Enzymes and enzyme inhibitors in Diagnosis and Therapy

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1 Enzymes and enzyme inhibitors in Diagnosis and Therapy Dr Phaedra Eleftheriou Department of Medical Laboratory Studies School of Health and Medical Care Alexander Technological Educational Institute of Thessaloniki, Greece

2 Enzymes: carbohydrates catalyze all reaction in human body proteins aminoacids NH 3 Carbon backbone CΗ 2OH Η Η Ο Η OH Η OH OH Η OH glucose glycolytic cycle pyrouvate triglycerides glycerol Fatty acids Enzyme Deficiency/ Overactivation Disease urea CH 3 CO-S-CoA Krebs cycle Respiratory chain Oxydative phosphorylation ΑΤΡ energy Disease Tissue Tissue lesions lesions Enzyme overexpression Enzyme Increased Serum Enzyme concentration

3 Enzymes: carbohydrates catalyze all reaction in human body proteins aminoacids NH 3 urea Carbon backbone CΗ 2OH Η Η Ο Η OH Η OH OH Η OH glucose glycolytic cycle pyrouvate CH 3 CO-S-CoA Krebs cycle Respiratory chain Oxidative phosphorylation ΑΤΡ energy triglycerides glycerol Fatty acids Enzyme Deficiency/ Overactivation Increase of intermediate metabolite Decrease in final product Disease Undesired side products and effects

4 pentose Enzymes: catalyze all reaction in human body phosphogluconate Phosphogluconate (pentose phosphate) pathway G6PD glucose Enzyme Deficiency G6PD Disease Hemolysis (in oxidative conditions) NADPH 2 a valuable product for mature erythrocyte which lacks mitochondrions and Krebs cycle Lack of NAD(P)H 2 Hb oxidation hemolysis

5 Enzymes: catalyze all reaction in human body phenylacetate phenylpyruvate Phenylalanine tyrosine Phenylalanine hydroxylase Enzyme Deficiency G6PD Protein metabolism Phenylalanine hydroxylase Disease Hemolysis (in oxidative conditions) hyperphenylalaninemia. Phenylketonuria (mental retardation) normal metabolism

6 Enzymes: catalyze all reaction in human body tyrosin Enzyme Deficiency Disease homogentisic acid Homogentisate oxidase G6PD Protein metabolism Hemolysis (in oxidative conditions) Phenylalanine hydroxylase hyperhenylalaninemia. Phenylketonuria (mental retardation) Omogentisate oxidase Homogentisuria, alkaptonuria, (kidney stones, heart valve impairment, acidosis, arthritis, blueblack skin spots) Fumarate, acetoacetate

7 Enzymes: catalyze all reaction in human body 6 CΗ 2 OH 5 ΟΗ Ο Η 4 1 OH Η H 3 2 Η Η OH β-d-galactose O lactose 6 CΗ 2 OH 5 Η Ο ΟΗ Η 4 1 OH Η 3 2 H Η OH β-d-glucose lactase Enzyme Deficiency Carbohydrate metabolism Disease Η CΗ 2 OH Η Ο Η OH OH Η OH Η OH glucose CΗ 2 OH OΗ Η Ο Η H OH Η OH Η OH galactose lactase Lactose intolerance galactose-1-phosphate uridyl transferase, galactosemia galactokinase galactokinase galactose-1-p uridyl transferase UDP-glucose

8 Enzymes: catalyze all reaction in human body O 3 SO COO Iduronic acid dermatane NHCOCH 3 Enzyme Deficiency Disease COO O 3 SO Carbohydrate metabolism OSO3 ηπαρίνη heparane HNSO 3 lactase Lactose intolerance galactose-1-phosphate uridyl transferase, galactosemia galactokinase alpha-l iduronidase mucopolysaccharidosis type I (MPS I), Hurler's disease (accumulation of heparane sulfate & dermatane sulfate in lysosomes Hepatosplenomegaly, mental retardation)

9 Enzymes: catalyze all reaction in human body O 3 SO COO Iduronic acid dermatane NHCOCH 3 Enzyme Deficiency Disease COO O 3 SO Carbohydrate metabolism OSO3 ηπαρίνη heparane HNSO 3 lactase Lactose intolerance galactose-1-phosphate uridyl transferase, galactosemia galactokinase alpha-l iduronidase mucopolysaccharidosis type I (MPS I), Hurler's disease (accumulation of heparane sulfate & dermatane sulfate in lysosomes Hepatosplenomegaly, mental retardation) Heparane-N-sulfatase L-iduronate-2-sulfatase β-galactosidase... accumulation of heparane, dermatane, keratane in lysosomes

