Conditions

1 Two components two sets of lecturers

2 Lectures 1-5 Prof F Hudecz Lectures 6-10 Dr Gy Domaacuteny 3 Examination two parts determined by the lecturers and one mark - option A written test - option B presentation based on literature - option C oral examination 4 Participation at lectures gt 70 fhudeczeltehu

First generatioin Second generation New generation

Oxytocin (L) Carbetocin (S) Abarelix (GnRH) (L) ACTH (1-24) amp (1-39) (LS) Terlipressin (LS) Cetrorelix (GnRH) (L) Vasopressin (LS) Felypressin (LS) Ganirelix (GnRH) (L) Insulin (ESS R) Buserelin (LS) Eptifibatide Glucagon (ESR) Deslorelin (LS) Bivalirudin (L) Calcitonins (LSR) Goserelin (L) Copaxone (L) TRH (L) Histrelin (L) Techtide P-289(S) Gonadorelin (LS) Leuprolide (LS) Cubicin (F) Somatostatin (LS) Nafarelin (S) Fuzeon (antiHIV (H) GHRH (1-29) amp (1-44) (S) Tryptorelin (LS) Ziconotide (pain) (S) CRF (Human amp Ovine) (S) Lecirelin (S) Pramlintide (diabetes) (S) Cyclosporin (F) Lanreotide (S) Exenatide (diabetes) (S) Thymopentin (L) Octreotide (LS) Icatibant (brady-rec) Thymosin Alpha-1 (S) Atosiban (L) Romiplostim (hormon) Secretins (Human amp Porcine) (ES) Desmopressin (LS) Degarelix (GnRH) Parathyroid Hormone (1-34) amp (1-84)(S) Lypressin (L) Mifamurtide (raacutek adj) Vasoactive Intestinal Polypeptide (S) Ornipressin Ecallantide (oumldeacutema) Brain Natriuretic Peptide (R) Pitressin (L) Liraglutide (diabetes) Cholecystokinin (L) ACE Inhibitors (Enalapril Lisinopril) (L) Tesamorelin Tetragastrin (L) HIV Protease Inhibitors (L) Surfaxin Pentagastrin (L) Peginesatide Eledoisin (L) Carfilzomib Linaclotide (enzinh)

L = in solution S = on solid phase E = extraction F = fermentation H = hybrid synthesis R = recombinant SS = semi-synthesis

Some Approved Peptide Pharmaceuticals and their Methods of Manufacture

Oxytocin

Structure 1953 V du Vigneaud Synthesis 1954 V du Vigneaud

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2

Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH2

V du Vigneaud (Nobel prize 1955)

Vasopressin

Hormones hypophysis

ACTH (Adrenocorticotropic hormone

corticotropin) (1-39 1-24)

H-Ser1-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly- -Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys- -Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Leu-Ala- -Glu-Ala-Phe-Pro-Leu-Glu-Phe39-OH

Synthesis (1971) S Bajusz L Kisfaludy K Medzihradszky

First generation the first ones

Hormones hypothalamus

CRH (Corticotropin-releasing hormone corticotropin-releasing factor CRF corticoliberin)

H-Ser1-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- -Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln- -Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala41

GHRH (growth hormone releasing hormone 1-44) HO-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile- -Met-SerndashArg29-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Glyndash -AlandashArg-Ala-Arg-Leu-NH2 Sermorelin ( GHRH 1-29 GRF 1-29)

The first generation

TRHthyrotropin-releasing hormone (thyrotropin-releasing factor TRF)

(pyro)Glu-His-Pro-NH2

(pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2

GhRH gonadotropin-releasing hormone Gonadorelin Luteinizing-hormone-releasing hormone LHRH)

A long-acting derivative of somatostatin

Sandostatin (Peptidomimetic)

Hormones

Calcitonin (32 amino acids) linear polypeptide produced in humans primarily by the parafollicular cells (also known as C-cells) of the thyroid Calcitonin can be used therapeutically for the treatment of hypercalcemia or osteoporosis Its structure comprises a single alpha helix

Parathyroid hormone (PTH) is secreted by the chief cells of the parathyroid glands as a

polypeptide with 84 amino acids It acts to increase the concentration of Ca2+ in the blood

whereas calcitonin acts to decrease calcium concentration

hPTH-(1-34) crystallizes as a slightly bent long helical dimer

The extended helical conformation of hPTH-(1-34) is

the likely bioactive conformation

The first generation

salmon Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro human Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe- His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro

Hormones pancreas The first generation

Glucagon (29 amino acids) Insulin

Vasoactive intestinal peptide (VIP) (28 amino acids)

H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- -Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- -Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH

Isolation 1922 F Banting (Nd 1923 32-year old) Structure 1953 F Sanger Synthesis 1969 H Zahn PG Katsoyannis Conformation 1965 D Hodgkin

Secreted by the pancreas raises blood glucose levels Its effect is opposite that of insulin which lowers

blood glucose levels

Produced by tissues of vertebrates (gut pancreas suprachiasmatic nuclei of the hypothalamus in the brain The highest levels are normally found

in the nervous system and gut It is a neuromodulatorneurotransmitter Regulates muscle activity epithelial cell secretion and blood flow in the

gastrointestinal tract

Cholecystokinin (CCK-8) C-terminal octapeptide

Pentagastrin (Peptavlon) 5 amino acids of the C-terminus end of

gastrin

Hormones digestion

Secretin (27 amino acids) HndashHis-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu- -Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu- -Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-CONH2

The first generation

Gastrin is a linear pepetide stimulating secretion of gastric acid (HCl) by the parietal cells of the stomach It is released by G cells in the antrum of the stomach duodenum and the pancreas into the bloodstream Gastrin is found primarily in three forms 1-34 (big gastrin) 1-17(little) 1-14 (mini)

From Greek chole bile cysto sac kinin move hence move the bile-sac (gallbladder) Responsible for stimulating

the digestion of fat and protein CCK is composed of varying numbers of amino acids depending on post-translational modification of the CCK gene product

eg CCK58 CCK33 and CCK8 CCK58 Kinevac (Sincalide for Injection)

First generatioin Second generation New generation

Oxytocin (L) Carbetocin (S) Abarelix (GnRH) (L) ACTH (1-24) amp (1-39) (LS) Terlipressin (LS) Cetrorelix (GnRH) (L) Vasopressin (LS) Felypressin (LS) Ganirelix (GnRH) (L) Insulin (ESS R) Buserelin (LS) Eptifibatide Glucagon (ESR) Deslorelin (LS) Bivalirudin (L) Calcitonins (LSR) Goserelin (L) Copaxone (L) TRH (L) Histrelin (L) Techtide P-289(S) Gonadorelin (LS) Leuprolide (LS) Cubicin (F) Somatostatin (LS) Nafarelin (S) Fuzeon (antiHIV (H) GHRH (1-29) amp (1-44) (S) Tryptorelin (LS) Ziconotide (pain) (S) CRF (Human amp Ovine) (S) Lecirelin (S) Pramlintide (diabetes) (S) Cyclosporin (F) Lanreotide (S) Exenatide (diabetes) (S) Thymopentin (L) Octreotide (LS) Icatibant (brady-rec) Thymosin Alpha-1 (S) Atosiban (L) Romiplostim (hormon) Secretins (Human amp Porcine) (ES) Desmopressin (LS) Degarelix (GnRH) Parathyroid Hormone (1-34) amp (1-84)(S) Lypressin (L) Mifamurtide (raacutek adj) Vasoactive Intestinal Polypeptide (S) Ornipressin Ecallantide (oumldeacutema) Brain Natriuretic Peptide (R) Pitressin (L) Liraglutide (diabetes) Cholecystokinin (L) ACE Inhibitors (Enalapril Lisinopril) (L) Tesamorelin Tetragastrin (L) HIV Protease Inhibitors (L) Surfaxin Pentagastrin (L) Peginesatide Eledoisin (L) Carfilzomib Linaclotide (enzinh)

L = in solution S = on solid phase E = extraction F = fermentation H = hybrid synthesis R = recombinant SS = semi-synthesis

