1. Lecture 8 Bioinformatics of TCM Y.Z. Chen Department of Pharmacy National University of Singapore Tel: 65-6616-6877; Email: phacyz@nus.edu.sg ; Web: http://bidd.nus.edu.sg phacyz@nus.edu.sghttp://bidd.nus.edu.sgphacyz@nus.edu.sghttp://bidd.nus.edu.sgContent Overview of TCMOverview of TCM TCM and systems biologyTCM and systems biology Computer methods and databases of TCMComputer methods and databases of TCM Bioinformatics and TCMBioinformatics and TCM

2. TCM History  The history of TCM can be summarized by a list of important doctors and books.  Unknown, Huángdì nèijīng ( 黃帝內經 / 黄帝内经 ) (Yellow Emperor's Inner Canon) - Sùwèn ( 素问 / 素問 ) and Língshū ( 灵枢 / 靈樞 ). The earliest classic of TCM passed on to the present.  Warring States Period (5th century BC to 221 BC): Silk manuscripts recording channels and collaterals, Zubi shiyi mai jiu jing ( 足臂十一脉灸经 / 足臂十一脈灸經 ) (Moxibustion Classic of the Eleven Channels of Legs and Arms), and Yinyang shiyi mai jiu jing ( 阴阳十一脉灸经 / 陰 陽十一脈灸經 ) (Moxibustion Classic on the Eleven Yin and Yang Channels). The latter was part of a cache of texts found in Mawangdui in the 1970s.

3. TCM History  Han Dynasty (206 BC–AD 220) to Three Kingdoms Period (220 - 280 AD):  Zhenjiu zhenzhong jing ( 针灸枕中经 / 鍼灸枕中經 ) (Classic of Moxibustion and Acupuncture Preserved in a Pillow) by Huà Tuó ( 华佗 / 華佗 ).  Shanghan zabing lun ( 伤寒杂病论 / 傷寒雜病論 ), which has since been split into two texts: the Shānghán lùn ( 伤寒论 / 傷寒論 ) ("Treatise on Cold Damage [Disorders]" - focusing on febrile conditions attributed to "Cold") and the Jingui yaolue ( 金匱要略 ) ("Essentials of the Golden Cabinet" - focusing on "miscellaneous illnesses") by Zhāng Zhòngjǐng ( 张仲景 / 張仲景 ).  Jìn Dynasty (265-420): Zhēnjiǔ jiǎyǐ jīng ( 针灸甲乙经 / 鍼 灸甲乙經 ) (Systematic Classic of Acupuncture and Moxibustion) by Huángfǔ Mì ( 皇甫谧 / 皇甫謐 ).

4. TCM History  Tang Dynasty (618–907)  Beiji qianjin yaofang ( 备急千金要方 / 備急千金要方 ) (Emergency Formulas Worth a Thousand in Gold) and Qianjin yifang ( 千金翼 方 ) (Supplement to the Formulas Worth a Thousand in Gold) by Sūn Sīmiǎo ( 孙思邈 / 孫思邈 ).  Waitai miyao ( 外台秘要 / 外臺秘要 ) (Arcane Essentials from the Imperial Library) by Wang Tao ( 王焘 / 王燾 ).  Song Dynasty (960 – 1279):  Tóngrén shūxué zhēnjiǔ tújīng ( 铜人腧穴针灸图经 / 銅人腧穴鍼灸 圖經 ) (Illustrated Manual of the Practice of Acupuncture and Moxibustion at (the Transmission) (and other) Acu-points, for use with the Bronze Figure) by Wáng Wéiyī ( 王惟一 ).  Yuan Dynasty (1271 to 1368): Shísì jīng fāhuī ( 十四经发 挥 / 十四經發揮 ) (Exposition of the Fourteen Channels) by Huá Shòu ( 滑寿 / 滑壽 ).

5. TCM History  Ming Dynasty (1368 to 1644): golden age of acupuncture and moxibustion. Many famous doctors and books. To name only a few:  Zhēnjiǔ dàquan ( 针灸大全 / 鍼灸大全 ) (A Complete Collection of Acupuncture and Moxibustion) by Xu Feng ( 徐凤 / 徐鳳 ).  Zhēnjiǔ jùyīng fāhuī ( 针灸聚英发挥 / 鍼灸聚英發揮 ) (An Exemplary Collection of Acupuncture and Moxibustion and their Essentials) by Gāo Wǔ ( 高武 ).  Zhēnjiǔ dàchéng ( 针灸大成 / 鍼灸大成 ) (Compendium of Acupuncture and Moxibustion) by Yáng Jìzhōu ( 杨继洲 / 楊繼洲 ), completed in 1601.  Běncǎo gāngmù ( 本草纲目 / 本草綱目 ) (Compendium of Materia Medica) by Lǐ Shízhēn ( 李时珍 / 李時珍 ), the most complete and comprehensive pre-modern herbal book (completed in 1578).  Wenyi lun ( 温疫论 / 溫疫論 ), by Wu Youxing 吴有性 (1642).