10 Enzymes: catalyze all reaction in human body O o P O CH 2 CH 2 NH 2 1 OH H H 2 C H φωσφοαιθανολαμίνη 2 ΗC ΝH- CO-R 3 HC OH 4 CH 5 CH 6 CH 2 7 CH 2 8 CH 2 9 CH 2 10 CH 2 11 CH 2 12 CH 2 13 CH 2 14 CH 2 15 CH 2 16 CH 2 17 CH 2 18 CH 3 σφιγγοσίνη Enzyme Deficiency Disease Lipid metabolism sphigomyelinase Sphingomyelin storage in lysosomes (Niemann-Pick disease) (ataxia, disarthria, disphagia, demedia etc. ) ceramidase Lipid storage in lysosomes (Farber disease) (moderately impaired mental ability, swallowing problems)

11 apoε receptors / Chylomicron remnant receptors apoβ Ε / LDL receptors Enzymes: insulin catalyze all reaction in human body 70% LPL VLDL chylomicrons Chylomicron remnants + + insulin αποcii (HDL) insulin αποcii (HDL) LPL IDL insulin LPL LDL + apoβ Ε / LDL receptors other tissues Adrenal gland adipose tissue Enzyme Deficiency Lipid metabolism Disease Lipoprotein lipase (LPL) Hyperlipidemia type I

12 apoε receptors / Chylomicron remnant receptors apoβ Ε / LDL receptors Enzymes: insulin catalyze all reaction in human body 70% LPL VLDL chylomicrons Chylomicron remnants + + insulin αποcii (HDL) insulin αποcii (HDL) LPL IDL insulin LPL LDL + apoβ Ε / LDL receptors other tissues Adrenal gland adipose tissue Enzyme Deficiency Lipid metabolism Disease apoε receptors Lipoprotein lipase (LPL) Hyperlipidemia type I macrophages μακροφάγα apoα I receptors Lecithin-cholesterol acyltransferase (LCAT) Hyperlipidemia HDL 1 apoa-i, apoe apoc-i, apoc-ii Εστέρας χολεστερόλης HDL 1 απο A1 HDL 2 εστέρες Cholesterol χοληστερόλης esters LCAT από-α1 apo apoc-i, apoc-ii apoe LCAT (lecithin-cholesterole acyltransferase) Cholesterol esters HDL 3 Ελεύθερη χολεστερόλη Υπολειματικά χυλομικρά IDL Ελεύθερη cholesterol χολεστερόλη Dr Phaedra Eleftheriou, κυτταρική μεμβράνη Department of Medical Laboratory Studies, Alexander TEITH, Greece

13 Enzymes: catalyze all reaction in human body Enzyme oneractivation SHP-2 a phosphatase involved in cell-cycle regulation Disease Noonan Syndrom Juvenil Myelomonocytic Leuchemias HDL 1

14 Enzymes: catalyze all reaction in human body Enzyme Inhibitor defficiency Disease A1-antitrypsin (serum protease, mainly elastase, inhibitor) emphysema HDL 1

15 Enzyme /enzyme inhibitor deficiency Administration of the enzyme/inhibitor Therapy? Insertion of gene (gene therapy)

16 Enzyme /enzyme inhibitor deficiency Administration of the enzyme/inhibitor Therapy

17 Enzyme /enzyme inhibitor deficiency Therapy Administration of the enzyme/inhibitor

18 Enzyme /enzyme inhibitor deficiency Therapy Production of enzyme/inhibitor Administration of the enzyme/inhibitor

19 Enzyme /enzyme inhibitor deficiency Transgenic animals Therapy Production of enzyme/inhibitor Administration of the enzyme/inhibitor

20 Enzyme /enzyme inhibitor deficiency Transgenic animals Therapy Transgenic goat carrying human gene for a1-antitrypsin Production of enzyme/inhibitor A1-antitripsin Administration of the enzyme/inhibitor

21 Enzyme enzyme inhibitor deficiency Therapy Gene therapy

22 Enzyme enzyme inhibitor deficiency Therapy Production of enzyme/inhibitor in the body Gene therapy

23 Enzyme enzyme inhibitor deficiency Insertion of gene into appropriate carrier Therapy gene administration Production of enzyme/inhibitor in the body Gene therapy