Some Approved Peptide Pharmaceuticals and their Methods of Manufacture

Oxytocin

Structure 1953 V du Vigneaud Synthesis 1954 V du Vigneaud

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2

Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH2

V du Vigneaud (Nobel prize 1955)

Vasopressin

Hormones hypophysis

ACTH (Adrenocorticotropic hormone

corticotropin) (1-39 1-24)

H-Ser1-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly- -Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys- -Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Leu-Ala- -Glu-Ala-Phe-Pro-Leu-Glu-Phe39-OH

Synthesis (1971) S Bajusz L Kisfaludy K Medzihradszky

First generation the first ones

Hormones hypothalamus

CRH (Corticotropin-releasing hormone corticotropin-releasing factor CRF corticoliberin)

H-Ser1-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- -Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln- -Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala41

GHRH (growth hormone releasing hormone 1-44) HO-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile- -Met-SerndashArg29-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Glyndash -AlandashArg-Ala-Arg-Leu-NH2 Sermorelin ( GHRH 1-29 GRF 1-29)

The first generation

TRHthyrotropin-releasing hormone (thyrotropin-releasing factor TRF)

(pyro)Glu-His-Pro-NH2

(pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2

GhRH gonadotropin-releasing hormone Gonadorelin Luteinizing-hormone-releasing hormone LHRH)

A long-acting derivative of somatostatin

Sandostatin (Peptidomimetic)

Hormones

Calcitonin (32 amino acids) linear polypeptide produced in humans primarily by the parafollicular cells (also known as C-cells) of the thyroid Calcitonin can be used therapeutically for the treatment of hypercalcemia or osteoporosis Its structure comprises a single alpha helix

Parathyroid hormone (PTH) is secreted by the chief cells of the parathyroid glands as a

polypeptide with 84 amino acids It acts to increase the concentration of Ca2+ in the blood

whereas calcitonin acts to decrease calcium concentration

hPTH-(1-34) crystallizes as a slightly bent long helical dimer

The extended helical conformation of hPTH-(1-34) is

the likely bioactive conformation

The first generation

salmon Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro human Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe- His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro

Hormones pancreas The first generation

Glucagon (29 amino acids) Insulin

Vasoactive intestinal peptide (VIP) (28 amino acids)

H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- -Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- -Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH

Isolation 1922 F Banting (Nd 1923 32-year old) Structure 1953 F Sanger Synthesis 1969 H Zahn PG Katsoyannis Conformation 1965 D Hodgkin

Secreted by the pancreas raises blood glucose levels Its effect is opposite that of insulin which lowers

blood glucose levels

Produced by tissues of vertebrates (gut pancreas suprachiasmatic nuclei of the hypothalamus in the brain The highest levels are normally found

in the nervous system and gut It is a neuromodulatorneurotransmitter Regulates muscle activity epithelial cell secretion and blood flow in the

gastrointestinal tract

Cholecystokinin (CCK-8) C-terminal octapeptide

Pentagastrin (Peptavlon) 5 amino acids of the C-terminus end of

gastrin

Hormones digestion

Secretin (27 amino acids) HndashHis-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu- -Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu- -Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-CONH2

The first generation

Gastrin is a linear pepetide stimulating secretion of gastric acid (HCl) by the parietal cells of the stomach It is released by G cells in the antrum of the stomach duodenum and the pancreas into the bloodstream Gastrin is found primarily in three forms 1-34 (big gastrin) 1-17(little) 1-14 (mini)

From Greek chole bile cysto sac kinin move hence move the bile-sac (gallbladder) Responsible for stimulating

the digestion of fat and protein CCK is composed of varying numbers of amino acids depending on post-translational modification of the CCK gene product

eg CCK58 CCK33 and CCK8 CCK58 Kinevac (Sincalide for Injection)

Oxytocin

Structure 1953 V du Vigneaud Synthesis 1954 V du Vigneaud

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2

Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH2

V du Vigneaud (Nobel prize 1955)

Vasopressin

Hormones hypophysis

ACTH (Adrenocorticotropic hormone

corticotropin) (1-39 1-24)

H-Ser1-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly- -Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys- -Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Leu-Ala- -Glu-Ala-Phe-Pro-Leu-Glu-Phe39-OH

Synthesis (1971) S Bajusz L Kisfaludy K Medzihradszky

First generation the first ones

Hormones hypothalamus

CRH (Corticotropin-releasing hormone corticotropin-releasing factor CRF corticoliberin)

H-Ser1-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- -Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln- -Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala41

GHRH (growth hormone releasing hormone 1-44) HO-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile- -Met-SerndashArg29-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Glyndash -AlandashArg-Ala-Arg-Leu-NH2 Sermorelin ( GHRH 1-29 GRF 1-29)

The first generation

TRHthyrotropin-releasing hormone (thyrotropin-releasing factor TRF)

(pyro)Glu-His-Pro-NH2

(pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2

GhRH gonadotropin-releasing hormone Gonadorelin Luteinizing-hormone-releasing hormone LHRH)

A long-acting derivative of somatostatin

Sandostatin (Peptidomimetic)

Hormones

Calcitonin (32 amino acids) linear polypeptide produced in humans primarily by the parafollicular cells (also known as C-cells) of the thyroid Calcitonin can be used therapeutically for the treatment of hypercalcemia or osteoporosis Its structure comprises a single alpha helix

Parathyroid hormone (PTH) is secreted by the chief cells of the parathyroid glands as a

polypeptide with 84 amino acids It acts to increase the concentration of Ca2+ in the blood

whereas calcitonin acts to decrease calcium concentration

hPTH-(1-34) crystallizes as a slightly bent long helical dimer

The extended helical conformation of hPTH-(1-34) is

the likely bioactive conformation

The first generation

salmon Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro human Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe- His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro

Hormones pancreas The first generation

Glucagon (29 amino acids) Insulin

Vasoactive intestinal peptide (VIP) (28 amino acids)

H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- -Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- -Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH

Isolation 1922 F Banting (Nd 1923 32-year old) Structure 1953 F Sanger Synthesis 1969 H Zahn PG Katsoyannis Conformation 1965 D Hodgkin

Secreted by the pancreas raises blood glucose levels Its effect is opposite that of insulin which lowers

blood glucose levels

Produced by tissues of vertebrates (gut pancreas suprachiasmatic nuclei of the hypothalamus in the brain The highest levels are normally found

in the nervous system and gut It is a neuromodulatorneurotransmitter Regulates muscle activity epithelial cell secretion and blood flow in the

gastrointestinal tract

Cholecystokinin (CCK-8) C-terminal octapeptide

Pentagastrin (Peptavlon) 5 amino acids of the C-terminus end of

gastrin

Hormones digestion

Secretin (27 amino acids) HndashHis-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu- -Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu- -Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-CONH2

The first generation

Gastrin is a linear pepetide stimulating secretion of gastric acid (HCl) by the parietal cells of the stomach It is released by G cells in the antrum of the stomach duodenum and the pancreas into the bloodstream Gastrin is found primarily in three forms 1-34 (big gastrin) 1-17(little) 1-14 (mini)

From Greek chole bile cysto sac kinin move hence move the bile-sac (gallbladder) Responsible for stimulating

the digestion of fat and protein CCK is composed of varying numbers of amino acids depending on post-translational modification of the CCK gene product

eg CCK58 CCK33 and CCK8 CCK58 Kinevac (Sincalide for Injection)

Hormones hypothalamus

CRH (Corticotropin-releasing hormone corticotropin-releasing factor CRF corticoliberin)

H-Ser1-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- -Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln- -Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala41

GHRH (growth hormone releasing hormone 1-44) HO-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile- -Met-SerndashArg29-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Glyndash -AlandashArg-Ala-Arg-Leu-NH2 Sermorelin ( GHRH 1-29 GRF 1-29)

The first generation

TRHthyrotropin-releasing hormone (thyrotropin-releasing factor TRF)

(pyro)Glu-His-Pro-NH2

(pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2

GhRH gonadotropin-releasing hormone Gonadorelin Luteinizing-hormone-releasing hormone LHRH)

A long-acting derivative of somatostatin

Sandostatin (Peptidomimetic)

