[PPT] - Enzymes and enzyme inhibitors in Diagnosis and Therapy Dr Phaedra PowerPoint Presentation

SLIDE 1

Enzymes and enzyme inhibitors in Diagnosis and Therapy

Dr Phaedra Eleftheriou Department of Medical Laboratory Studies

http://www.MedLabstudies.webs.com

School of Health and Medical Care Alexander Technological Educational Institute of Thessaloniki, Greece

SLIDE 2

carbohydrates

glucose glycolytic cycle pyrouvate CH3CO-S-CoA

Krebs cycle

Respiratory chain Oxydative phosphorylation

ΑΤΡ

energy

triglycerides

Fatty acids glycerol

proteins

aminoacids NH3 urea Carbon backbone

Η C Η

2OH

Ο Η Η Η Η OH OH OH OH Η C Η

2OH

Ο Η Η Η Η OH OH OH OH

Enzymes: catalyze all reaction in human body Enzyme Deficiency/ Overactivation

Disease Disease

Tissue lesions Tissue lesions

Increased Serum Enzyme concentration

Enzyme

verexpression

Enzyme

verexpression

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 3

carbohydrates

glucose glycolytic cycle pyrouvate CH3CO-S-CoA

Krebs cycle

Respiratory chain Oxidative phosphorylation

ΑΤΡ

energy

triglycerides

Fatty acids glycerol

proteins

aminoacids NH3 urea Carbon backbone

Η C Η

2OH

Ο Η Η Η Η OH OH OH OH Η C Η

2OH

Ο Η Η Η Η OH OH OH OH

Enzymes: catalyze all reaction in human body Enzyme Deficiency/ Overactivation

Disease

Increase of intermediate metabolite Decrease in final product Undesired side products and effects

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 4

Enzymes: catalyze all reaction in human body Enzyme Deficiency Enzyme Deficiency

Disease

G6PD

glucose

Phosphogluconate (pentose phosphate) pathway

G6PD G6PD G6PD

phosphogluconate

pentose

NADPH2

a valuable product for mature erythrocyte which lacks mitochondrions and Krebs cycle

Lack of NAD(P)H2 Hb oxidation hemolysis

Hemolysis

(in oxidative conditions)

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 5

Enzymes: catalyze all reaction in human body Enzyme Deficiency Enzyme Deficiency

Disease

G6PD Hemolysis

(in oxidative conditions) Protein metabolism Protein metabolism Phenylalanine hydroxylase hyperphenylalaninemia. Phenylketonuria (mental retardation)

Phenylalanine tyrosine

normal metabolism

Phenylalanine hydroxylase

phenylpyruvate phenylacetate

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 6

Enzymes: catalyze all reaction in human body Enzyme Deficiency Enzyme Deficiency

Disease

G6PD Hemolysis

(in oxidative conditions) Protein metabolism Protein metabolism Phenylalanine hydroxylase hyperhenylalaninemia. Phenylketonuria (mental retardation) Omogentisate oxidase

Homogentisuria, alkaptonuria,

(kidney stones, heart valve impairment, acidosis, arthritis, blue- black skin spots)

tyrosin homogentisic acid

Homogentisate Homogentisate

xidase
xidase

Fumarate, acetoacetate

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 7

Enzymes: catalyze all reaction in human body Enzyme Deficiency Enzyme Deficiency

Disease

Carbohydrate metabolism

lactase lactase lactase

Lactose intolerance galactose-1-phosphate uridyl transferase, galactokinase galactose galactose-

1

1-

phosphate uridyl transferase,

phosphate uridyl transferase, galactokinase galactokinase galactosemia

lactose

Η CΗ2OH Ο ΟΗ Η Η OH OH H Η CΗ2OH Ο Η Η Η ΟΗ OH H OH O

1 4 3 2 5 6 1 2 3 4 5 6

β-D- β-D- Η Η CΗ2OH Ο ΟΗ Η Η OH OH H Η CΗ2OH Ο Η Η Η ΟΗ OH H OH O

1 4 3 2 5 6 1 2 3 4 5 6

β-D- β-D- Η Η CΗ2OH Ο ΟΗ Η Η OH OH H Η CΗ2OH Ο Η Η Η ΟΗ OH H OH O

1 4 3 2 5 6 1 2 3 4 5 6

β-D-galactose β-D-glucose Η Η CΗ2OH Ο ΟΗ Η Η OH OH H Η CΗ2OH Ο Η Η Η ΟΗ OH H OH O

1 4 3 2 5 6 1 2 3 4 5 6

β-D- β-D- Η Η CΗ2OH Ο ΟΗ Η Η OH OH H Η CΗ2OH Ο Η Η Η ΟΗ OH H OH O

1 4 3 2 5 6 1 2 3 4 5 6

β-D- β-D- Η Η CΗ2OH Ο ΟΗ Η Η OH OH H Η CΗ2OH Ο Η Η Η ΟΗ OH H OH O

1 4 3 2 5 6 1 2 3 4 5 6

β-D-galactose β-D-glucose Η

lactase lactase

Η CΗ2OH Ο Η Η Η Η OH OH OH OH Η CΗ2OH Ο Η Η Η Η OH OH OH OH Η CΗ2OH Ο OΗ Η Η Η OH OH OH H Η CΗ2OH Ο OΗ Η Η Η OH OH OH H

glucose galactose galactokinase galactokinase

galactose galactose-

1

1-

P

P uridyl transferase uridyl transferase

UDP-glucose

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 8

Enzymes: catalyze all reaction in human body Enzyme Deficiency Enzyme Deficiency

Disease

Carbohydrate metabolism lactase lactase lactase Lactose intolerance galactose-1-phosphate uridyl transferase, galactokinase galactose galactose-