24 Enzyme inhibitors in therapy Enzymes as drug targets Many drugs are Enzyme Inhibitors Anti-inflammatory drugs COX-1, COX-2 inhibitors Arachidonic acid prostaglandin COX-1 COX-2 pain

25 Enzyme inhibitors in therapy Enzymes as drug targets Many drugs are Enzyme Inhibitors Hypolipidemic drugs Inhibitors of of endogenous cholesterol synthesis HMG-CoA reductase inhibitors:statines Acetyl-CoA HMG-CoA reductase mevalonic acid cholesterole synthesis chylomicrons liver Squalene Synthase inhibitors Squalene VLDL cholesterol

26 Enzyme inhibitors in therapy Enzymes as drug targets Many drugs are Enzyme Inhibitors Hypolipidemic drugs Inhibitors of of endogenous cholesterol synthesis (statines : : simvastatin..) LPL LPLactivators LCAT activators resins antioxudants LPL apoε receptors / Chylomicron remnant receptors VLDL chylomicrons Chylomicron remnants + + insulin insulin αποcii (HDL) αποcii (HDL) apoβ Ε / LDL receptors insulin LPL apoa-i, apoe apoc-i, apoc-ii Cholesterol Εστέρας χολεστερόλης esters HDL 1 αποa1 HDL 2 LCAT (lecithin-cholesterole acyltransferase) Ελεύθερη χολεστερόλη Υπολειματικά χυλομικρά IDL LPL εστέρες χοληστερόλης από-α1 apo 70% IDL LDL + insulin apoβ Ε / LDL receptors other tissues Adrenal gland adipose tissue apoε receptors macrophages μακροφάγα apoα I receptors apoc-i, apoc-ii apoe Cholesterol esters HDL 1 cholesterol Ελεύθερη χολεστερόλη κυτταρική μεμβράνη HDL 3

27 Enzyme inhibitors in therapy Enzymes as drug targets Many drugs are Phosphatase Inhibitors Κinases phosphatases: biochemical pathways regulation kinases + + ATP OH kinase O O-P-OH OH + ADP phosphatases O O-P-OH OH phosphatase + H 2 O OH + O HO-P- OH OH biochemical pathways regulation

28 Phosphatases with possible pharmaceutical interest Enzyme inhibitors in therapy Enzymes as drug targets Many drugs are Phosphatase Inhibitors Phosphatases: potential drug targets PTP1B Glucose uptake πρόσληψη γλυκόζης Treatment of χρήση των αναστολέων της PTP1B για θεραπεία διαβήτη diabetes type II τύπου II SHP-2 Cell πολλαπλασιασμό proliferation και μετανάστευση κυττάρων αυξημένη δραστηριότητα Noonan Syndrom εξαιτίας μεταλλάξεων σε σύνδρομο certain Νοonan leucemias και τύπους καρκίνου CD45 Anticancer action αντικαρκινική δράση EPM2A laforin Accumulation of συσσώρευση Lafora σωματιδίων Inclusion Lafora bodies Mutations μεταλλαγμένη found στην in ασθένεια Lafora disease Lafora PTP-ε Osteoclastic process οστεοκλαστική διαδικασία RPTP-β/ζ Dendritic επιβίωση ολιγο- cell δενδροκυττάρων survival LAR -RPTP Εισαγωγή γλυκόζης Glucose στα κύτταρα uptake

29 Enzyme inhibitors in therapy Enzymes as drug targets Diabetes therapy PTP1B, LAR Inhibitors Diabetes Type Ι inefficient synthesis of insulin Type ΙΙ Inefficient interaction with insulin receptors Type ΙΙΙ Other reasons

30 Enzyme inhibitors in therapy Enzymes as drug targets Diabetes therapy PTP1B, LAR Inhibitors Mechanism of glucose uptake glucose Insulin receptor (inactive)

31 Enzyme inhibitors in therapy Enzymes as drug targets Diabetes therapy PTP1B, LAR Inhibitors Mechanism of glucose uptake insulin insulin receptor (active) active (kinase activity) glucose gene expression active Glycogen, lipid, Protein synthesis

32 Enzyme inhibitors in therapy Enzymes as drug targets Diabetes therapy PTP1B, LAR Inhibitors Mechanism of glucose uptake insulin insulin receptor (active) active (kinase activity) glucose phosphatase activity gene expression active Glycogen, lipid, Protein synthesis

