoxyl acetate with AChE, resulting in the improved analytical performance of the sensor. The absence or decrease in blue color produced by the AChE hydrolysis of indoxyl acetate can be observed in the presence of OPs and CMs. Under optimized conditions and using image analysis, the limit of detection (LOD) of carbofuran, dichlorvos, carbaryl, paraoxon, and pirimicarb are 0.003, 0.3, 0.5, 0.6, and 0.6 ppm, respectively. The assay could be applied to determine OP and CM residues in spiked food samples. Visual interpretation of the color signal was clearly observed at the concentration of 5 mg/kg. Furthermore, a self-contained sample pre-concentration approach greatly enhanced the detection sensitivity. The paper-based device developed here is low-cost, requires minimal reagents and is easy to handle. As such, it would be practically useful for pesticide screening by nonprofessional end-users.Publication Open Access Investigation of aromatase inhibitory activity of metal complexes of 8-hydroxyquinoline and uracil derivatives(2014-08-14) Veda Prachayasittikul; Ratchanok Pingaew; Virapong Prachayasittikul; Chanin Nantasenamat; Somsak Ruchirawat; Supaluk Prachayasittikul; Mahidol University. Faculty of Medical Technology. Department of Clinical Microbiology and Applied Technology; Mahidol University. Faculty of Medical Technology. Center of Data Mining and Biomedical InformaticsPurpose: Estrogens play important roles in the pathogenesis and progression of breast cancer as well as estrogen-related diseases. Aromatase is a key enzyme in the rate-limiting step of estrogen production, in which its inhibition is one strategy for controlling estrogen levels to improve prognosis of estrogen-related cancers and diseases. Herein, a series of metal (Mn, Cu, and Ni) complexes of 8-hydroxyquinoline (8HQ) and uracil derivatives (4–9) were investigated for their aromatase inhibitory and cytotoxic activities. Methods: The aromatase inhibition assay was performed according to a Gentest™ kit using CYP19 enzyme, wherein ketoconazole and letrozole were used as reference drugs. The cytotoxicity was tested on normal embryonic lung cells (MRC-5) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: Only Cu complexes (6 and 9) exhibited aromatase inhibitory effect with IC50 0.30 and 1.7 µM, respectively. Cytotoxicity test against MRC-5 cells showed that Mn and Cu complexes (5 and 6), as well as free ligand 8HQ, exhibited activity with IC50 range 0.74–6.27 µM. Conclusion: Cu complexes (6 and 9) were found to act as a novel class of aromatase inhibitor. Our findings suggest that these 8HQ–Cu–uracil complexes are promising agents that could be potentially developed as a selective anticancer agent for breast cancer and other estrogen-related diseases.Publication Open Access Quinoline-based clioquinol and nitroxoline exhibit anticancer activity inducing FoxM1 inhibition in cholangiocarcinoma cells(2015-04-08) Waraporn Chan-on; Nguyen Thi Bich Huyen; Napat Songtawee; Wilasinee Suwanjang; Supaluk Prachayasittikul; Virapong Prachayasittikul; Mahidol University. Faculty of Medical Technology. Department of Clinical Microbiology and Applied Technology; Mahidol University. Faculty of Medical Technology. Center of Data Mining and Biomedical InformaticsPurpose: Fork head box M1 (FoxM1) is an oncogenic transcription factor frequently elevated in numerous cancers, including cholangiocarcinoma (CCA). A growing body of evidence documents its diverse functions contributing to tumorigenesis and cancer progression. As such, discovery of agents that can target FoxM1 would be valuable for the treatment of CCA. The quinoline-based compounds, namely clioquinol (CQ) and nitroxoline (NQ), represent a new class of anticancer drug. However, their efficacy and underlying mechanisms have not been elucidated in CCA. In this study, anticancer activities and inhibitory effects of CQ and NQ on FoxM1 signaling were explored using CCA cells. Methods: The effects of CQ and NQ on cell viability and proliferation were evaluated using the colorimetric 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-(4-sulfophenyl)-2H-tetrazolium (MTS assay). Colony formation and cell migration affected by CQ and NQ were investigated using a clonogenic and a wound healing assay, respectively. To demonstrate the agents’ effects on FoxM1 signaling, expression levels of the target genes were quantitatively determined using real-time polymerase chain reaction. Results: CQ and NQ significantly inhibited cell survival of HuCCT1 and Huh28 in a dose- and a time-dependent fashion. Further investigations using the rapidly proliferating HuCCT1 cells revealed significant suppression of cell proliferation and colony formation induced by low doses of the compounds. Treatment of CQ and NQ repressed expression of cyclin D1 but enhanced expression of p21. Most importantly, upon CQ and NQ treatment, expression of oncogenic FoxM1 was markedly decreased concomitant with downregulation of various FoxM1’s downstream targets including cdc25b, CENP-B, and survivin. In addition, the compounds distinctly impaired HuCCT1 migration