1

1-

phosphate uridyl transferase,

phosphate uridyl transferase, galactokinase galactokinase galactosemia alpha-L iduronidase

mucopolysaccharidosis type I (MPS I), Hurler's disease (accumulation of heparane sulfate & dermatane sulfate in lysosomes Hepatosplenomegaly, mental retardation)

COO¯ NHCOCH3 ¯O3SO Iduronic acid COO¯ NHCOCH3 ¯O3SO Iduronic acid

COO¯ HNSO3¯ ¯O3SO OSO3¯

ηπαρίνη

COO¯ HNSO3¯ ¯O3SO OSO3¯

ηπαρίνη

dermatane heparane Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 9

Enzymes: catalyze all reaction in human body Enzyme Deficiency Enzyme Deficiency

Disease

Carbohydrate metabolism lactase lactase lactase Lactose intolerance galactose-1-phosphate uridyl transferase, galactokinase galactose galactose-

1

1-

phosphate uridyl transferase,

phosphate uridyl transferase, galactokinase galactokinase galactosemia alpha-L iduronidase

mucopolysaccharidosis type I (MPS I), Hurler's disease (accumulation of heparane sulfate & dermatane sulfate in lysosomes Hepatosplenomegaly, mental retardation)

COO¯ NHCOCH3 ¯O3SO Iduronic acid COO¯ NHCOCH3 ¯O3SO Iduronic acid

COO¯ HNSO3¯ ¯O3SO OSO3¯

ηπαρίνη

COO¯ HNSO3¯ ¯O3SO OSO3¯

ηπαρίνη

dermatane heparane

Heparane-N-sulfatase L-iduronate-2-sulfatase β-galactosidase ......... accumulation of heparane, dermatane, keratane in lysosomes

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 10

Enzymes: catalyze all reaction in human body Enzyme Deficiency Enzyme Deficiency

Disease

Lipid metabolism sphigomyelinase Sphingomyelin storage in lysosomes (ataxia, disarthria, disphagia, demedia etc. ) (Niemann-Pick disease) ceramidase Lipid storage in lysosomes (Farber disease) (moderately impaired mental ability, swallowing problems)

CH2 CH2 NH2 P O O OH φωσφοαιθανολαμίνη CH2 CH2 NH2 P O O OH P O O OH φωσφοαιθανολαμίνη H2C ΗC ΝH- CO-R HC OH CH CH CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3 σφιγγοσίνη

1 2 3 4 5 9 7 6 8 10 11 12 13 14 15 16 17 18

H

H

CH2 CH2 NH2 P O O OH φωσφοαιθανολαμίνη CH2 CH2 NH2 P O O OH P O O OH φωσφοαιθανολαμίνη H2C ΗC ΝH- CO-R HC OH CH CH CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3 σφιγγοσίνη

1 2 3 4 5 9 7 6 8 10 11 12 13 14 15 16 17 18

H2C ΗC ΝH- CO-R HC OH CH CH CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3 H2C ΗC ΝH- CO-R HC OH H2C ΗC ΝH- CO-R HC OH CH CH CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3 σφιγγοσίνη

1 2 3 4 5 9 7 6 8 10 11 12 13 14 15 16 17 18

H

H

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 11

Enzymes: catalyze all reaction in human body Enzyme Deficiency Enzyme Deficiency

Disease

Lipid metabolism

chylomicrons Chylomicron remnants

VLDL IDL LDL

apoΕ receptors / Chylomicron remnant receptors apoΒ –Ε / LDL receptors apoΒ –Ε / LDL receptors

ther tissues

LPL LPL LPL

insulin

αποCII (HDL) + +

insulin

αποCII (HDL) +

insulin insulin

70%

Adrenal gland adipose tissue

chylomicrons Chylomicron remnants

VLDL IDL LDL

apoΕ receptors / Chylomicron remnant receptors apoΒ –Ε / LDL receptors apoΒ –Ε / LDL receptors

ther tissues

LPL LPL LPL

insulin

αποCII (HDL) + +

insulin

αποCII (HDL) +

insulin insulin

70%

Adrenal gland adipose tissue

Lipoprotein lipase (LPL)

Hyperlipidemia type I

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 12

HDL 1 HDL 2

Εστέρας χολεστερόλης Ελεύθερη χολεστερόλη

Υπο λειματικά χυλομικρά IDL

LCAT (lecithin-cholesterole

acyltransferase ) αποA1 Ελεύθερη χολεστερόλη κυτταρική μεμβράνη

μακροφάγα HDL 1 HDL 2

Εστέρας χολεστερόλης Ελεύθερη χολεστερόλη

Υπο λειματικά χυλομικρά IDL

Ελεύθερη χολεστερόλη

Υπο λειματικά χυλομικρά IDL

LCAT (lecithin-cholesterole

acyltransferase ) αποA1 Ελεύθερη χολεστερόλη κυτταρική μεμβράνη Ελεύθερη χολεστερόλη κυτταρική μεμβράνη

μακροφάγα μακροφάγα

εστέρες χοληστερόλης LCAT

από-Α1

HDL 1 HDL 2

Εστέρας χολεστερόλης Ελεύθερη χολεστερόλη

Υπο λειματικά χυλομικρά IDL

LCAT (lecithin-cholesterole

acyltransferase ) αποA1 Ελεύθερη χολεστερόλη κυτταρική μεμβράνη

μακροφάγα

apoΕ receptors

HDL 1 HDL 2

Εστέρας χολεστερόλης Ελεύθερη χολεστερόλη

Υπο λειματικά χυλομικρά IDL

Ελεύθερη χολεστερόλη

Υπο λειματικά χυλομικρά IDL

LCAT (lecithin-cholesterole

acyltransferase ) αποA1 Ελεύθερη χολεστερόλη κυτταρική μεμβράνη Ελεύθερη χολεστερόλη κυτταρική μεμβράνη