33 Enzyme inhibitors in therapy Enzymes as drug targets Diabetes therapy PTP1B, LAR Inhibitors Mechanism of glucose uptake insulin insulin receptor (active) active (kinase activity) glucose phosphatase activity PTP1B LAR gene expression active Glycogen, lipid, Protein synthesis

34 Enzyme inhibitors in therapy Enzymes as drug targets Diabetes therapy PTP1B, LAR Inhibitors Mechanism of glucose uptake glucose Insulin receptor (inactive)

35 Enzyme inhibitors in therapy Enzymes as drug targets Diabetes therapy PTP1B, LAR Inhibitors Mechanism of action of PTP1B/LAR Inhibitors Inhibition insulin receptor (active) insulin PTP1B LAR inactivation active (kinase activity) glucose active gene Glycogen, lipid, expression Protein synthesis Extention of glucose intake period

36 Enzyme inhibitors in therapy Enzymes as drug targets Anti-microbial // anti-viral therapy anti-hiv agents HIV / AIDS HIV infection Acquired Immunodeficiency Syndrome(AIDS)

37 Enzyme inhibitors in therapy Enzymes as drug targets Anti-microbial // anti-viral therapy anti-hiv agents HIV / AIDS HIV types: HIV-1 (responsible for AIDS epidemic) HIV-2

38 Enzyme inhibitors Κύκλος ζωής in therapy του ιού Enzymes as drug targets Anti-microbial // anti-viral therapy anti-hiv agents Retrovirus Genetic information carrier: RNA virus cycle

39 Enzyme inhibitors Κύκλος ζωής in therapy του ιού Enzymes as drug targets Anti-microbial // anti-viral therapy anti-hiv agents Retrovirus :Genetic information carrier: RNA virus cycle 1. Introduction into the host cell (CD4+)

40 Enzyme inhibitors Κύκλος ζωής in therapy του ιού Enzymes as drug targets Anti-microbial // anti-viral therapy anti-hiv agents Retrovirus :Genetic information carrier: RNA virus cycle 1. Introduction into the host cell (CD4+) 2. Reverse transcription (RNA DNA)

41 Enzyme inhibitors Κύκλος ζωής in therapy του ιού Enzymes as drug targets Anti-microbial // anti-viral therapy anti-hiv agents Retrovirus :Genetic information carrier: RNA virus cycle 1. Introduction into the host cell (CD4+) 2. Reverse transcription (RNA DNA) 3. Integration of genetic material with host DNA

42 Enzyme inhibitors Κύκλος ζωής in therapy του ιού Enzymes as drug targets Anti-microbial // anti-viral therapy anti-hiv agents Retrovirus :Genetic information carrier: RNA virus cycle 1. Introduction into the host cell (CD4+) 2. Reverse transcription (RNA DNA) 3. Integration of genetic material with host DNA 4. Transcription

43 Enzyme inhibitors Κύκλος ζωής in therapy του ιού Enzymes as drug targets Anti-microbial // anti-viral therapy anti-hiv agents Retrovirus :Genetic information carrier: RNA virus cycle 1. Introduction into the host cell (CD4+) 2. Reverse transcription (RNA DNA) 3. Integration of genetic material with host DNA 4. Transcription 5. translation

44 Enzyme inhibitors Κύκλος ζωής in therapy του ιού Enzymes as drug targets Anti-microbial // anti-viral therapy anti-hiv agents Retrovirus :Genetic information carrier: RNA virus cycle 1. Introduction into the host cell (CD4+) 2. Reverse transcription (RNA DNA) 3. Integration of genetic material with host DNA Viral proteins & enzymes 4. Transcription 5. Translation 6. Proteolysis of the polyprotein produced

45 Enzyme inhibitors Κύκλος ζωής in therapy του ιού Enzymes as drug targets Anti-microbial // anti-viral therapy anti-hiv agents Retrovirus :Genetic information carrier: RNA virus cycle 1. Introduction into the host cell (CD4+) 2. Reverse transcription (reverse transcriptase) (RNA DNA) (integrase) 3. Integration of genetic material with host DNA Viral proteins & enzymes 4. Transcription 5. Translation (protease) 6. Proteolysis of the polyprotein produced

46 Enzyme inhibitors Κύκλος ζωής in therapy του ιού Enzymes as drug targets Anti-microbial // anti-viral therapy anti-hiv agents Retrovirus :Genetic information carrier: RNA virus cycle 1. Introduction into the host cell (CD4+) 2. Reverse transcription (reverse transcriptase) (RNA DNA) (integrase) 3. Integration of genetic material with host DNA Drug Targets 4. Transcription 5. Translation (protease) 6. Proteolysis of the polyprotein produced