6. TCM History  Qing Dynasty (1644-1912):  Yizong jinjian ( 医宗金鉴 / 醫宗金鑒 ) (Golden Mirror of the Medical Tradition) compiled by Wu Qian ( 吴谦 / 吴謙 ) under imperial commission.  Zhenjiu fengyuan ( 针灸逢源 / 鍼灸逢源 ) (The Source of Acupuncture and Moxibustion) by Li Xuechuan ( 李学川 / 李學川 ).  Wenre lun ( 温热论 / 溫熱論 ), by Ye Tianshi ( 叶天士 / 業天士 ).  Wenbing tiaobian ( 温病条辨 / 溫病條辨 ) (Systematized Identification of Warm-factor disorders) compiled by Wu Jutong ( 吴鞠通 ) in 1798.

7. Classification of TCM herbs based on traditional therapeutic classes

10. Classification of TCM herbs in traditional therapeutic classes

11. Classification of TCM herbs based on traditional therapeutic classes

12. TCM Therapeutics  Potentially novel therapeutic approaches  Existing markets in certain countries and potential market in others Pharmacology & Therapeutics 2000, 86:191-198

13. TCM Therapeutics  Need for standardization, validation, & further development  Markers for standardization  Molecular mechanism  Safety and efficacy tests  New recipes, combination drug mimics Pharmacology & Therapeutics 2000, 86:191-198

14. TCM Therapeutics  Multiple herb therapies  Collective synergistic actions, maintenance and balance.  Mutual accentuation mutual enhancement mutual counteraction mutual suppression mutual antagonism mutual incompatibility Pharmacology & Therapeutics 2000, 86:191-198

15. TCM Therapeutics  Multiple targets:  therapeutic effects  symptom treatment  toxicity modulation  Immune regulation  drug delivery  energy metabolism  harmonization Pharmacology & Therapeutics 2000, 86:191-198

16. TCM Therapeutics

18. Systems: the way for TCM to produce clinical effects Fact1: Weak effect of components Fact2: Validated effect of TCM by clinical practice Inference: Interaction among components Direct Interaction (seldom) Indirect Interaction (often) TCM related Biological system Systems are the targets of TCM function TCM function

19. Targeting systems: interpreting the mechanism of TCM function Direct Interaction (seldom) Indirect Interaction (often) TCM related Biological system Mechanism of TCM function

20. Targeting systems: bridging biology and TCM theory Direct Interaction (seldom) Indirect Interaction (often) TCM related Biological system TCM function TCM theory

21. Targeting systems: providing new cues for the treatment of complex disease Direct Interaction (seldom) Indirect Interaction (often) TCM related Biological system TCM function TCM theoryValidated clinical prescription

22. Targeting systems: providing new resource for drug development Direct Interaction (seldom) Indirect Interaction (often) TCM related Biological system TCM function TCM theoryValidated clinical prescription Impact based molecular target selection “Remote” interruption of systems Complex drugs

23. A Proposed Platform for System- targeting TCM research Database TCM pathway mol targets Active compnts bio systems

24. Biological Systems related with TCM Prescriptions that Promoting Blood Circulation and Removing Blood Stasis Map00590:prostaglandin and leukotriene metabolism Map00230: purine metablism Map00100: biosynthesis of steroids Map00531: glycosaminoglycan degradation Map00010: glycolysis /gluconeogenesis

25. Gaëlle Hardy, et. al. Urinary leukotriene E4 excretion is increased in type 1 diabetic patients A quantification by liquid chromatography–tandem mass spectrometry. Prostaglandins Other Lipid Mediat. 2005. 78(1-4):291-299 220 paper’s title in 中国期 刊网 CNKI containing: promoting blood circulation and removing blood stasis + diabetes Biological Systems related with TCM Prescriptions that Promoting Blood Circulation and Removing Blood Stasis

26. Computer Analysis of Molecular Mechanism of TCM Y.Z. Chen and D.G. Zhi Proteins 2001;43: 217 Traditional Medicine Database  Herbal Ingredient & Content Database  Therapeutic Target Database   Drug Adverse Reaction Target Database   Drug Absorption Distribution Metabolism Excretion Database   Computer Match-Making Software  Herbal Ingredient Therapeutic Target Toxicity Target ADME protein  Collective therapeutic and maintenance effects  Toxicity / side effects and modulation  Drug delivery and clearance Matched Targets Mutual Enhancement ? Mutual Counteraction ? Maintenance or Balance ? Delivery or Clearance ?