μακροφάγα

macrophages

apoΑ I receptors

cholesterol Cholesterol esters LCAT

από-Α1

LCAT

apo -Α1 Cholesterol esters

HDL3

apoA-I, apoE apoC-I, apoC-II apoC-I, apoC-II apoE

HDL 1 HDL 2

Εστέρας χολεστερόλης Ελεύθερη χολεστερόλη

Υπο λειματικά χυλομικρά IDL

LCAT (lecithin-cholesterole

acyltransferase ) αποA1 Ελεύθερη χολεστερόλη κυτταρική μεμβράνη

μακροφάγα HDL 1 HDL 2

Εστέρας χολεστερόλης Ελεύθερη χολεστερόλη

Υπο λειματικά χυλομικρά IDL

Ελεύθερη χολεστερόλη

Υπο λειματικά χυλομικρά IDL

LCAT (lecithin-cholesterole

acyltransferase ) αποA1 Ελεύθερη χολεστερόλη κυτταρική μεμβράνη Ελεύθερη χολεστερόλη κυτταρική μεμβράνη

μακροφάγα μακροφάγα

εστέρες χοληστερόλης LCAT

από-Α1

HDL 1 HDL 2

Εστέρας χολεστερόλης Ελεύθερη χολεστερόλη

Υπο λειματικά χυλομικρά IDL

LCAT (lecithin-cholesterole

acyltransferase ) αποA1 Ελεύθερη χολεστερόλη κυτταρική μεμβράνη

μακροφάγα

apoΕ receptors

HDL 1 HDL 2

Εστέρας χολεστερόλης Ελεύθερη χολεστερόλη

Υπο λειματικά χυλομικρά IDL

Ελεύθερη χολεστερόλη

Υπο λειματικά χυλομικρά IDL

LCAT (lecithin-cholesterole

acyltransferase ) αποA1 Ελεύθερη χολεστερόλη κυτταρική μεμβράνη Ελεύθερη χολεστερόλη κυτταρική μεμβράνη

μακροφάγα

macrophages

apoΑ I receptors

cholesterol Cholesterol esters LCAT

από-Α1

LCAT

apo -Α1 Cholesterol esters

HDL3

apoA-I, apoE apoC-I, apoC-II apoC-I, apoC-II apoE

Enzymes: catalyze all reaction in human body Enzyme Deficiency Enzyme Deficiency

Disease

Lipid metabolism

chylomicrons Chylomicron remnants

VLDL IDL LDL

apoΕ receptors / Chylomicron remnant receptors apoΒ –Ε / LDL receptors apoΒ –Ε / LDL receptors

ther tissues

LPL LPL LPL

insulin

αποCII (HDL) + +

insulin

αποCII (HDL) +

insulin insulin

70%

Adrenal gland adipose tissue

chylomicrons Chylomicron remnants

VLDL IDL LDL

apoΕ receptors / Chylomicron remnant receptors apoΒ –Ε / LDL receptors apoΒ –Ε / LDL receptors

ther tissues

LPL LPL LPL

insulin

αποCII (HDL) + +

insulin

αποCII (HDL) +

insulin insulin

70%

Adrenal gland adipose tissue

Lipoprotein lipase (LPL) Hyperlipidemia type I

HDL1

Lecithin-cholesterol acyltransferase (LCAT) Hyperlipidemia

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 13

Enzymes: catalyze all reaction in human body Enzyme

neractivation

Enzyme

neractivation

Disease

HDL1

SHP-2

a phosphatase involved

in cell-cycle regulation

Noonan Syndrom Juvenil Myelomonocytic Leuchemias

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 14

Enzymes: catalyze all reaction in human body Enzyme Inhibitor defficiency Enzyme Inhibitor defficiency

Disease

HDL1

A1-antitrypsin

(serum protease, mainly elastase, inhibitor)

emphysema

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 15

Enzyme /enzyme inhibitor deficiency

SLIDE 24

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Many drugs are Enzyme Inhibitors Anti-inflammatory drugs

Arachidonic acid

prostaglandin

COX-1, COX-2 inhibitors

COX-1 COX-2

pain

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 25

Squalene Synthase inhibitors

Acetyl-CoA Squalene cholesterol

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Many drugs are Enzyme Inhibitors Hypolipidemic Hypolipidemic drugs drugs

:statines Inhibitors of endogenous cholesterol synthesis Inhibitors Inhibitors of endogenous cholesterol synthesis

f endogenous cholesterol synthesis

HMG-CoA reductase inhibitors

liver

chylomicrons

VLDL

cholesterole synthesis

mevalonic acid

HMG-CoA reductase

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 26

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Many drugs are Enzyme Inhibitors Hypolipidemic Hypolipidemic drugs drugs