47 Integrase activity retroviral DNA (donor DNA) In all retrovirouses, the viral DNA which is formed by Reverse Transcriptase ( donor DNA) is integrated into the host DNA (target DNA) via the action of integrase. DNA ξενιστή (Target DNA)

48 Commercially available Integrase Inhibitors raltegravir (ISENTRESS) : the first integrase inhibitor firstly approved: 2007 approved for use at the first drug combination: 2009

49 Participation of the Department of Medical Laboratory Studies of Alexander Technological Educational Institute of Thessaloniki in Drug Research In collaboration with School of Pharmacy of Aristotle University of Thessaloniki Anti-inflammatory drugs COX-1, COX-2 inhibitors O N N S N S 2-(thiazole-2-ylamino)-5-phenylidene-4-thiazolidinones R O Journal of Medicinal Chemistry (2008) O R C H 3 N 4 O O CH 3 N-substituted pyrazolo-1,3- oxazin-2-ones N N Ph Bioorganic & Medicinal Chemistry (2008) S N NHCO(CH 2 )nx Adamantane derivatives of Thiazolyl N-substituted amides European Journal of Medicinal Chemistry (2009)

50 Participation of the Department of Medical Laboratory Studies of Alexander Technological Educational Institute of Thessaloniki in Drug Research In collaboration with School of Pharmacy of Aristotle University of Thessaloniki Hypolipidemic drugs Inhibitors of of endogenous cholesterol synthesis O O R 2 Squalen Synthase Inhibitors Chemistry and Physics of Lipids (2006, 2007) N LCAT activators R 1 Morpholin/thiomorpholin derivatives Natural products quercetin Guai-azulen

51 Participation of the Department of Medical Laboratory Studies of Alexander Technological Educational Institute of Thessaloniki in Drug Research In collaboration with School of Pharmacy of Aristotle University of Thessaloniki Phosphatase Inhibitors SHP-2 Inhibitors Treatment of of Noonan Syndrom and and certain leukemias Z NH S N O CH R Journal of Medicinal Chemistry (2008) N-substituted 2-amino-5-aryliden-thiazolidin-4-ones

52 Participation of the Department of Medical Laboratory Studies of Alexander Technological Educational Institute of Thessaloniki in Drug Research In collaboration with School of Pharmacy of Aristotle University of Thessaloniki Phosphatase Inhibitors PTP1B PTP1B Inhibitors LAR LAR Inhibitors Treatment of of diabetes type type II II 10 th Ibn Sina International Conference 2007, Egypt PTP1B inhibitors 3-[(furan-2-yl) methyl]-2-phenyl-thiazolidin-4-ones R O O LAR inhibitors 34 o FEBS Conference, 2009 O 6H-5,8-dioxa-phenanthren Dr Phaedra Eleftheriou, derivatives Department of Medical Laboratory Studies, Alexander TEITH, Greece

53 Participation of the Department of Medical Laboratory Studies of Alexander Technological Educational Institute of Thessaloniki in Drug Research In collaboration with School of Pharmacy of Aristotle University of Thessaloniki anti-hiv agents Reverse transcriptase inhibitors R 1 N S O N R R 3 2 R 4 S XVIII International AIDS Conference Vienna, July 2010

54 Participation of the Department of Medical Laboratory Studies of Alexander Technological Educational Institute of Thessaloniki in Drug Research In collaboration with School of Pharmacy of Aristotle University of Thessaloniki anti-hiv agents Integrase inhibitors R 1 R 2 H R 3 O N H structure n Spyro-isatino-cyclopropan derivatives XVIII International AIDS Conference Vienna, July 2010

55 Enzymes: carbohydrates catalyze all reaction in human body proteins aminoacids NH 3 Carbon backbone CΗ 2OH Η Η Ο Η OH Η OH OH Η OH glucose glycolytic cycle pyrouvate triglycerides glycerol Fatty acids Enzyme Deficiency/ Overactivation Disease urea CH 3 CO-S-CoA Krebs cycle Respiratory chain Oxydative phosphorylation ΑΤΡ energy Disease Tissue Tissue lesions lesions Enzyme overexpression Enzyme Increased Serum Enzyme concentration