27. Medicinal Herb Databases at BIDD Comparison with existing TCM databases: Formula: TCM-ID: 1000 TCHFL: 270 Herb: TCM-ID: 1200 TCSHL: 520 TCMD: 1500 Compound: TCM-ID: 9000 CNPD: 3000 TCMD: 6800 TCM Formula Herb Compound Protein Function Structure TCMD TCHF Library CNPD TCSH Library TCM-ID: Traditional Chinese Medicine - Information Database   Only database providing integrated and comprehensive info about: TCM formula, constituent herbs, herbal ingredients, effect on proteins Molecular structure Function at the formula, herb and compound levels

28. What bioinformatics can do in TCM 1R4L from PDB Pathway form KEGG

29. General Protein–Ligand Binding Ligand - Molecule that binds with a protein - DNA, drug lead compounds, etc. Protein active site(s) - Allosteric binding - Competitive binding Function of binding interaction - Natural and artificial

30. . Design of HIV-1 Protease Inhibitor

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34. Scoring in Ligand-Protein Docking Potential Energy Description:

35. DAHAHA/DAH/HE ACE177981886563525E-040.7916.02 AngII177981532491724900.0031.003.33 PDE4177981285312971680.0020.543.35 5-HT3177981278407222000.0020.493.14 HMG-CoA177981215337181360.0010.403.34 D ： number of molecules in database A ： number of active molecules in database H ： number of hit molecules AH ： number of active molecules in hitlist Virtual Screening

36. HMG -CoA T-cell PDE TNF ɑ COX2 AngII

37. INVDOCK Strategy for Match Making Science 1992;257: 1078 Proteins 1999; 36:1

38. INVDOCK Test on INVDOCK Test on Targets of Chinese Medicinal Herbal Ingredients (Am. J. Chin. Med. 2002, 30, 139) Chinese Natural Product Number of Identified Putative and Known Therapeutic Targets Number Confirmed or Implicated Therapeutic Targets by experiment Number of Identified Putative and Known Toxicity/Side effect Targets Number Confirmed or Implicated Toxicity/Side Effect Targets by experiment Acronycine314- Allicin5211 Baicalin1446- Catechin17125- Camptothecine9632 Dicoumarin7131 Emodin6351 Genistin227121

39. INVDOCK Test on TCM Target Prediction INVDOCK Test on TCM Target Prediction Therapeutic targets of Camptothecine (Am. J. Chin. Med. 2002, 30, 139) PDBProteinExperimental FindingTarget Status Theraputic EffectRef 1adsAldose Reductase Diabetes treatment 2gssGlutathione S- Transferase p1-1 Increases intracellular glutathione ImplicatedEnhance radical scavenging activities that may useful in cancer treatment Matsumoto 7iceDNA Polymerase Beta Anti-cancer 1a25Protein Kinase CInhibitorConfirmedInduction of apoptosis in tumor. Martelli Nieve-Neira 1cdkCAMP-Dependent Protein Kinase Anti-cancer 3bctBeta-CateninInduces apoptosis in leukemic cells. 1dviCalpainInhibition of calpain activities.Anti-cancerEymin 1yfoReceptor Protein Tyrosine Phosphatase Causes elevation of PTPase in the cytosol and the nucleus which play a critical role in the induction of the differentiation of IW32 erythroleukemia cells. Anti-cancerWang M 1a35Topoisomerase IInhibitorWang MC

40. INVDOCK Test on TCM Target Prediction INVDOCK Test on TCM Target Prediction Toxicity and side effect targets of Camptothecine (Am. J. Chin. Med. 2002, 30, 139) PDBProteinExperimental Finding Target Status Toxicity/Side Effect Ref. 2cljAcetylcholinesteraseReversible inhibition ConfirmedCauses Cholinergic ToxicityDodds 1aqbRetinol-Binding Protein Interfering with retinol transport that may cause complication in cancer and cardiovascular disease 5rlaArginaseIncrease blood urea level Implicate d Hyperammonemia that may cause nerve toxicity Yu ZJ

41. Network Regulation by Anticancer TCM Anticancer Effect of 贯叶连翘 St.John's Wort Drug Discov Today 2009, 14, 579 Hypericin Hyperforin Cancer Counteraction