Inhibitors of endogenous cholesterol synthesis (statines : simvastatin..) Inhibitors Inhibitors of

f endogenous cholesterol synthesis

endogenous cholesterol synthesis (statines : simvastatin..) (statines : simvastatin..) LPL activators LPL LPL activators activators LCAT activators LCAT LCAT activators activators resins resins resins antioxudants

antioxudants

antioxudants

chylomicrons Chylomicron remnants

VLDL IDL LDL

apoΕ receptors / Chylomicron remnant receptors apoΒ –Ε / LDL receptors apoΒ –Ε / LDL receptors

ther tissues

LPL LPL LPL

insulin

αποCII (HDL) + +

insulin

αποCII (HDL) +

insulin insulin

70%

Adrenal gland adipose tissue

chylomicrons Chylomicron remnants

VLDL IDL LDL

apoΕ receptors / Chylomicron remnant receptors apoΒ –Ε / LDL receptors apoΒ –Ε / LDL receptors

ther tissues

LPL LPL LPL

insulin

αποCII (HDL) + +

insulin

αποCII (HDL) +

insulin insulin

70%

Adrenal gland adipose tissue

HDL 1 HDL 2

Εστέρας χολεστερόλης Ελεύθερη χολεστερόλη Υπο λειματικά χυλομικρά IDL

LCAT (lecithin-cholesterole

acyltransferase ) αποA1 Ελεύθερη χολεστερόλη κυτταρική μεμβράνη

μακροφάγα HDL 1 HDL 2

Εστέρας χολεστερόλης Ελεύθερη χολεστερόλη Υπο λειματικά χυλομικρά IDL Ελεύθερη χολεστερόλη Υπο λειματικά χυλομικρά IDL

LCAT (lecithin-cholesterole

acyltransferase ) αποA1 Ελεύθερη χολεστερόλη κυτταρική μεμβράνη Ελεύθερη χολεστερόλη κυτταρική μεμβράνη

μακροφάγα μακροφάγα

εστέρες χοληστερόλης LCAT

από-Α1

HDL 1 HDL 2

Εστέρας χολεστερόλης Ελεύθερη χολεστερόλη Υπο λειματικά χυλομικρά IDL

LCAT (lecithin-cholesterole

acyltransferase ) αποA1 Ελεύθερη χολεστερόλη κυτταρική μεμβράνη

μακροφάγα

apoΕ receptors

HDL 1 HDL 2

Εστέρας χολεστερόλης Ελεύθερη χολεστερόλη Υπο λειματικά χυλομικρά IDL Ελεύθερη χολεστερόλη Υπο λειματικά χυλομικρά IDL

LCAT (lecithin-cholesterole

acyltransferase ) αποA1 Ελεύθερη χολεστερόλη κυτταρική μεμβράνη Ελεύθερη χολεστερόλη κυτταρική μεμβράνη

μακροφάγα

macrophages

apoΑ I receptors

cholesterol Cholesterol esters LCAT

από-Α1

LCAT

apo -Α1 Cholesterol esters

HDL3

apoA-I, apoE apoC-I, apoC-II apoC-I, apoC-II apoE

HDL 1 HDL 2

Εστέρας χολεστερόλης Ελεύθερη χολεστερόλη Υπο λειματικά χυλομικρά IDL

LCAT (lecithin-cholesterole

acyltransferase ) αποA1 Ελεύθερη χολεστερόλη κυτταρική μεμβράνη

μακροφάγα HDL 1 HDL 2

Εστέρας χολεστερόλης Ελεύθερη χολεστερόλη Υπο λειματικά χυλομικρά IDL Ελεύθερη χολεστερόλη Υπο λειματικά χυλομικρά IDL

LCAT (lecithin-cholesterole

acyltransferase ) αποA1 Ελεύθερη χολεστερόλη κυτταρική μεμβράνη Ελεύθερη χολεστερόλη κυτταρική μεμβράνη

μακροφάγα μακροφάγα

εστέρες χοληστερόλης LCAT

από-Α1

HDL 1 HDL 2

Εστέρας χολεστερόλης Ελεύθερη χολεστερόλη Υπο λειματικά χυλομικρά IDL

LCAT (lecithin-cholesterole

acyltransferase ) αποA1 Ελεύθερη χολεστερόλη κυτταρική μεμβράνη

μακροφάγα

apoΕ receptors

HDL 1 HDL 2

Εστέρας χολεστερόλης Ελεύθερη χολεστερόλη Υπο λειματικά χυλομικρά IDL Ελεύθερη χολεστερόλη Υπο λειματικά χυλομικρά IDL

LCAT (lecithin-cholesterole

acyltransferase ) αποA1 Ελεύθερη χολεστερόλη κυτταρική μεμβράνη Ελεύθερη χολεστερόλη κυτταρική μεμβράνη

μακροφάγα

macrophages

apoΑ I receptors

cholesterol Cholesterol esters LCAT

από-Α1

LCAT

apo -Α1 Cholesterol esters

HDL3

apoA-I, apoE apoC-I, apoC-II apoC-I, apoC-II apoE

HDL1

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 27

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Many drugs are Phosphatase Inhibitors

Κinases – phosphatases: biochemical pathways regulation phosphatases kinases

+

biochemical pathways regulation

OH OH OH OH

+

O-P-OH OH O O-P-OH OH O

+

OH OH OH OH

+ ATP

O-P-OH OH O O-P-OH OH O O-P-OH OH O O-P-OH OH O

+ ADP

kinase kinase kinase

+ H2O

OH OH HO-P-OH OH O HO-P-OH OH O O-P-OH OH O O-P-OH OH O

+ H2O

OH OH HO-P-OH OH O HO-P-OH OH O

+

O-P-OH OH O O-P-OH OH O O-P-OH OH O O-P-OH OH O

phosphatase phosphatase phosphatase

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 28

Phosphatases with possible pharmaceutical interest Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Many drugs are Phosphatase Inhibitors

PTP1B

πρόσληψη γλυκόζης

SHP-2 CD45

πολλαπλασιασμό και μετανάστευση κυττάρων χρήση των αναστολέων της PTP1B για θεραπεία διαβήτη τύπου II αυξημένη δραστηριότητα εξαιτίας μεταλλάξεων σε σύνδρομο Νοonan και τύπους καρκίνου