56 Enzymes: carbohydrates catalyze all reaction in human body proteins aminoacids NH 3 Carbon backbone CΗ 2OH Η Η Ο Η OH Η OH OH Η OH glucose glycolytic cycle pyrouvate triglycerides glycerol Fatty acids Enzyme Deficiency/ Overactivation Disease urea CH 3 CO-S-CoA Krebs cycle Respiratory chain Oxydative phosphorylation ΑΤΡ energy Disease Tissue Tissue lesions lesions Enzyme overexpression Enzyme Increased Serum Enzyme concentration

57 Enzymes: carbohydrates catalyze all reaction in human body proteins aminoacids NH 3 urea Carbon backbone CΗ 2OH Η Η Ο Η OH Η OH OH Η OH glucose glycolytic cycle pyrouvate CH 3 CO-S-CoA Krebs cycle triglycerides glycerol Fatty acids Disease Tissue Tissue lesions lesions Enzyme Enzyme overexpression Increased Serum Enzyme concentration Respiratory chain Oxydative phosphorylation ΑΤΡ energy

58 Enzymes: carbohydrates catalyze all reaction in human body proteins aminoacids NH 3 urea Carbon backbone CΗ 2OH Η Η Ο Η OH Η OH OH Η OH glucose glycolytic cycle pyrouvate CH 3 CO-S-CoA Krebs cycle Respiratory chain Oxydative phosphorylation ΑΤΡ energy triglycerides glycerol Fatty acids Enzymes in Clinical Chemistry

59 Serum enzymes useful tools in Clinical Chemistry amylase lipase alkaline phosphatases acid phosphatases LDH Transaminases γ-gt CPK aldolase G6PD

60 Isoenzymes: catalyze the same reaction different gene products Alkaline phosphatases Liver/bone/kidney isoenzyme Intestinal isoenzyme Placental isoenzyme

61 Serum isoenzymes amylase lipase alkaline phosphatases acid phosphatases LDH Transaminases γ-gt CPK aldolase G6PD S (saliva) isoenzymes P (pancrease) isoenzymes LDH1 (heart, erythrocytes, kidneys) Liver Bone Intestine placenta bone Liver prostate LDH2 (heart, erythrocytes, kidneys, pancreas, lymphocytes) LDH3 (pancreas, lymphocytes) LDH4 (liver, skeletal muscles, lungs) LDH5 (liver, skeletal muscles, lungs) CPK1 (brain, muscle) CPK2 (heart, muscle) CPK3 (muscle, heart)

62 Methods of isoenzyme differential identification Electrophoresis amylase LDH 1 LDH LDH 2 LDH 3 LDH 4 LDH 5 νεφροί καρδιά Serum ορός kidneys heart liver ήπαρ muscles μυς CPK1 CPK CPK2 CPK3

63 Methods of isoenzyme differential identification Electrophoresis alkaline phosphatases acid phosphatases 1 2 (+) (-) φ loading adult serum with intestinal isoenzymes adult serum child serum placenta intestine bone liver Alkaline phosphatase isoenzymes (Celulose acetate, 200V, 18 min) 1: serum of patient with prostate cancer 2: serum of patient with prostate cancer with bone metastasis acid phosphatases (polyacrylamide gel)

64 Methods of isoenzyme differential identification Electrophoresis alkaline phosphatases acid phosphatases 1 2 (+) (-) φ loading adult serum with intestinal isoenzymes adult serum child serum placenta intestine bone liver Alkaline phosphatase isoenzymes (Celulose acetate, 200V, 18 min) 1: serum of patient with prostate cancer 2: serum of patient with prostate cancer with bone metastasis acid phosphatases (polyacrylamide gel)

65 Methods of isoenzyme differential identification Electrophoresis alkaline phosphatases acid phosphatases 1 2 (+) (-) φ loading adult serum with intestinal isoenzymes adult serum child serum placenta intestine bone liver Alkaline phosphatase isoenzymes (Celulose acetate, 200V, 18 min) 1: serum of patient with prostate cancer 2: serum of patient with prostate cancer with bone metastasis acid phosphatases (polyacrylamide gel)

66 Methods of isoenzyme differential identification Electrophoresis alkaline phosphatases acid phosphatases 1 2 (+) (-) φ loading adult serum with intestinal isoenzymes adult serum child serum placenta intestine bone liver Alkaline phosphatase isoenzymes (Celulose acetate, 200V, 18 min) 1: serum of patient with prostate cancer 2: serum of patient with prostate cancer with bone metastasis acid phosphatases (polyacrylamide gel)