Hormones

Calcitonin (32 amino acids) linear polypeptide produced in humans primarily by the parafollicular cells (also known as C-cells) of the thyroid Calcitonin can be used therapeutically for the treatment of hypercalcemia or osteoporosis Its structure comprises a single alpha helix

Parathyroid hormone (PTH) is secreted by the chief cells of the parathyroid glands as a

polypeptide with 84 amino acids It acts to increase the concentration of Ca2+ in the blood

whereas calcitonin acts to decrease calcium concentration

hPTH-(1-34) crystallizes as a slightly bent long helical dimer

The extended helical conformation of hPTH-(1-34) is

the likely bioactive conformation

The first generation

salmon Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro human Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe- His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro

Hormones pancreas The first generation

Glucagon (29 amino acids) Insulin

Vasoactive intestinal peptide (VIP) (28 amino acids)

H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- -Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- -Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH

Isolation 1922 F Banting (Nd 1923 32-year old) Structure 1953 F Sanger Synthesis 1969 H Zahn PG Katsoyannis Conformation 1965 D Hodgkin

Secreted by the pancreas raises blood glucose levels Its effect is opposite that of insulin which lowers

blood glucose levels

Produced by tissues of vertebrates (gut pancreas suprachiasmatic nuclei of the hypothalamus in the brain The highest levels are normally found

in the nervous system and gut It is a neuromodulatorneurotransmitter Regulates muscle activity epithelial cell secretion and blood flow in the

gastrointestinal tract

Cholecystokinin (CCK-8) C-terminal octapeptide

Pentagastrin (Peptavlon) 5 amino acids of the C-terminus end of

gastrin

Hormones digestion

Secretin (27 amino acids) HndashHis-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu- -Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu- -Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-CONH2

The first generation

Gastrin is a linear pepetide stimulating secretion of gastric acid (HCl) by the parietal cells of the stomach It is released by G cells in the antrum of the stomach duodenum and the pancreas into the bloodstream Gastrin is found primarily in three forms 1-34 (big gastrin) 1-17(little) 1-14 (mini)

From Greek chole bile cysto sac kinin move hence move the bile-sac (gallbladder) Responsible for stimulating

the digestion of fat and protein CCK is composed of varying numbers of amino acids depending on post-translational modification of the CCK gene product

eg CCK58 CCK33 and CCK8 CCK58 Kinevac (Sincalide for Injection)

TRHthyrotropin-releasing hormone (thyrotropin-releasing factor TRF)

(pyro)Glu-His-Pro-NH2

(pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2

GhRH gonadotropin-releasing hormone Gonadorelin Luteinizing-hormone-releasing hormone LHRH)

A long-acting derivative of somatostatin

Sandostatin (Peptidomimetic)

Hormones

Calcitonin (32 amino acids) linear polypeptide produced in humans primarily by the parafollicular cells (also known as C-cells) of the thyroid Calcitonin can be used therapeutically for the treatment of hypercalcemia or osteoporosis Its structure comprises a single alpha helix

Parathyroid hormone (PTH) is secreted by the chief cells of the parathyroid glands as a

polypeptide with 84 amino acids It acts to increase the concentration of Ca2+ in the blood

whereas calcitonin acts to decrease calcium concentration

hPTH-(1-34) crystallizes as a slightly bent long helical dimer

The extended helical conformation of hPTH-(1-34) is

the likely bioactive conformation

The first generation

salmon Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro human Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe- His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro

Hormones pancreas The first generation

Glucagon (29 amino acids) Insulin

Vasoactive intestinal peptide (VIP) (28 amino acids)

H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- -Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- -Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH

Isolation 1922 F Banting (Nd 1923 32-year old) Structure 1953 F Sanger Synthesis 1969 H Zahn PG Katsoyannis Conformation 1965 D Hodgkin

Secreted by the pancreas raises blood glucose levels Its effect is opposite that of insulin which lowers

blood glucose levels

Produced by tissues of vertebrates (gut pancreas suprachiasmatic nuclei of the hypothalamus in the brain The highest levels are normally found

in the nervous system and gut It is a neuromodulatorneurotransmitter Regulates muscle activity epithelial cell secretion and blood flow in the

gastrointestinal tract

Cholecystokinin (CCK-8) C-terminal octapeptide

Pentagastrin (Peptavlon) 5 amino acids of the C-terminus end of

gastrin

Hormones digestion

Secretin (27 amino acids) HndashHis-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu- -Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu- -Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-CONH2

The first generation

Gastrin is a linear pepetide stimulating secretion of gastric acid (HCl) by the parietal cells of the stomach It is released by G cells in the antrum of the stomach duodenum and the pancreas into the bloodstream Gastrin is found primarily in three forms 1-34 (big gastrin) 1-17(little) 1-14 (mini)

From Greek chole bile cysto sac kinin move hence move the bile-sac (gallbladder) Responsible for stimulating

the digestion of fat and protein CCK is composed of varying numbers of amino acids depending on post-translational modification of the CCK gene product

eg CCK58 CCK33 and CCK8 CCK58 Kinevac (Sincalide for Injection)

Hormones

Calcitonin (32 amino acids) linear polypeptide produced in humans primarily by the parafollicular cells (also known as C-cells) of the thyroid Calcitonin can be used therapeutically for the treatment of hypercalcemia or osteoporosis Its structure comprises a single alpha helix

Parathyroid hormone (PTH) is secreted by the chief cells of the parathyroid glands as a

polypeptide with 84 amino acids It acts to increase the concentration of Ca2+ in the blood

whereas calcitonin acts to decrease calcium concentration

hPTH-(1-34) crystallizes as a slightly bent long helical dimer

The extended helical conformation of hPTH-(1-34) is

the likely bioactive conformation

The first generation

salmon Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro human Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe- His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro

Hormones pancreas The first generation

Glucagon (29 amino acids) Insulin

Vasoactive intestinal peptide (VIP) (28 amino acids)

H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- -Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- -Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH

Isolation 1922 F Banting (Nd 1923 32-year old) Structure 1953 F Sanger Synthesis 1969 H Zahn PG Katsoyannis Conformation 1965 D Hodgkin

Secreted by the pancreas raises blood glucose levels Its effect is opposite that of insulin which lowers

blood glucose levels

Produced by tissues of vertebrates (gut pancreas suprachiasmatic nuclei of the hypothalamus in the brain The highest levels are normally found

in the nervous system and gut It is a neuromodulatorneurotransmitter Regulates muscle activity epithelial cell secretion and blood flow in the

gastrointestinal tract

Cholecystokinin (CCK-8) C-terminal octapeptide

Pentagastrin (Peptavlon) 5 amino acids of the C-terminus end of

gastrin

Hormones digestion

Secretin (27 amino acids) HndashHis-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu- -Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu- -Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-CONH2

The first generation

Gastrin is a linear pepetide stimulating secretion of gastric acid (HCl) by the parietal cells of the stomach It is released by G cells in the antrum of the stomach duodenum and the pancreas into the bloodstream Gastrin is found primarily in three forms 1-34 (big gastrin) 1-17(little) 1-14 (mini)

From Greek chole bile cysto sac kinin move hence move the bile-sac (gallbladder) Responsible for stimulating

the digestion of fat and protein CCK is composed of varying numbers of amino acids depending on post-translational modification of the CCK gene product

eg CCK58 CCK33 and CCK8 CCK58 Kinevac (Sincalide for Injection)

Hormones pancreas The first generation

Glucagon (29 amino acids) Insulin

Vasoactive intestinal peptide (VIP) (28 amino acids)

H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- -Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- -Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH

Isolation 1922 F Banting (Nd 1923 32-year old) Structure 1953 F Sanger Synthesis 1969 H Zahn PG Katsoyannis Conformation 1965 D Hodgkin

Secreted by the pancreas raises blood glucose levels Its effect is opposite that of insulin which lowers

blood glucose levels

Produced by tissues of vertebrates (gut pancreas suprachiasmatic nuclei of the hypothalamus in the brain The highest levels are normally found

in the nervous system and gut It is a neuromodulatorneurotransmitter Regulates muscle activity epithelial cell secretion and blood flow in the

gastrointestinal tract

Cholecystokinin (CCK-8) C-terminal octapeptide

Pentagastrin (Peptavlon) 5 amino acids of the C-terminus end of

gastrin

Hormones digestion

Secretin (27 amino acids) HndashHis-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu- -Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu- -Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-CONH2