αντικαρκινική δράση

PTP-ε

οστεοκλαστική διαδικασία

RPTP-β/ζ

επιβίωση ολιγο- δενδροκυττάρων

LAR -RPTP

Εισαγωγή γλυκόζης στα κύτταρα EPM2A laforin συσσώρευση σωματιδίων Lafora

μεταλλαγμένη στην ασθένεια Lafora

Glucose uptake Treatment of diabetes type II Cell proliferation Noonan Syndrom certain leucemias Anticancer action Mutations found in Lafora disease Osteoclastic process Glucose uptake Accumulation of Lafora Inclusion bodies Dendritic cell survival PTP1B

πρόσληψη γλυκόζης

SHP-2 CD45

πολλαπλασιασμό και μετανάστευση κυττάρων χρήση των αναστολέων της PTP1B για θεραπεία διαβήτη τύπου II αυξημένη δραστηριότητα εξαιτίας μεταλλάξεων σε σύνδρομο Νοonan και τύπους καρκίνου

αντικαρκινική δράση

PTP-ε

οστεοκλαστική διαδικασία

RPTP-β/ζ

επιβίωση ολιγο- δενδροκυττάρων

LAR -RPTP

Εισαγωγή γλυκόζης στα κύτταρα EPM2A laforin συσσώρευση σωματιδίων Lafora

μεταλλαγμένη στην ασθένεια Lafora

Glucose uptake Treatment of diabetes type II Cell proliferation Noonan Syndrom certain leucemias Anticancer action Mutations found in Lafora disease Osteoclastic process Glucose uptake Accumulation of Lafora Inclusion bodies Dendritic cell survival

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

Phosphatases: potential drug targets

SLIDE 29

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets 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

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 30

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Diabetes therapy – PTP1B, LAR Inhibitors

Mechanism of glucose uptake

Insulin receptor (inactive) glucose Insulin receptor (inactive) glucose

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 31

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Diabetes therapy – PTP1B, LAR Inhibitors

Mechanism of glucose uptake

insulin receptor (active) insulin glucose

active

(kinase activity) active

gene expression

Glycogen, lipid, Protein synthesis

insulin receptor (active) insulin glucose

active

(kinase activity) active

gene expression

Glycogen, lipid, Protein synthesis

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 32

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Diabetes therapy – PTP1B, LAR Inhibitors

Mechanism of glucose uptake

insulin receptor (active) insulin glucose

active

(kinase activity) active

gene expression

Glycogen, lipid, Protein synthesis

phosphatase activity

insulin receptor (active) insulin glucose

active

(kinase activity) active

gene expression

Glycogen, lipid, Protein synthesis

phosphatase activity

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 33

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Diabetes therapy – PTP1B, LAR Inhibitors

Mechanism of glucose uptake

insulin receptor (active) insulin glucose

active

(kinase activity) active

gene expression

Glycogen, lipid, Protein synthesis

phosphatase activity

PTP1B LAR insulin receptor (active) insulin glucose

active

(kinase activity) active

gene expression

Glycogen, lipid, Protein synthesis

phosphatase activity

PTP1B LAR

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 34

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Diabetes therapy – PTP1B, LAR Inhibitors

Mechanism of glucose uptake

Insulin receptor (inactive) glucose Insulin receptor (inactive) glucose

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 35

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Diabetes therapy – PTP1B, LAR Inhibitors

insulin receptor (active) insulin glucose

active

(kinase activity) active

gene expression

Glycogen, lipid, Protein synthesis

insulin receptor (active) insulin glucose

active

(kinase activity) active

gene expression

Glycogen, lipid, Protein synthesis

Inhibition

Extention of glucose intake period

PTP1B LAR

inactivation

Mechanism of action of PTP1B/LAR Inhibitors

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 36

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

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 37

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

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 38

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

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 39

Κύκλος ζωής του ιού Κύκλος Κύκλος ζωής ζωής του του ιού ιού

Retrovirus :Genetic information carrier: RNA

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Anti-microbial / anti-viral therapy Anti-microbial / anti-viral therapy anti-HIV agents anti-HIV agents

virus cycle virus cycle virus cycle

1. Introduction into the host cell (CD4+)

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 40

Κύκλος ζωής του ιού Κύκλος Κύκλος ζωής ζωής του του ιού ιού

Retrovirus :Genetic information carrier: RNA

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Anti-microbial / anti-viral therapy Anti-microbial / anti-viral therapy anti-HIV agents anti-HIV agents

virus cycle virus cycle virus cycle

1. Introduction into the host cell (CD4+)
2. Reverse transcription

(RNA DNA)

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 41

Κύκλος ζωής του ιού Κύκλος Κύκλος ζωής ζωής του του ιού ιού

Retrovirus :Genetic information carrier: RNA

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Anti-microbial / anti-viral therapy Anti-microbial / anti-viral therapy anti-HIV agents anti-HIV agents

virus cycle virus cycle virus cycle

1. Introduction into the host cell (CD4+)
2. Reverse transcription

(RNA DNA)

3. Integration of genetic material with host DNA

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 42

Κύκλος ζωής του ιού Κύκλος Κύκλος ζωής ζωής του του ιού ιού

Retrovirus :Genetic information carrier: RNA

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Anti-microbial / anti-viral therapy Anti-microbial / anti-viral therapy anti-HIV agents anti-HIV agents

virus cycle virus cycle virus cycle

1. Introduction into the host cell (CD4+)
2. Reverse transcription

(RNA DNA)