67 Electrophoresis Methods of isoenzyme differential identification Not efficient in all cases Relatively expensive Time consuming Not suitable for many samples

68 Methods of isoenzyme differential identification Cholinesterase + CH 3 CH 3 -CO-O-CH -CH 2 -N CH 3 CH 3 CH 3 Acetyl-methyl-choline Use of different substrates pseudocholinesterase + CH 3 CH 3 -CH 2 -CH 2 -CO-O-CH 2 -CH 2 -N CH 3 CH 3 Butyryl-thiocholine LDL LDH1, LDH2, LDH3 LDH4, LDH5 LDH1, LDH2 lactate β-hydroxy-butyrate Use of special inhibitors Immunologic technics

69 Methods of isoenzyme differential identification Cholinesterase + CH 3 CH 3 -CO-O-CH -CH 2 -N CH 3 CH 3 CH 3 Acetyl-methyl-choline Use of different substrates pseudocholinesterase + CH 3 CH 3 -CH 2 -CH 2 -CO-O-CH 2 -CH 2 -N CH 3 CH 3 Butyryl-thiocholine LDL LDH1, LDH2, LDH3 LDH4, LDH5 LDH1, LDH2 lactate β-hydroxy-butyrate Use of special inhibitors Prostatic acid phosphatase Use of tartrate Immunologic techniques

70 Methods of isoenzyme differential identification Use of special inhibitors Prostatic acid phosphatase Use of tartrate Immunologic techniques

71 Methods of isoenzyme differential identification Use of special inhibitors Prostatic acid phosphatase Use of tartrate Immunologic techniques CPK MB inhibition by specific antibody Placental alkaline phosphatase ELISA method

72 Recent research results of our Laboratory Combination of two compounds Compound 1 Compound 2 Inhibits all other serum alkaline phosphatase isoenzymes except of the placental isoenzyme 95% activation of Placental isoenzyme Probable good combination for colorimetric determination of placental isoenzyme

73 Need for specific inhibitors for treatment and diagnosis Valuable tools: Computer Activity Prediction Programmes (PASS) Docking Studies

74 Need for specific inhibitors for treatment and diagnosis Valuable tools: Compound structures Activities Computer Activity Prediction Programmes (PASS) Data Base Comparison With Data base structures Your Compound structure Predicted activity

75 Need for specific inhibitors for treatment and diagnosis Valuable tools: Computer Activity Prediction Programmes (PASS) Sructure design According to the literature Computer prediction Final desision

76 Need for specific inhibitors for treatment and diagnosis Valuable tools: Modeling Crystalographic data of enzyme complex with substrate or other inhibitors Special computer programme Complex free energy

77 Need for specific inhibitors for treatment and diagnosis Valuable tools: Crystalographic data of enzyme complex with substrate or other inhibitors Special computer programme Docking Studies enzyme Complex free energy Potential inhibitor The lower the energy The most stable complex The most potent inhibitor

78 Other uses of of Enzymes Bioelectrodes-biosensors Need for: a highly active, stable appropriate enzyme, immobilized on a membrane attached to the appropriate electrode

79 Other uses of of Enzymes Bioelectrodes-biosensors glucose oxidase Glucose electrode Glucose + O 2 gluconic acid + H 2 O 2 H 2 O 2 Immobilised glucose oxidase glucose

80 Other uses of of Enzymes Bioelectrodes-biosensors Biosensors in Critical Care Manufacturer System Biosensor Technology Bayer RapidLab 800 Glucose, lactate Electrochemical thick film RapidPoint 400 Glucose Nova Biomedical Critical Care Express, Nova 16 Glucose, lactate, urea, creatinine Electrochemical PHOx Glucose, lactate Radiometer ABL 725 Glucose, lactate Electrochemical i-stat I-STAT 1 and PCA Glucose, lactate, urea, creatinine diametrics IRMA Glucose, lactate, urea, Electrochemical, thin film Electrochemical, thick film, strip Roche OMNI 9, OPTI CCA Glucose, lactate, urea, creatinine Electrochemical, thick film, optical

81 Immunosensors Other uses of of Enzymes Aplications in microbiology Herpes virus Chlamidia in urin Staphylococcal Enterotoxin etc.