as well as inhibited expression of matrix metalloproteinase (MMP)-2 and MMP-9. Conclusion: Collectively, this study reports for the first time the anticancer effects of CQ and NQ against CCA cells, and highlights new insights into the mechanism of actions of the quinoline-based compounds to disrupt FoxM1 signaling.Publication Open Access Bioactive triterpenoids, antimicrobial, antioxidant and cytotoxic activities of Eclipta prostrata Linn(2015-03) Rungrot Cherdtrakulkiat; Somchai Boonpangrak; Somsak Ruchirawat; Ratchanok Pingaew; Virapong Prachayasittikul; Supaluk Prachayasittikul; Mahidol University. Faculty of Medical Technology. Department of Clinical Microbiology and Applied Technology; Mahidol University. Faculty of Medical Technology. Center for Innovation Development and Technology Transfer; Mahidol University. Faculty of Medical Technology. Center of Data Mining and Biomedical InformaticsBioactive triterpenoids; 3-acetylaleuritolic acid, stigmasterol, a mixture of triterpenoids, fatty esters and aromatic components were isolated from the aerial parts of Eclipta prostrata by column chromatography. The plant extracts were investigated for their antimicrobial activity (agar dilution method) against twenty-eight strains of gram-positive and gram-negative bacteria, including diploid fungus. In addition, antioxidant and cytotoxic activities were also evaluated.The extracts and isolated fractions exhibited antimicrobial activity against Morexella catarrhalis, Corynebacterium diphtheriae NCTC 10356 and Streptococcus pyogenes with the MIC of 64 g/mL including Saccharomyces cerevisiae ATCC 2601 (MIC 256 g/mL). The ethyl acetate extract and isolated fractions displayed antioxidant effect. In addition, the plant extracts showed cytotoxic activity (ED50 > 100 g/mL) toward HuCCA-1 and KB cells. The results demonstrate beneficial effects of E. prostrata as the antimicrobials and bioactive compounds for medicinal usages.Publication Open Access Antimicrobial and cytotoxic acetogenin from Polyalthia debilis(2015-03) Somchai Boonpangrak; Rungrot Cherdtrakulkiat; Ratchanok Pingaew; Patumporn Manam; Supaluk Prachayasittikul; Somsak Ruchirawat; Virapong Prachayasittikul; Mahidol University. Faculty of Medical Technology. Center for Innovation Development and Technology Transfer; Mahidol University. Faculty of Medical Technology. Department of Clinical Microbiology and Applied Technology; Mahidol University. Faculty of Medical Technology. Center of Data Mining and Biomedical InformaticsObjective: To isolate bioactive acetogenin compound and to investigate antimicrobial, antioxidant as well as cytotoxic activities of the isolate, fractions and extracts of Polyalthia debilis. Methods: The P. debilis (roots) extracts and isolated compound were tested for their antimicrobial (agar dilution method) against twenty-seven strains of microorganisms (gram positive and gram negative bacteria, and diploid fungus), antioxidant (DPPH assay) and cytotoxic activities. The plant extracts were isolated by column chromatography and structure of compound was confirmed by spectral data . Result: The plant extracts and isolated fractions exhibited antioxidant and cytotoxic activities. The isolated acetogenin 1 (debilisome E) displayed antimicrobial activity against Morexella catarrhalis with the MIC of 64 microgram/mL, Corynebacterium diphtheriae NCTC 10356 and Streptococcus pyogenes with partial inhibition (50-75%) at 128 microgram/mL. The compound 1 exerted cytotoxic activity against 5 cancer cells (HepG2, A549, HCC-S102,HL-60 and P388) with IC50 values 18.4 - 40.3 microgram/mL. Conclusion: The results demonstrate novel bioactivities of P. debilis as antimicrobials and anticancer agents.Publication Open Access Bioactive azafluorenone alkaloids from polyalthia debilis (Pierre) finet and gagnep(2009) Supaluk Prachayasittikul; Patumporn Manam; Maneekarn Chinworrungsee; Chartchalerm Isarankura-Na-Ayudhya; Somsak Ruchirawat; Virapong PrachayasittikulThis study investigated bioactive extracts of Polyalthia debilis (Annonaceae) with antimicrobial, antimalarial and cytotoxic activities. Extensive chromatographic isolations provided azafluorenone alkaloids; onychine (1) and 7-methoxyonychine (2) together with a mixture of β–sitosterol and stigmasterol. The two alkaloids were isolated from the P. debilis for the first time. Isolated fractions containing a mixture of triterpenoids (C7, C8 and C9) exhibited the most potent antimicrobial activity against many bacterial strains with minimum inhibitory concentration of 64 μg/mL. Fractions with antimalarial and cytotoxic activities were also observed. The findings suggest the potential use of P. debilis in medicinal applications.Publication Open Access Synthesis of a “clickable” Angiopep-conjugated p-coumaric acid for brain-targeted delivery(2014-08-19) Thummaruk Suksrichavalit; Supaluk Prachayasittikul; Virapong Prachayasittikul; Chartchalerm Isarankura Na Ayudhya; Mahidol University. Faculty of Medical Technology. Center of Data Mining and Biomedical Informatics; Mahidol University. Faculty of Medical Technology. Department