3. Integration of genetic material with host DNA
4. Transcription

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 43

Κύκλος ζωής του ιού Κύκλος Κύκλος ζωής ζωής του του ιού ιού

Retrovirus :Genetic information carrier: RNA

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Anti-microbial / anti-viral therapy Anti-microbial / anti-viral therapy anti-HIV agents anti-HIV agents

virus cycle virus cycle 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

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 44

Κύκλος ζωής του ιού Κύκλος Κύκλος ζωής ζωής του του ιού ιού

Retrovirus :Genetic information carrier: RNA

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Anti-microbial / anti-viral therapy Anti-microbial / anti-viral therapy anti-HIV agents anti-HIV agents

virus cycle virus cycle 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
6. Proteolysis of the polyprotein produced

Viral proteins & enzymes

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 45

Κύκλος ζωής του ιού Κύκλος Κύκλος ζωής ζωής του του ιού ιού

Retrovirus :Genetic information carrier: RNA

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Anti-microbial / anti-viral therapy Anti-microbial / anti-viral therapy anti-HIV agents anti-HIV agents

virus cycle virus cycle 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
6. Proteolysis of the polyprotein produced

Viral proteins & enzymes

(reverse transcriptase)

(integrase) (protease)

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 46

Κύκλος ζωής του ιού Κύκλος Κύκλος ζωής ζωής του του ιού ιού

Retrovirus :Genetic information carrier: RNA

Enzyme Enzyme inhibitors inhibitors in therapy in therapy – – Enzymes as drug targets Enzymes as drug targets Anti-microbial / anti-viral therapy Anti-microbial / anti-viral therapy anti-HIV agents anti-HIV agents

virus cycle virus cycle 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
6. Proteolysis of the polyprotein produced

Drug Targets

(reverse transcriptase)

(integrase) (protease)

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 47

Integrase activity

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. retroviral DNA (donor DNA) DNA ξενιστή (Target DNA)

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 48

raltegravir (ISENTRESS) : the first integrase inhibitor firstly approved: 2007 approved for use at the first drug combination: 2009

Commercially available Integrase Inhibitors

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 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

S N S N N O O

C H3 N N N O CH3 Ph O O R

4

N-substituted pyrazolo-1,3- oxazin-2-ones 2-(thiazole-2-ylamino)-5-phenylidene-4-thiazolidinones R

N S NHCO(CH2)nX

Adamantane derivatives of Thiazolyl N-substituted amides

European Journal of Medicinal Chemistry (2009)

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

Journal of Medicinal Chemistry (2008) Bioorganic & Medicinal Chemistry (2008)

SLIDE 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 Hypolipidemic drugs

Inhibitors of endogenous cholesterol synthesis Inhibitors of endogenous cholesterol synthesis Inhibitors of endogenous cholesterol synthesis

N O O N O O

R1 R2 Morpholin/thiomorpholin derivatives

Natural products

quercetin Guai-azulen

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

Squalen Synthase Inhibitors LCAT activators Chemistry and Physics of Lipids (2006, 2007)

SLIDE 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 Phosphatase Inhibitors SHP-2 Inhibitors SHP-2 Inhibitors

Treatment of Noonan Syndrom and certain leukemias Treatment of Noonan Syndrom and certain leukemias

Z N H N O S CH R Z N H N O S CH R

N-substituted 2-amino-5-aryliden-thiazolidin-4-ones

Journal of Medicinal Chemistry (2008)

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 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 Phosphatase Inhibitors

PTP1B Inhibitors LAR Inhibitors PTP1B Inhibitors LAR Inhibitors

Treatment of diabetes type II Treatment of diabetes type II

PTP1B inhibitors

3-[(furan-2-yl) methyl]-2-phenyl-thiazolidin-4-ones

O O O R

6H-5,8-dioxa-phenanthren derivatives

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

LAR inhibitors

34o FEBS Conference, 2009 10th Ibn Sina International Conference 2007, Egypt

SLIDE 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 anti-HIV agents

Reverse transcriptase inhibitors Reverse transcriptase inhibitors

N S O N S R1 R2 R3 R4 N S O N S N S R1 R2 R3 R4

XVIII International AIDS Conference Vienna, July 2010

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 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 anti-HIV agents