82 ELISA- EIA methods Other uses of of Enzymes EIA : Enzyme ImmunoAssay ELISA : Enzyme-Linked ImmunoSorbent Assay Antigen detection Υ ΥΥ A A A Y Y Y Y Ab-Enzyme αντίσωμα-ιχνηλάτης ορμόνη antigen αντίσωμα Ab Enzyme μετρούμενη reaction ποσότητα product ιχνηλάτη antigen concentration συγκέντρωση ορμόνης A A A A A A A A A A A Y Y Y Y antigen ορμόνη Antigen-Enzyme ορμόνη-ιχνηλάτης Ab αντίσωμα Enzyme reaction product μετρούμενη ποσότητα ιχνηλάτη antigen concentration συγκέντρωση ορμόνης

83 Other uses of of Enzymes ELISA- EIA methods EIA : Enzyme ImmunoAssay ELISA : Enzyme-Linked ImmunoSorbent Assay Υ : προς antibody προσδιορισμό αντίσωμα A : antigen-enzyme επισημασμένο αντιγόνο IgG ανοσοσφαιρίνες IgG A Y Y Y A Y anti-igg Ab EE EY Y Y Y Y Y anti-igg-enzyme antigen

84 ELISA- EIA methods Other uses of of Enzymes EIA : Enzyme ImmunoAssay ELISA : Enzyme-Linked ImmunoSorbent Assay

85 An application 2009 Α (Η1Ν1) hemaglutinin Ion channel RΝΑ capside nucleoproteins Lipid envelop neuraminidase

86 human Η3Ν Α (Η1Ν1) avian Shanta M. Zimmer, M.D., and Donald S. Burke, M.D, Historical Perspective Emergence of Influenza A (H1N1) Viruses. New England Journal of Medicine, 2009.

87 2009 Α (Η1Ν1) 11 genes: ΝΑ, Μ1, Μ2 ΗΑ, ΝΡ, ΝS1, NS2 PB2, PA PB1 Swine Eurasian Classical Swine (1918) Swine (triple reasortion) (avian) Swine (triple reasortion) (human)

88 2009 Α (Η1Ν1) Blood samples Blood samples were collected from November 1 st 2009 till January 30 th 2010 from 43 residents of Thessaloniki who had experienced flue symptoms from October 1 st 2009 till January 10 th All samples were collected 20 to 30 days after experiencing the symptoms. Samples from 25 individuals inoculated with the new influenza A (H1N1) vaccine were collected 20 to 40 days after vaccination. amples from 23 adult residents of Thessaloniki, collected on February 2009, before first identification of the new virus were also tested for comparison reasons. Serum was kept at 20 o C until determination.

89 2009 Α (Η1Ν1) Method ABTS POD Υ Υ Coloured product SecondaryAntibody sample (1:400) antigen (15μg/ml) - Swine-origin hemaglutinin peptide segments (5237P and 5241P) derived from A/California/14/2009 (H1N1) new influenza virus were purchased by ΨProSci inc. - Swine-origin Neuraminidase peptide 5249P was also purchased by ΨProSci inc. - Rabbit antibodies recognizing the swine origin peptides Cat. No 5237, 5241 and 5249 (ΨProSci) - Horseradish peroxidase (HRP)-conjugated goat anti human IgM antibody and - HRP-conjugated goat anti human IgG antibody were purchased by AbD serotec.

90 Immunologic response to 2009 A(H1N1) HA and NA peptides Anti- 5237P IgM A B μg/ml μg/ml 1, Anti- 5237P IgG C D Figure Distribution of antibody concentrations against swine-origin hemaglutinin peptide 5237P in vaccinated individuals ( ) and not vaccinated persons who had experienced flue symptoms ( ) In comparison to the old samples of infected/vaccinated population ( ). A,B: IgM concentration. C,D: IgG concentration.

91 Immunologic response to 2009 A(H1N1) HA and NA peptides μg/ml Anti IgM A B μg/ml 1, , Anti IgG C D Figure Distribution of antibody concentrations against swine-origin hemaglutinin peptide 5241P in vaccinated individuals ( ) and not vaccinated persons who had experienced flue symptoms ( ) in comparison to the old samples of infected/ vaccinated population ( ). A,B: IgM concentration. C,D: IgG concentration.

92 Immunologic response to 2009 A(H1N1) HA and NA peptides Anti IgM 60 A 60 B μg/ml Anti IgG C D μg/ml Figure 3. Distribution of antibody concentrations against swine-origin neuraminidase peptide 5249P in vaccinated individuals ( ) and not vaccinated persons who had experienced flue symptoms ( ) in comparison to the old samples of infected/vaccinated population ( ). A,B: IgM concentration. C,D: IgG concentration.

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