The first generation

Gastrin is a linear pepetide stimulating secretion of gastric acid (HCl) by the parietal cells of the stomach It is released by G cells in the antrum of the stomach duodenum and the pancreas into the bloodstream Gastrin is found primarily in three forms 1-34 (big gastrin) 1-17(little) 1-14 (mini)

From Greek chole bile cysto sac kinin move hence move the bile-sac (gallbladder) Responsible for stimulating

the digestion of fat and protein CCK is composed of varying numbers of amino acids depending on post-translational modification of the CCK gene product

eg CCK58 CCK33 and CCK8 CCK58 Kinevac (Sincalide for Injection)

Cholecystokinin (CCK-8) C-terminal octapeptide

Pentagastrin (Peptavlon) 5 amino acids of the C-terminus end of

gastrin

Hormones digestion

Secretin (27 amino acids) HndashHis-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu- -Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu- -Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-CONH2

The first generation

Gastrin is a linear pepetide stimulating secretion of gastric acid (HCl) by the parietal cells of the stomach It is released by G cells in the antrum of the stomach duodenum and the pancreas into the bloodstream Gastrin is found primarily in three forms 1-34 (big gastrin) 1-17(little) 1-14 (mini)

From Greek chole bile cysto sac kinin move hence move the bile-sac (gallbladder) Responsible for stimulating

the digestion of fat and protein CCK is composed of varying numbers of amino acids depending on post-translational modification of the CCK gene product

eg CCK58 CCK33 and CCK8 CCK58 Kinevac (Sincalide for Injection)

Cyclosporin (cyclosporin A) immunesuppressantfungi natural product

11 amino acids cyclic D-amino acids

Peptides acting on the immune system

Arg-Lys-Asp-Val-Tyr

Thymosin α1 T-cell immune stimulant 28 amino acids fragment

Thymopentin (TP-5) T-cell immune stimulant

The first generation

Isolation 1971 fromTolypocladium inflatum Medical use 1983

2009 treatment of hepatitis BC

Eledoisin 11 amino acids octopus (Eledone) origin

Belonging to the tachykinin family of neuropeptides it has vasodilator hypotensive and extravascular smooth muscle stimulant properties The amino acid sequence

pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2

Brain natriuretic peptide (BNP) 32 amino acids cyclic

Antihypertensive drugs

The first generation

Secreted by the ventricles of the heart in response to excessive stretching of heart muscle cells The

physiologic actions include decrease in systemic vascular resistance and central venous pressure

as well as an increase in natriuresis The net effect a decrease in blood volume

which lowers systemic blood pressure

New generation

Cetrorelix (synthetic)

Hormones GnRH antatogists - peptidomimetics

Abarelix (Plenaxis)

Ganirelix Degarelix

New generation

In oncology to reduce the amount of testosterone in patients with advanced

symptomatic prostate cancer

In assisted reproduction to control ovulation

Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2

httpwwwusermedscom

Enfuvirtide 36 amino acids

HIV fusion inhibitor antiviral

(binding to gp41 protein)

Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)

13 amino acids D-amino acid non-natural amino acid

N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone

Antibacterial drugs inhibitors of interactions

New generation

poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10

Ziconotide ω-conotoxin peptide bdquoConus magusrdquo

Ca channel blocking (non-opioid non-NSAID) pain killer

H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2

J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85

Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis

random copolymer polymer

Mifamurtide (Mepact) muramyl tripeptide

phosphatidylethanolamine osteosarcoma

httpwwwmedkoocomAnticancer-trialsMifamurtidehtm

New generation

Carfilzomib tetrapeptide multiple myeloma

proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012

httpwwwlookchemcomcas-868868540-17-4html

N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide

Enzyme inhibitors

Linaclotide (Linzess) 14 amino acids 3 cycles

guanlylate cyclase 2C inhibitor

irritable bowel syndrome FDA 30 August 2012

HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH

New generation

Peptide

Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )

40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10

18 16 14 12 10 8 6 4 2 0

Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002

SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT

Product Indication Length Status

Degludec Type 1 and type 2 diabetes -- NDA Pending

Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending

Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)

Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)

MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections

Pasireotide Cushingrsquos disease 6 Phase III

Albiglutide Type 2 diabetes -- Phase III

E75 Breast cancer (therapeutic vaccine) -- Phase IIIII

Pexiganan Diabetic foot infections 22 Phase IIIII

Cilengitide Glioblastoma 5 Phase III

KAI-4169 Seconday hyperparathyroidism -- Phase II

TRV120027 Acute heart failure -- Phase II

MIM-D3 Dry eye -- Phase II

API Date Manufacturing Method Indication

Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]

Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes

PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]

Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome

Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome

RECENTLY APPROVED PEPTIDES

AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines

Main fields of applications

Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414

Number of Peptides Approved or in Active Development

Eledoisin 11 amino acids octopus (Eledone) origin

Belonging to the tachykinin family of neuropeptides it has vasodilator hypotensive and extravascular smooth muscle stimulant properties The amino acid sequence

pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2

Brain natriuretic peptide (BNP) 32 amino acids cyclic

Antihypertensive drugs

The first generation

Secreted by the ventricles of the heart in response to excessive stretching of heart muscle cells The

physiologic actions include decrease in systemic vascular resistance and central venous pressure

as well as an increase in natriuresis The net effect a decrease in blood volume

which lowers systemic blood pressure

New generation

Cetrorelix (synthetic)

Hormones GnRH antatogists - peptidomimetics

Abarelix (Plenaxis)

Ganirelix Degarelix

New generation

In oncology to reduce the amount of testosterone in patients with advanced

symptomatic prostate cancer

In assisted reproduction to control ovulation

Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2

httpwwwusermedscom

Enfuvirtide 36 amino acids

HIV fusion inhibitor antiviral

(binding to gp41 protein)

Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)

13 amino acids D-amino acid non-natural amino acid

N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone

Antibacterial drugs inhibitors of interactions

New generation

poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10

Ziconotide ω-conotoxin peptide bdquoConus magusrdquo

Ca channel blocking (non-opioid non-NSAID) pain killer

H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2

J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85

Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis

random copolymer polymer

Mifamurtide (Mepact) muramyl tripeptide

phosphatidylethanolamine osteosarcoma

httpwwwmedkoocomAnticancer-trialsMifamurtidehtm

New generation

Carfilzomib tetrapeptide multiple myeloma

proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012

httpwwwlookchemcomcas-868868540-17-4html

N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide

Enzyme inhibitors

Linaclotide (Linzess) 14 amino acids 3 cycles

guanlylate cyclase 2C inhibitor

irritable bowel syndrome FDA 30 August 2012

HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH

New generation

Peptide

Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )

40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10

18 16 14 12 10 8 6 4 2 0

Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002

SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT

Product Indication Length Status

Degludec Type 1 and type 2 diabetes -- NDA Pending

Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending

Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)

Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)

MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections

Pasireotide Cushingrsquos disease 6 Phase III

Albiglutide Type 2 diabetes -- Phase III

E75 Breast cancer (therapeutic vaccine) -- Phase IIIII

Pexiganan Diabetic foot infections 22 Phase IIIII

Cilengitide Glioblastoma 5 Phase III

KAI-4169 Seconday hyperparathyroidism -- Phase II

TRV120027 Acute heart failure -- Phase II

MIM-D3 Dry eye -- Phase II

API Date Manufacturing Method Indication

Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]

Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes

PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]

Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome

Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome

RECENTLY APPROVED PEPTIDES

AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines

Main fields of applications

Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414

Number of Peptides Approved or in Active Development

New generation

Cetrorelix (synthetic)

Hormones GnRH antatogists - peptidomimetics

Abarelix (Plenaxis)

Ganirelix Degarelix

New generation

In oncology to reduce the amount of testosterone in patients with advanced

symptomatic prostate cancer

In assisted reproduction to control ovulation

Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2

httpwwwusermedscom

Enfuvirtide 36 amino acids

HIV fusion inhibitor antiviral

(binding to gp41 protein)

Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)

13 amino acids D-amino acid non-natural amino acid

N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone

Antibacterial drugs inhibitors of interactions

New generation

poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10

Ziconotide ω-conotoxin peptide bdquoConus magusrdquo

Ca channel blocking (non-opioid non-NSAID) pain killer

H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2

J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85

Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis

random copolymer polymer

Mifamurtide (Mepact) muramyl tripeptide

phosphatidylethanolamine osteosarcoma

httpwwwmedkoocomAnticancer-trialsMifamurtidehtm

New generation

Carfilzomib tetrapeptide multiple myeloma

proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012

httpwwwlookchemcomcas-868868540-17-4html

N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide

Enzyme inhibitors

Linaclotide (Linzess) 14 amino acids 3 cycles

guanlylate cyclase 2C inhibitor

irritable bowel syndrome FDA 30 August 2012

HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH

New generation

Peptide

Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )

40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10

18 16 14 12 10 8 6 4 2 0

Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002

SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT

Product Indication Length Status

Degludec Type 1 and type 2 diabetes -- NDA Pending

Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending

Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)

Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)

MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections

Pasireotide Cushingrsquos disease 6 Phase III

Albiglutide Type 2 diabetes -- Phase III

E75 Breast cancer (therapeutic vaccine) -- Phase IIIII

Pexiganan Diabetic foot infections 22 Phase IIIII

Cilengitide Glioblastoma 5 Phase III

KAI-4169 Seconday hyperparathyroidism -- Phase II

TRV120027 Acute heart failure -- Phase II

MIM-D3 Dry eye -- Phase II

API Date Manufacturing Method Indication

Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]

Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes

PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]

Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome

Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome

RECENTLY APPROVED PEPTIDES

AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines

Main fields of applications

Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414

Number of Peptides Approved or in Active Development

Cetrorelix (synthetic)

Hormones GnRH antatogists - peptidomimetics

Abarelix (Plenaxis)

Ganirelix Degarelix

New generation

In oncology to reduce the amount of testosterone in patients with advanced

symptomatic prostate cancer

In assisted reproduction to control ovulation

Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2

httpwwwusermedscom

Enfuvirtide 36 amino acids

HIV fusion inhibitor antiviral

(binding to gp41 protein)

Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)

13 amino acids D-amino acid non-natural amino acid

N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone

Antibacterial drugs inhibitors of interactions

New generation

poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10

Ziconotide ω-conotoxin peptide bdquoConus magusrdquo

Ca channel blocking (non-opioid non-NSAID) pain killer

H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2

J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85

Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis

random copolymer polymer

Mifamurtide (Mepact) muramyl tripeptide

phosphatidylethanolamine osteosarcoma

httpwwwmedkoocomAnticancer-trialsMifamurtidehtm

New generation

Carfilzomib tetrapeptide multiple myeloma

proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012

httpwwwlookchemcomcas-868868540-17-4html

N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide

Enzyme inhibitors

Linaclotide (Linzess) 14 amino acids 3 cycles

guanlylate cyclase 2C inhibitor

irritable bowel syndrome FDA 30 August 2012

HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH

New generation

Peptide

Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )

40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10

18 16 14 12 10 8 6 4 2 0

Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002

SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT

Product Indication Length Status

Degludec Type 1 and type 2 diabetes -- NDA Pending

Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending

Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)

Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)

MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections

Pasireotide Cushingrsquos disease 6 Phase III

Albiglutide Type 2 diabetes -- Phase III

E75 Breast cancer (therapeutic vaccine) -- Phase IIIII

Pexiganan Diabetic foot infections 22 Phase IIIII

Cilengitide Glioblastoma 5 Phase III

KAI-4169 Seconday hyperparathyroidism -- Phase II

TRV120027 Acute heart failure -- Phase II

MIM-D3 Dry eye -- Phase II

API Date Manufacturing Method Indication

Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]

Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes

PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]

Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome

Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome

RECENTLY APPROVED PEPTIDES

AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines

Main fields of applications

Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414

Number of Peptides Approved or in Active Development

Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu- -Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu--Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn- -Trp-Phe-NH2

httpwwwusermedscom

Enfuvirtide 36 amino acids

HIV fusion inhibitor antiviral

(binding to gp41 protein)

Daptomycin (Cubicin) Lipopeptide antibiotics (Gram positive)

13 amino acids D-amino acid non-natural amino acid

N-decanoyl-L-Trp-L-Asn-L-Asp-L-Thr-Gly-L-Orn-L-Asp-D-Ala-L-Asp-Gly-D-Ser-threo -3-methyl-L-Glu-3-anthraniloyl-L-Ala[egr]1-lactone

Antibacterial drugs inhibitors of interactions

New generation

poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10

Ziconotide ω-conotoxin peptide bdquoConus magusrdquo

Ca channel blocking (non-opioid non-NSAID) pain killer

H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2

J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85

Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis

random copolymer polymer

Mifamurtide (Mepact) muramyl tripeptide

phosphatidylethanolamine osteosarcoma

httpwwwmedkoocomAnticancer-trialsMifamurtidehtm

New generation

Carfilzomib tetrapeptide multiple myeloma

proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012

httpwwwlookchemcomcas-868868540-17-4html

N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide

Enzyme inhibitors

Linaclotide (Linzess) 14 amino acids 3 cycles

guanlylate cyclase 2C inhibitor

irritable bowel syndrome FDA 30 August 2012

HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH

New generation

Peptide

Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )

40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10

18 16 14 12 10 8 6 4 2 0

Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002

SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT

Product Indication Length Status

Degludec Type 1 and type 2 diabetes -- NDA Pending

Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending

Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)

Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)

MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections

Pasireotide Cushingrsquos disease 6 Phase III

Albiglutide Type 2 diabetes -- Phase III

E75 Breast cancer (therapeutic vaccine) -- Phase IIIII

Pexiganan Diabetic foot infections 22 Phase IIIII

Cilengitide Glioblastoma 5 Phase III

KAI-4169 Seconday hyperparathyroidism -- Phase II

TRV120027 Acute heart failure -- Phase II

MIM-D3 Dry eye -- Phase II

API Date Manufacturing Method Indication

Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]

Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes

PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]

Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome

Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome

RECENTLY APPROVED PEPTIDES

AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines

Main fields of applications

Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414

Number of Peptides Approved or in Active Development

poly(Glu14-18- Ala41-58-Lys32-42-Tyr 10

Ziconotide ω-conotoxin peptide bdquoConus magusrdquo

Ca channel blocking (non-opioid non-NSAID) pain killer

H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2

J G McGivern Neuropsychiatr Dis Treat 2007 3 69ndash85

Glatiramer acetate (Copolymer 1 Copaxone) immunomodulator multiple sclerosis

random copolymer polymer

Mifamurtide (Mepact) muramyl tripeptide

phosphatidylethanolamine osteosarcoma

httpwwwmedkoocomAnticancer-trialsMifamurtidehtm

New generation

Carfilzomib tetrapeptide multiple myeloma

proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012

httpwwwlookchemcomcas-868868540-17-4html

N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide

Enzyme inhibitors

Linaclotide (Linzess) 14 amino acids 3 cycles

guanlylate cyclase 2C inhibitor

irritable bowel syndrome FDA 30 August 2012

HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH

New generation

Peptide

Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )

40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10

18 16 14 12 10 8 6 4 2 0

Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002

SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT

Product Indication Length Status

Degludec Type 1 and type 2 diabetes -- NDA Pending

Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending

Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)

Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)

MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections

Pasireotide Cushingrsquos disease 6 Phase III

Albiglutide Type 2 diabetes -- Phase III

E75 Breast cancer (therapeutic vaccine) -- Phase IIIII

Pexiganan Diabetic foot infections 22 Phase IIIII

Cilengitide Glioblastoma 5 Phase III

KAI-4169 Seconday hyperparathyroidism -- Phase II

TRV120027 Acute heart failure -- Phase II

MIM-D3 Dry eye -- Phase II

API Date Manufacturing Method Indication

Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]

Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes

PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]

Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome

Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome

RECENTLY APPROVED PEPTIDES

AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines

Main fields of applications

Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414

Number of Peptides Approved or in Active Development

Carfilzomib tetrapeptide multiple myeloma

proteosome inhibitor chymotripsine-like enzyme FDA 20 July 2012

httpwwwlookchemcomcas-868868540-17-4html

N-(2S)-2-[(4-Morpholinylacetyl)amino]-4-phenylbutanoyl- L-Leu-N-(2S)-4-methyl-1-[(2R)-2-methyl-2-oxiranyl]-1-oxo- 2-pentanyl-L-Phe-amide

Enzyme inhibitors

Linaclotide (Linzess) 14 amino acids 3 cycles

guanlylate cyclase 2C inhibitor

irritable bowel syndrome FDA 30 August 2012

HndashCysndashCysndashGlundashTyrndashCysndashCysndashAsnndashProndashAlandashCysndashThrndashGlyndashCysndashTyr14ndashOH

New generation

Peptide

Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )

40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10

18 16 14 12 10 8 6 4 2 0

Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002

SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT

Product Indication Length Status

Degludec Type 1 and type 2 diabetes -- NDA Pending

Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending

Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)

Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)

MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections

Pasireotide Cushingrsquos disease 6 Phase III

Albiglutide Type 2 diabetes -- Phase III

E75 Breast cancer (therapeutic vaccine) -- Phase IIIII

Pexiganan Diabetic foot infections 22 Phase IIIII

Cilengitide Glioblastoma 5 Phase III

KAI-4169 Seconday hyperparathyroidism -- Phase II

TRV120027 Acute heart failure -- Phase II

MIM-D3 Dry eye -- Phase II

API Date Manufacturing Method Indication

Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]

Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes

PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]

Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome

Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome

RECENTLY APPROVED PEPTIDES

AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines

Main fields of applications

Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414

Number of Peptides Approved or in Active Development

Peptide

Year of clinical studies (1940 - ) Number of peptide drugs on the market (1970 - )

40rsquos 50rsquos 60rsquos 70rsquos 80rsquos 90rsquos 00-10

18 16 14 12 10 8 6 4 2 0

Peptide Therapeutics Foundations 2010 appr 50 approved peptide (API) until 2002

SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT

Product Indication Length Status

Degludec Type 1 and type 2 diabetes -- NDA Pending

Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending

Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)

Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)

MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections

Pasireotide Cushingrsquos disease 6 Phase III

Albiglutide Type 2 diabetes -- Phase III

E75 Breast cancer (therapeutic vaccine) -- Phase IIIII

Pexiganan Diabetic foot infections 22 Phase IIIII

Cilengitide Glioblastoma 5 Phase III

KAI-4169 Seconday hyperparathyroidism -- Phase II

TRV120027 Acute heart failure -- Phase II

MIM-D3 Dry eye -- Phase II

API Date Manufacturing Method Indication

Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]

Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes

PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]

Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome

Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome

RECENTLY APPROVED PEPTIDES

AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines

Main fields of applications

Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414

Number of Peptides Approved or in Active Development

SOME EXAMPLES OF PEPTIDES IN LATE STAGE CLINICAL DEVELOPMENT

Product Indication Length Status

Degludec Type 1 and type 2 diabetes -- NDA Pending

Teduglutide Short bowel syndrome (GLP-2 analog) 33 NDA Pending

Lixisenatide (ZP10) Type 2 diabetes 44 Phase III (GLP-1 Agonist)

Stimuvax Non-small cell lung carcinoma (therapeutic vaccine) 25 Phase III (BLP-25 lipopeptide)

MX-226 Topical antimicrobial for catheter- 12 Phase III (Omiganan) related infections

Pasireotide Cushingrsquos disease 6 Phase III

Albiglutide Type 2 diabetes -- Phase III

E75 Breast cancer (therapeutic vaccine) -- Phase IIIII

Pexiganan Diabetic foot infections 22 Phase IIIII

Cilengitide Glioblastoma 5 Phase III

KAI-4169 Seconday hyperparathyroidism -- Phase II

TRV120027 Acute heart failure -- Phase II

MIM-D3 Dry eye -- Phase II

API Date Manufacturing Method Indication

Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]

Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes

PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]

Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome

Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome

RECENTLY APPROVED PEPTIDES

AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines

Main fields of applications

Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414

Number of Peptides Approved or in Active Development

API Date Manufacturing Method Indication

Teriparatide 112602 Recombinant Osteoporosis [PTH (1-34)]

Fuzeon (T20) 31303 Hybrid synthesis AIDS Cubicin 91203 Fermentation Bacterial skin infections Abarelix 112503 Solution-phase synthesis Prostate cancer Human Secretin 4904 Solid-phase synthesis Diagnostic for pancreatic function Prialt (Ziconotide) 122904 Solid-phase synthesis Chronic pain Symlin (Pramlintide) 31605 Solid-phase synthesis Types 1 and 2 diabetes Byetta (Exenatide) 42905 Solid-phase synthesis Type 2 diabetes

PreosPreotact 42606 Recombinant Osteoporosis [PTH (1-84)]

Romiplostim 82208 Recombinant Chronic idiopathic thrombocytopenia Degarelix 122408 Solution-phase synthesis Prostate cancer Mefamurtide 3609 Solution-phase synthesis Osteosarcoma Ecallantide 112709 Recombinant Hereditary angioedema Liraglutide 22510 Recombinant Type 2 diabetes Tesamorelin 111210 Solid-phase synthesis HIV lipodystrophy Surfaxin 31612 Solid-phase synthesis Respiratory distress syndrome

Peginesatide 32712 Solid-phase synthesis Anemia Carfilzomib 72012 Solution-phase synthesis Multiple myeloma Linaclotide 83012 Solid-phase synthesis Irritable bowel syndrome

RECENTLY APPROVED PEPTIDES

AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines

Main fields of applications

Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414

Number of Peptides Approved or in Active Development

AIDS Gynecological Disorders Allergies Hypertension Analgesia IBDIBS Arthritis Immune Deficiencies Birth Control Infections (anti-viral anti-microbial) Cardiovascular Diseases Inflammation CNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes Oncology Epilepsy Ophthalmology Gastrointestinal Disorders Osteoporosis Growth Deficiencies Urology Vaccines

Main fields of applications

Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414

Number of Peptides Approved or in Active Development

Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414

Number of Peptides Approved or in Active Development

generic

name

year of

approval

therapeutic

area

country of

approval

generic name year of

approval

therapeutic area country

of

approval

atosiban 2000 obstetrics EU mifamurtide 2009 oncology EU

taltirelin 2000 CNS JP liraglutide 2009 metabolic disease EU

aviptadil 2000 urology EU tesamorelin 2010 antiinfective US

carbetocin 2001 obstetrics EU lucinactant 2012 pulmonary US

nesiritide 2001 cardiovascular US peginesatide 2012 hematology US

teriparatide 2002 osteoporosis US pasireotide 2012 endocinology EU

enfuvirtide 2003 antiinfective US carfilzomib 2012 oncology US

abarelix 2003 oncology US linaclotide 2012 gastroenterology US

ziconotide 2004 pain US teduglutide 2012 gastroenterology EU

pramlintide 2005 metabolic

disease

US lixisenatide 2013 metabolic disease EU

exenatide 2005 metabolic

disease

US albiglutide 2014 metabolic disease EU

icatibant 2008 hematology EU oritavancin 2014 antiinfective US

romiplostim 2008 hematology US dulaglutide 2014 metabolic disease US

degarelix 2008 oncology US afamelanotide 2014 dermatology

Non-insulin Peptides Approved in the Years 2000ndash2016 Including Region of Launch

Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414

Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414

Top Selling Non-Insulin Peptide Sales 2015

Extracellular - receptors (eg G-protein-coupled receptors ) - enzymes - protein-protein interactions

Targets

Intracellular (10 of the compounds under development - Cell penetration - Reductive cytosol (Cys-Cys)

Advantages - Disadvantages

Specificity Potency Low toxicity

Low stability Short half-life Enzymatic decomposition Lack of oral application

Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav

Sources

Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)