Integrase inhibitors Integrase inhibitors

XVIII International AIDS Conference Vienna, July 2010

R1 N O H R3 H R2 structure n R1 N O H R3 H R2 structure n

Spyro-isatino-cyclopropan derivatives

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 55

carbohydrates

glucose glycolytic cycle pyrouvate CH3CO-S-CoA

Krebs cycle

Respiratory chain Oxydative phosphorylation

ΑΤΡ

energy

triglycerides

Fatty acids glycerol

proteins

aminoacids NH3 urea Carbon backbone

Η C Η

2OH

Ο Η Η Η Η OH OH OH OH Η C Η

2OH

Ο Η Η Η Η OH OH OH OH

Enzymes: catalyze all reaction in human body Enzyme Deficiency/ Overactivation

Disease Disease

Tissue lesions Tissue lesions

Increased Serum Enzyme concentration

Enzyme

verexpression

Enzyme

verexpression

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 56

carbohydrates

glucose glycolytic cycle pyrouvate CH3CO-S-CoA

Krebs cycle

Respiratory chain Oxydative phosphorylation

ΑΤΡ

energy

triglycerides

Fatty acids glycerol

proteins

aminoacids NH3 urea Carbon backbone

Η C Η

2OH

Ο Η Η Η Η OH OH OH OH Η C Η

2OH

Ο Η Η Η Η OH OH OH OH

Enzymes: catalyze all reaction in human body Enzyme Deficiency/ Overactivation

Disease Disease Disease

Tissue lesions Tissue lesions

Increased Increased Serum Serum Enzyme Enzyme concentration concentration

Enzyme

verexpression

Enzyme

verexpression

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 57

carbohydrates

glucose glycolytic cycle pyrouvate CH3CO-S-CoA

Krebs cycle

Respiratory chain Oxydative phosphorylation

ΑΤΡ

energy

triglycerides

Fatty acids glycerol

proteins

aminoacids NH3 urea Carbon backbone

Η C Η

2OH

Ο Η Η Η Η OH OH OH OH Η C Η

2OH

Ο Η Η Η Η OH OH OH OH

Enzymes: catalyze all reaction in human body

Disease Disease

Tissue lesions Tissue lesions Tissue lesions

Increased Increased Serum Serum Enzyme Enzyme concentration concentration

Enzyme

verexpression

Enzyme Enzyme

verexpression
verexpression

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 58

carbohydrates

glucose glycolytic cycle pyrouvate CH3CO-S-CoA

Krebs cycle

Respiratory chain Oxydative phosphorylation

ΑΤΡ

energy

triglycerides

Fatty acids glycerol

proteins

aminoacids NH3 urea Carbon backbone

Η C Η

2OH

Ο Η Η Η Η OH OH OH OH Η C Η

2OH

Ο Η Η Η Η OH OH OH OH

Enzymes: catalyze all reaction in human body

Enzymes in Enzymes in Clinical Chemistry Clinical Chemistry

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 59

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

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 60

Isoenzymes Isoenzymes: catalyze the same reaction same reaction different gene different gene products Alkaline phosphatases

Liver/bone/kidney isoenzyme Placental isoenzyme Intestinal isoenzyme

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 61

G6PD aldolase CPK

Serum isoenzymes

amylase lipase alkaline phosphatases acid phosphatases LDH Transaminases γ-GT

S (saliva) isoenzymes P (pancrease) isoenzymes Liver Bone Intestine placenta bone Liver prostate

LDH1 (heart, erythrocytes, kidneys) 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)

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 62

CPK Methods of isoenzyme differential identification amylase LDH Electrophoresis

LDH1 LDH2 LDH3 LDH4 LDH5

ορός νεφροί ήπαρ καρδιά μυς

LDH1 LDH2 LDH3 LDH4 LDH5

ορός νεφροί ήπαρ καρδιά μυς

Serum kidneys heart liver muscles

CPK1 CPK2 CPK3

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 63

Methods of isoenzyme differential identification alkaline phosphatases Electrophoresis

φ

loading

(+) (-)

adult serum child serum placenta intestine bone liver adult serum with intestinal isoenzymes Alkaline phosphatase isoenzymes (Celulose acetate, 200V, 18 min)

SLIDE 69

Methods of isoenzyme differential identification Use of different substrates Use of special inhibitors Immunologic techniques

CH3-CO-O-CH -CH2-N + CH3 CH3 CH3 CH3-CO-O-CH -CH2-N + CH3 CH3 CH3 CH3

Acetyl-methyl-choline

CH3-CH2-CH2-CO-O-CH2-CH2-N + CH3 CH3 CH3

Butyryl-thiocholine

Cholinesterase

pseudocholinesterase

LDL LDL

LDH1, LDH2, LDH3 LDH1, LDH2, LDH3 LDH4, LDH5 LDH4, LDH5 LDH1, LDH2 LDH1, LDH2

lactate

β-hydroxy-butyrate Prostatic Prostatic acid phosphatase

Use of tartrate tartrate

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 70

Methods of isoenzyme differential identification Use of special inhibitors Immunologic techniques

Prostatic Prostatic acid phosphatase

Use of tartrate tartrate

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 71

Methods of isoenzyme differential identification Use of special inhibitors Immunologic techniques

Prostatic Prostatic acid phosphatase

Use of tartrate tartrate

CPK MB inhibition

inhibition by specific antibody ntibody

Placental Placental alkaline

alkaline phosphatase

ELISA ELISA method

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 72

Recent research results of our Laboratory

Inhibits all other serum alkaline phosphatase isoenzymes except of the placental isoenzyme 95% activation of Placental isoenzyme Probable good combination for colorimetric determination

f placental

isoenzyme

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

Combination of two compounds Compound 1 Compound 2

SLIDE 73

Need for specific inhibitors for treatment and diagnosis Valuable tools: Valuable tools: Computer Activity Prediction Programmes

Computer Activity Prediction Programmes

Computer Activity Prediction Programmes (PASS) Docking Studies

Docking Studies

Docking Studies

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 74

Need for specific inhibitors for treatment and diagnosis Valuable tools: Valuable tools: Computer Activity Prediction Programmes

Computer Activity Prediction Programmes

Computer Activity Prediction Programmes (PASS) Data Base Compound structures Activities

Your Compound structure

Comparison With Data base structures

Predicted Predicted activity activity

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 75

Need for specific inhibitors for treatment and diagnosis Valuable tools: Valuable tools: Computer Activity Prediction Programmes

Computer Activity Prediction Programmes

Computer Activity Prediction Programmes (PASS) Sructure design According to the literature Computer prediction Final desision

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 76

Need for specific inhibitors for treatment and diagnosis Valuable tools: Valuable tools: Modeling

Modeling

Modeling

Crystalographic data

f enzyme complex

with substrate or

ther inhibitors

Special computer programme

Complex free energy

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 77

Need for specific inhibitors for treatment and diagnosis Valuable tools: Valuable tools: Docking Studies

Docking Studies

Docking Studies

Crystalographic data

f enzyme complex

with substrate or

ther inhibitors

Special computer programme

Complex free energy

The lower the energy The most stable complex

The most potent inhibitor Potential inhibitor enzyme

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 78

Other uses of Enzymes Other uses of Enzymes Bioelectrodes-biosensors Bioelectrodes-biosensors

Need for: a highly active, stable appropriate enzyme, immobilized on a membrane attached to the appropriate electrode

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 79

Other uses of Enzymes Other uses of Enzymes Bioelectrodes-biosensors Bioelectrodes-biosensors Glucose electrode Glucose electrode

Glucose + O2 gluconic acid + H2O2

glucose

xidase

glucose glucose

Immobilised Immobilised glucose glucose

xidase
xidase

H2O2 Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 80

Other uses of Enzymes Other uses of Enzymes Bioelectrodes-biosensors Bioelectrodes-biosensors Biosensors in Critical Care

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

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 81

Other uses of Enzymes Other uses of Enzymes Immunosensors Immunosensors

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

Aplications in microbiology Herpes virus Chlamidia in urin Staphylococcal Enterotoxin etc.