42. Synergistic Network Regulation by Herbal Product Synergistic Anti-HIV effect of Rosa damascena 突厥蔷薇 Drug Discov Today 2009, 14, 579 AIDS-058145: 2-Phenylethanol-O-(6-O-galloyl)-beta-D-glucopyranoside 8

43. Chinese Medicinal Herbs All Other Herbs All ingredients of the other herbs Interior Warming Herbs All ingredients of interior warming herbs Exclusive ingredients COMPARE Bioinformatics Study of Mechanism of TCM Herbs Interior Warming Herbs as an Example Pharmacological effects possibly linked to interior warming Other Pharmacological and regulatory effects Interior warming effects primarily come from these ingredients

44. Pharmacological Effects Possibly Linked to Interior Warming

45. Vasodilation and its Relation to Interior Warming Common mode of action (except Hua Jiao) –Inhibit Ca 2+ influx Other Modes of actions –Increase NO –Increase cGMP –Activate TRPV1 –Alpha-adrenoceptor blockade Why vasodiation produces warming effect or sensation? –Blushing A result of vasodilation Warm feeling –Cold extremeties due to loss of beta-adrenoceptor mediated vasodilation –Increased lumen size of blood vessels More blood brought to vasodilated area  Warm sensation 1 Rang HP, Dale MM, Ritter JM, P.K. M. Pharmacology. 5th ed. Edinburgh ; New York: Churchill Livingstone, 2003

46. Pharmacological Effects Possibly Linked to Interior Warming

47. TRPV1 Agonism and its Relation to Interior Warming Transient receptor potential ion channel of vanilloid type 1 –Highly expressed in a subset of primary sensory neurons of the trigeminal, vagal and dorsal root ganglia –Non-neuronal cells 1 1 Geppetti P, Materazzi S, Nicoletti P. Eur J Pharmacol 2006;533(1-3):207-14. Activation leads to many effects –Burning sensation –Vasodilation via release of Calcitonin gene related peptide (CGRP) –Inhibit gastric leisions –Thermogenesis Piperine, 6-gingerol, 10-gingerol, 6-shogaol Associated with vasoconstriction

48. TRPV1 Agonism and its Relation to Interior Warming Dual actions leading to the same warming effect or sensation? 1.TRPV1 causes both vasodilation and vasoconstriction under different conditions 1 –Directly induce vascular smooth muscle contraction, linked to thermogenesis that is abolished by vasodilators –But release neuropeptides e.g. CGRP that causes vasodilation 1 Eldershaw T, Colquhoun E, Bennett K et al. Life Sci 1994;55(5):389-97. VasodilationThermogenesis Bi BaBisabolenePiperine (Abolished by Bisabolene?) Gan Jiang 10-gingerol (Net-effect?) Effect Herb 6-shogaol6-gingerol 6-shogaol6-gingerol 2.TRPV1 agonists reported to result in hypothermia due to response to thermogenesis and activation of warm sensors 1, –False sense of warmth –Not truly ‘therapeutic’

49. Acknowledgement Current Group Members: Computer-Aided Drug Design: CY Ung, XH Ma, XH Liu, Pankaj Kumar, F Zhu, X Liu, J Jia Protein Function, Interaction, Network: HL Zhang, CY Ung, XH Ma, F Zhu, WK Teo, Z Shi Databases and Servers: J Jia Medicinal Herb: CY Ung, Pankaj Kumar, Cao Jinyi(undergraduate students) Microarray and biomarkers: J Jia, ZQ Tang Former Members: PhD: ZW Cao (Prof SCBIT, Tongji U), ZL Ji (Assoc Prof Xiamen U), X Chen (Assoc Prof Zhejiang U), CW Yap (Assist Prof NUS), LY Han (Postdoc NIH), CJ Zheng (Postdoc NIH), HH Lin (Postdoc Harvard ), J Cui (Postdoc U Georgia), H Li (Postdoc Einstein College Med) Research Fellow/Assistant: ZR Li (Assoc Prof SiChuan U), Y Xue (Prof SiChuan U), W Liu (Assoc Prof DUT), D Mi (Assoc Prof DUT), CZ Cai (Prof ChongQing U), DG Zhi (Postdoc, Berkeley), MSc: Y.J. Guo (Postdoc NIH), L.Z. Sun (RA, U Tenn.), J. F. Wang (MSU), L.X. Yao (Columbia), S Ong (Washington U), H Zhou (local company), B Xie (local company) BSc: W.K. Yeo (IMCB, Novartis)