of Clinical Chemistry; Mahidol University. Faculty of Medical Technology. Department of Clinical Microbiology and Applied TechnologyOverexpression of free radicals in the brain is emerging as important markers in the etiology of neurodegenerative diseases including Parkinson’s disease, Alzheimer’s disease, and stroke. Numerous antioxidants with protective effect on neuronal injuries under oxidative stress are often limited to penetrate the blood–brain barrier (BBB). Angiopep-2 is the ligand of low-density lipoprotein receptor-related protein expressed on the BBB possessing high transcytosis capacity and parenchymal accumulation. In this study, novel Angiopep-conjugated p-coumaric acid (3) was synthesized, using the Click chemistry, as a potential antioxidant for the protection of the brain under oxidative stress. The clickable Angiopep (3) was synthesized by Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction of the terminal acetylene-modified Angiopep and azide of p-coumaric acid. The Angiopep-conjugated compound (3) showed antioxidant potency and non-cytotoxic effect toward brain endothelial cells (BECs). Obviously, the penetration and BECs protection of 3 were higher than that of the unconjugated p-coumaric acid. The results establish the bio-conjugation of antioxidant and Angiopep with enhanced protective effect on the BECs under oxidative stress. The findings provide great potential for the development of neurotherapeutics with increased brain penetration.Publication Open Access Elucidating the structure-activity relationships of the vasorelaxation and antioxidation properties of thionicotinic acid derivatives(2010) Supaluk Prachayasittikul; Orapin Wongsawatkul; Apilak Worachartcheewan; Chanin Nantasenamat; Somsak Ruchirawat; Virapong PrachayasittikulNicotinic acid, known as vitamin B3, is an effective lipid lowering drug and intense cutaneous vasodilator. This study reports the effect of 2-(1-adamantylthio)nicotinic acid (6) and its amide 7 and nitrile analog 8 on phenylephrine-induced contraction of rat thoracic aorta as well as antioxidative activity. It was found that the tested thionicotinic acid analogs 6-8 exerted maximal vasorelaxation in a dose-dependent manner, but their effects were less than acetylcholine (ACh)-induced nitric oxide (NO) vasorelaxation. The vasorelaxations were reduced, apparently, in both NG-nitro-L-arginine methyl ester (L-NAME) and indomethacin (INDO). Synergistic effects were observed in the presence of L-NAME plus INDO, leading to loss of vasorelaxation of both the ACh and the tested nicotinic acids. Complete loss of the vasorelaxation was noted under removal of endothelial cells. This infers that the vasorelaxations are mediated partially by endothelium-induced NO and prostacyclin. The thionicotinic acid analogs all exhibited antioxidant properties in both 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide dismutase (SOD) assays. Significantly, the thionicotinic acid 6 is the most potent vasorelaxant with ED50 of 21.3 nM and is the most potent antioxidant (as discerned from DPPH assay). Molecular modeling was also used to provide mechanistic insights into the vasorelaxant and antioxidative activities. The findings reveal that the thionicotinic acid analogs are a novel class of vasorelaxant and antioxidant compounds which have potential to be further developed as promising therapeutics.Publication Open Access Origin of aromatase inhibitory activity via proteochemometric modeling(2016-04) Saw Simeon; Ola Spjuth; Maris Lapins; Chanin Nantasenamat; Jarl ES Wikberg; Virapong Prachayasittikul; Mahidol University. Faculty of Medical Technology. Center of Data Mining and Biomedical InformaticsAromatase, the rate-limiting enzyme that catalyzes the conversion of androgen to estrogen, plays an essential role in the development of estrogen-dependent breast cancer. Side effects due to aromatase inhibitors (AIs) necessitate the pursuit of novel inhibitor candidates with high selectivity, lower toxicity and increased potency. Designing a novel therapeutic agent against aromatase could be achieved computationally by means of ligand-based and structure-based methods. For over a decade, we have utilized both approaches to design potential AIs for which quantitative structure-activity relationships and molecular docking were used to explore inhibitory mechanisms of AIs towards aromatase. However, such approaches do not consider the effects that aromatase variants have on different AIs. In this study, proteochemometrics modeling was applied to analyze the interaction space between AIs and aromatase variants as a function of their substructural and amino acid features. Good predictive performance was achieved, as rigorously verified by 10-fold cross-validation, external validation, leave-one-compound-out cross-validation, leave-one-protein-out cross-validation and Y-scrambling tests. The investigations presented herein provide important insights into the mechanisms of aromatase inhibitory activity that could aid in the design of novel potent AIs as breast cancer therapeutic agents.