Advantages - Disadvantages

Specificity Potency Low toxicity

Low stability Short half-life Enzymatic decomposition Lack of oral application

Isolation Synthesis Peptide antibiotics Protein fragments Peptide conjugates Bispecific peptides Disulfide-rich peptidesrdquo (DRP) 8-40 aminosav

Sources

Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)

Suggested readings Henninot A Collins JC Nuss JM The Current State of Peptide Drug Discovery Back to the Future J Med Chem 2018 611382-1414 doi 101021acsjmedchem7b00318 Eder J Herrling PL Trends in Modern Drug Discovery Handb Exp Pharmacol 20162323-22 doi 101007164_2015_20 Gaspar R Aksu B Cuine A Danhof M Takac MJ Linden HH Link A Muchitsch EM Wilson CG Ohrngren P Dencker L Towards a European strategy for medicines research (2014-2020) The EUFEPS position paper on Horizon 2020 Eur J Pharm Sci 2012 47979-87 doi 101016jejps201209020 Parthasarathy A Anandamma SK Kalesh KA The Medicinal Chemistry of Therapeutic Peptides Recent Developments in Synthesis and Design Optimizations Curr Med Chem 2017 doi 1021740929867324666171012103559 (Epub ahead of print)

NUMBERS

Estimated number of proteins in the human body 100 000 Primary structure analysis (F Sanger 1953)

1953-1978 (25 years) 1081 1979-1991 (13 years) 16 000 1992- 1000year Three-dimensional (3D) structure (J Kendrew 1962)

1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100years

CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION

1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection

bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection

(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)

bull nerve impulses bull growth bull differentiation

6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support

bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)

RECOGNITION PHENOMENA

Interaction Kd[M]

1 Enzyme ndash substrate 10-3 ndash 10-5

2 Transporter ndash ligand 10-6 ndash 10-8

3 Hormone ndash receptor 10-9

4 Antibody ndash antigen 10-7 ndash 10-11

5 Storage protein ndash ligand

6 Toxin ndash receptor

7 Protein ndash protein (in a contractile super assembly)

8 Lectin ndash carbohydrate 10-4 ndash 10-7

9 Avidin ndash biotin 10-15

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

I Experimental methods

Chemical modification bull side chain modification bull conjugation Fragmentation

bull enzymatic (eg trysin) bull chemical (eg BrCN)

Separation bull centrifugation bull chromatography bull electrophoresis

Identification bull amino acid analysis bull sequencing bull mass spectrometry

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

I Experimental methods

Genetic engineering bull deletion bull chimeric proteins bull mutagenesis

ndash site directed ndash random

bull phage display libraries

Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries

An example Identification of epitope sequences in protein antigens

Whole virus

Viral envelope proteins (mixture)

1 Affinity chromatography 2 Gradient centrifugation

Amino acid sequence Immunodominant protein component komponens

Immuneprecipitation (Gelelectrophoresis)

Sequencing

Identification of peptid epitopes

protein

prediction of 3D structure

epitope bdquomaprdquo

Prediction of hydrophilicity

modeling

Identification of short sequences responsible for activity

bdquooverlappingrdquo strategy

bdquopredictedrdquo

Gaacutet

laacutes

TPTPTGTQ

PTGTQ

TGTQ

0

20

40

60

80

100

001 1 100 cpeptid (mmoll)

bdquoshorteningrdquo strategy

intuicioacute

bdquocombinatorialrdquo strategy

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

IITheoretical methods

Quantum chemistry bull molecular mechanics bull molecular dynamics

Predictions from the primary structure

bull Probabilistic (statistical) 1970 ndash

bull Physicochemical 1974 ndash

bull Information theory 1974 -

Approaches for the localisation of functionally relevant domains in proteins

Protein primary structure

Know Unknown

3D Structure

Know Unknown

Isolation

Fragmentation separation

Functional assay with fragments

Selection

Structure elucidation

Prediction of

bull secondary structure

bull functionally relevant domain

Fragmentation separation

Functional assay with fragments

Selection

3D Structure elucidation

Chemical modification

Genetic engineering

Chemical synthesis

Smallest functional domain

Strategies for determinations of 3D structures

Experimental methods Theoretical methods

X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)

bull crystal bull time-intensive

NMR

CD spectroscopy FT-IR spectroscopy

Quantum chemistry mechanics and dynamics

Empirical calculations

Relationship assay

Prediction of secondary structures (helix b-turn)

Prediction of hydrophobic ndash hydrophilic regions

Techniques for the detection of interactionrecognition phenomena

1 Molecular level

Detection with Separation Detection without Separation

Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis

Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation

Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy

Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)

Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy

Techniques for the detection of interactionrecognition phenomena

2 Cellular level

Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity

In vivo post-translational modifications

alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)

Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)

Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)

Ubiquitination

bull Ubiquitin protein (76 amino acids 85 kDa)

bull Almost all tissues of eukaryotic organisms

bull It can signal the degradation of the attached protein by transporting to proteasome

bull Isopeptide linkage (4)

bull Enzymes involved

ndash E1 (ub activation)

ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)

ndash E3 ( ub ligation)

Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose

NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)

bull Function

activationregulation of ubiquitin

SUMOylation (Small Ubiquitin-like Modifier)

bull SUMO proteins 100 aa 12 kDa 4 isoforms

bull Post-translational modification

bull Aktivation cleavage of 4 residues at the C-terminal

bull Attachment to target protein by using three enzymes

Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation

R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)

CLASSIFICATION OF PROTEINS ACCORDING TO THEIR FUNCTION

1 Enzymatic catalysis (eg Ser proteases) 2 Transport (eg transferrin for iron serum albumin for fatty acids) 3 Storage (eg ferrin for iron in liver casein in milk) 4 Protection

bull toxins (eg ricin [plant] diphteria [bacteria]) bull self and non-self discrimination immune protection

(eg antibodies antigenes) 5 Signal transduction (eg hormones receptors)

bull nerve impulses bull growth bull differentiation

6 Cell to cell communication (eg adhesion molecules factors acceptors) 7 Coordinated motion (eg muscle proteins) 8 Mechanical support

bull at cellular level (eg Membrane proteins) bull at tissue level (eg structural proteins eg collagen in skin bone)

RECOGNITION PHENOMENA

Interaction Kd[M]

1 Enzyme ndash substrate 10-3 ndash 10-5

2 Transporter ndash ligand 10-6 ndash 10-8

3 Hormone ndash receptor 10-9

4 Antibody ndash antigen 10-7 ndash 10-11

5 Storage protein ndash ligand

6 Toxin ndash receptor

7 Protein ndash protein (in a contractile super assembly)

8 Lectin ndash carbohydrate 10-4 ndash 10-7

9 Avidin ndash biotin 10-15

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

I Experimental methods

Chemical modification bull side chain modification bull conjugation Fragmentation

bull enzymatic (eg trysin) bull chemical (eg BrCN)

Separation bull centrifugation bull chromatography bull electrophoresis

Identification bull amino acid analysis bull sequencing bull mass spectrometry

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

I Experimental methods

Genetic engineering bull deletion bull chimeric proteins bull mutagenesis

ndash site directed ndash random

bull phage display libraries

Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries

An example Identification of epitope sequences in protein antigens

Whole virus

Viral envelope proteins (mixture)

1 Affinity chromatography 2 Gradient centrifugation

Amino acid sequence Immunodominant protein component komponens

Immuneprecipitation (Gelelectrophoresis)

Sequencing

Identification of peptid epitopes

protein

prediction of 3D structure

epitope bdquomaprdquo

Prediction of hydrophilicity

modeling

Identification of short sequences responsible for activity

bdquooverlappingrdquo strategy

bdquopredictedrdquo

Gaacutet

laacutes

TPTPTGTQ

PTGTQ

TGTQ

0

20

40

60

80

100

001 1 100 cpeptid (mmoll)

bdquoshorteningrdquo strategy

intuicioacute

bdquocombinatorialrdquo strategy

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

IITheoretical methods

Quantum chemistry bull molecular mechanics bull molecular dynamics

Predictions from the primary structure

bull Probabilistic (statistical) 1970 ndash

bull Physicochemical 1974 ndash

bull Information theory 1974 -

Approaches for the localisation of functionally relevant domains in proteins

Protein primary structure

Know Unknown

3D Structure

Know Unknown

Isolation

Fragmentation separation

Functional assay with fragments

Selection

Structure elucidation

Prediction of

bull secondary structure

bull functionally relevant domain

Fragmentation separation

Functional assay with fragments

Selection

3D Structure elucidation

Chemical modification

Genetic engineering

Chemical synthesis

Smallest functional domain

Strategies for determinations of 3D structures

Experimental methods Theoretical methods

X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)

bull crystal bull time-intensive

NMR

CD spectroscopy FT-IR spectroscopy

Quantum chemistry mechanics and dynamics

Empirical calculations

Relationship assay

Prediction of secondary structures (helix b-turn)