SLIDE 82

συγκέντρωση ορμόνης μετρούμενη ποσότητα ιχνηλάτη

Y Y Y Y

A A A αντίσωμα-ιχνηλάτης ορμόνη αντίσωμα

Υ Υ Υ ☼ ☼☼

συγκέντρωση ορμόνης μετρούμενη ποσότητα ιχνηλάτη συγκέντρωση ορμόνης μετρούμενη ποσότητα ιχνηλάτη

Y Y Y Y

A A A αντίσωμα-ιχνηλάτης ορμόνη αντίσωμα

Υ Υ Υ ☼ ☼☼ Y Y Y Y

A A A αντίσωμα-ιχνηλάτης ορμόνη αντίσωμα

Υ Υ Υ ☼ ☼☼

A

Y Y Y Y

A A

☼

A A A A

☼

A

☼

A A A

ορμόνη ορμόνη-ιχνηλάτης αντίσωμα

συγκέντρωση ορμόνης μετρούμενη ποσότητα ιχνηλάτη A

Y Y Y Y

A A

☼

A A A A

☼

A

☼

A A A A

Y Y Y Y

A A

☼

A A A A

☼

A

☼

A

☼

A

☼

A A A

ορμόνη ορμόνη-ιχνηλάτης αντίσωμα

συγκέντρωση ορμόνης μετρούμενη ποσότητα ιχνηλάτη συγκέντρωση ορμόνης μετρούμενη ποσότητα ιχνηλάτη

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

Ab-Enzyme antigen Ab antigen concentration Enzyme reaction product Enzyme reaction product antigen concentration antigen Ab Antigen-Enzyme

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 83

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

Y Y Y Y

A

☼

: επισημασμένο αντιγόνο

anti-IgG

Y Y Y Y

A

☼

ανοσοσφαιρίνες IgG

Υ : προς προσδιορισμό αντίσωμα

A

☼

Y Y Y Y

A

☼

A

☼

: επισημασμένο αντιγόνο

anti-IgG

Y Y Y Y

A

☼

A

☼

ανοσοσφαιρίνες IgG

Υ : προς προσδιορισμό αντίσωμα

A

☼

A

☼ antibody

IgG

antigen-Enzyme

Y Y Y Y Y Y

E E E Ab

anti-IgG-Enzyme

antigen

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 84

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

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 85

2009 Α (Η1Ν1)

Lipid envelop

neuraminidase capside hemaglutinin Ion channel

RΝΑ nucleoproteins

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

An application

SLIDE 86

2009 Α (Η1Ν1)

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

. avian human Η3Ν2

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 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)

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 88

2009 Α (Η1Ν1)

Blood samples Blood samples were collected from November 1st 2009 till January 30th 2010 from 43 residents of Thessaloniki who had experienced flue symptoms from October 1st 2009 till January 10th 2010. 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 –20oC until determination.

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 89

2009 Α (Η1Ν1) Υ Υ antigen (15μg/ml) sample (1:400) SecondaryAntibody

POD

ABTS Coloured product Method

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.

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 90

500 1,000 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 500 1000 1500 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 Anti- 5237P IgG 200 100 300 150

μg/ml

500 1000 1500 2000 2500 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 500 1000 1500 2000 2500 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 Anti- 5237P IgM 100 80 60 40 20

μg/ml

100 80 60 40 20

A B D C

500 1000 1500 2000 2500 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 500 1000 1500 2000 2500 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 Anti- 5237P IgM 100 80 60 40 20

μg/ml

100 80 60 40 20 500 1000 1500 2000 2500 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 Anti- 5237P IgM 500 1000 1500 2000 2500 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 Anti- 5237P IgM 100 80 60 40 20

μg/ml

100 80 60 40 20

A B D C Figure 1

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.

Immunologic response to 2009 A(H1N1) HA and NA peptides

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 91

0.2 0.4 0.6 0.8 1 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 Anti- 5241 IgM 400 200

μg/ml

400 200 500 1,000 1,500 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43

500 1000 1500 2000 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43

Anti- 5241 IgG 600 400 200 1000 500

μg/ml A B D C Figure 2 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.

Immunologic response to 2009 A(H1N1) HA and NA peptides

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 92

20 40 60 80 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 20 40 60 80 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49

Anti- 5249 IgG

20 40 60 80 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 20 40 60 80 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49

Anti- 5249 IgG

μg/ml

20 40 60 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 20 40 60 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49

Anti- 5249 IgM

μg/ml A B C D 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.

Immunologic response to 2009 A(H1N1) HA and NA peptides

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece

SLIDE 93

Similarity of immunologic response to hemaglutinin swine-origin peptides of 2009 A(H1N1) between individuals experiencing flue symptoms and vaccinated ones, Better immunologic response to neuraminidase following natural infection.

Immunologic response to 2009 A(H1N1) HA and NA peptides

Dr Phaedra Eleftheriou, Department of Medical Laboratory Studies, Alexander TEITH, Greece