Prediction of hydrophobic ndash hydrophilic regions

Techniques for the detection of interactionrecognition phenomena

1 Molecular level

Detection with Separation Detection without Separation

Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis

Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation

Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy

Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)

Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy

Techniques for the detection of interactionrecognition phenomena

2 Cellular level

Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity

In vivo post-translational modifications

alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)

Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)

Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)

Ubiquitination

bull Ubiquitin protein (76 amino acids 85 kDa)

bull Almost all tissues of eukaryotic organisms

bull It can signal the degradation of the attached protein by transporting to proteasome

bull Isopeptide linkage (4)

bull Enzymes involved

ndash E1 (ub activation)

ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)

ndash E3 ( ub ligation)

Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose

NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)

bull Function

activationregulation of ubiquitin

SUMOylation (Small Ubiquitin-like Modifier)

bull SUMO proteins 100 aa 12 kDa 4 isoforms

bull Post-translational modification

bull Aktivation cleavage of 4 residues at the C-terminal

bull Attachment to target protein by using three enzymes

Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation

R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)

RECOGNITION PHENOMENA

Interaction Kd[M]

1 Enzyme ndash substrate 10-3 ndash 10-5

2 Transporter ndash ligand 10-6 ndash 10-8

3 Hormone ndash receptor 10-9

4 Antibody ndash antigen 10-7 ndash 10-11

5 Storage protein ndash ligand

6 Toxin ndash receptor

7 Protein ndash protein (in a contractile super assembly)

8 Lectin ndash carbohydrate 10-4 ndash 10-7

9 Avidin ndash biotin 10-15

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

I Experimental methods

Chemical modification bull side chain modification bull conjugation Fragmentation

bull enzymatic (eg trysin) bull chemical (eg BrCN)

Separation bull centrifugation bull chromatography bull electrophoresis

Identification bull amino acid analysis bull sequencing bull mass spectrometry

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

I Experimental methods

Genetic engineering bull deletion bull chimeric proteins bull mutagenesis

ndash site directed ndash random

bull phage display libraries

Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries

An example Identification of epitope sequences in protein antigens

Whole virus

Viral envelope proteins (mixture)

1 Affinity chromatography 2 Gradient centrifugation

Amino acid sequence Immunodominant protein component komponens

Immuneprecipitation (Gelelectrophoresis)

Sequencing

Identification of peptid epitopes

protein

prediction of 3D structure

epitope bdquomaprdquo

Prediction of hydrophilicity

modeling

Identification of short sequences responsible for activity

bdquooverlappingrdquo strategy

bdquopredictedrdquo

Gaacutet

laacutes

TPTPTGTQ

PTGTQ

TGTQ

0

20

40

60

80

100

001 1 100 cpeptid (mmoll)

bdquoshorteningrdquo strategy

intuicioacute

bdquocombinatorialrdquo strategy

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

IITheoretical methods

Quantum chemistry bull molecular mechanics bull molecular dynamics

Predictions from the primary structure

bull Probabilistic (statistical) 1970 ndash

bull Physicochemical 1974 ndash

bull Information theory 1974 -

Approaches for the localisation of functionally relevant domains in proteins

Protein primary structure

Know Unknown

3D Structure

Know Unknown

Isolation

Fragmentation separation

Functional assay with fragments

Selection

Structure elucidation

Prediction of

bull secondary structure

bull functionally relevant domain

Fragmentation separation

Functional assay with fragments

Selection

3D Structure elucidation

Chemical modification

Genetic engineering

Chemical synthesis

Smallest functional domain

Strategies for determinations of 3D structures

Experimental methods Theoretical methods

X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)

bull crystal bull time-intensive

NMR

CD spectroscopy FT-IR spectroscopy

Quantum chemistry mechanics and dynamics

Empirical calculations

Relationship assay

Prediction of secondary structures (helix b-turn)

Prediction of hydrophobic ndash hydrophilic regions

Techniques for the detection of interactionrecognition phenomena

1 Molecular level

Detection with Separation Detection without Separation

Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis

Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation

Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy

Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)

Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy

Techniques for the detection of interactionrecognition phenomena

2 Cellular level

Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity

In vivo post-translational modifications

alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)

Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)

Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)

Ubiquitination

bull Ubiquitin protein (76 amino acids 85 kDa)

bull Almost all tissues of eukaryotic organisms

bull It can signal the degradation of the attached protein by transporting to proteasome

bull Isopeptide linkage (4)

bull Enzymes involved

ndash E1 (ub activation)

ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)

ndash E3 ( ub ligation)

Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose

NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)

bull Function

activationregulation of ubiquitin

SUMOylation (Small Ubiquitin-like Modifier)

bull SUMO proteins 100 aa 12 kDa 4 isoforms

bull Post-translational modification

bull Aktivation cleavage of 4 residues at the C-terminal

bull Attachment to target protein by using three enzymes

Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation

R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

I Experimental methods

Chemical modification bull side chain modification bull conjugation Fragmentation

bull enzymatic (eg trysin) bull chemical (eg BrCN)

Separation bull centrifugation bull chromatography bull electrophoresis

Identification bull amino acid analysis bull sequencing bull mass spectrometry

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

I Experimental methods

Genetic engineering bull deletion bull chimeric proteins bull mutagenesis

ndash site directed ndash random

bull phage display libraries

Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries

An example Identification of epitope sequences in protein antigens

Whole virus

Viral envelope proteins (mixture)

1 Affinity chromatography 2 Gradient centrifugation

Amino acid sequence Immunodominant protein component komponens

Immuneprecipitation (Gelelectrophoresis)

Sequencing

Identification of peptid epitopes

protein

prediction of 3D structure

epitope bdquomaprdquo

Prediction of hydrophilicity

modeling

Identification of short sequences responsible for activity

bdquooverlappingrdquo strategy

bdquopredictedrdquo

Gaacutet

laacutes

TPTPTGTQ

PTGTQ

TGTQ

0

20

40

60

80

100

001 1 100 cpeptid (mmoll)

bdquoshorteningrdquo strategy

intuicioacute

bdquocombinatorialrdquo strategy

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

IITheoretical methods

Quantum chemistry bull molecular mechanics bull molecular dynamics

Predictions from the primary structure

bull Probabilistic (statistical) 1970 ndash

bull Physicochemical 1974 ndash

bull Information theory 1974 -

Approaches for the localisation of functionally relevant domains in proteins

Protein primary structure

Know Unknown

3D Structure

Know Unknown

Isolation

Fragmentation separation

Functional assay with fragments

Selection

Structure elucidation

Prediction of

bull secondary structure

bull functionally relevant domain

Fragmentation separation

Functional assay with fragments

Selection

3D Structure elucidation

Chemical modification

Genetic engineering

Chemical synthesis

Smallest functional domain

Strategies for determinations of 3D structures

Experimental methods Theoretical methods

X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)

bull crystal bull time-intensive

NMR

CD spectroscopy FT-IR spectroscopy

Quantum chemistry mechanics and dynamics

Empirical calculations

Relationship assay

Prediction of secondary structures (helix b-turn)

Prediction of hydrophobic ndash hydrophilic regions

Techniques for the detection of interactionrecognition phenomena

1 Molecular level

Detection with Separation Detection without Separation

Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis

Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation

Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy

Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)

Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy

Techniques for the detection of interactionrecognition phenomena

2 Cellular level

Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity

In vivo post-translational modifications

alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)

Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)

Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)

Ubiquitination

bull Ubiquitin protein (76 amino acids 85 kDa)

bull Almost all tissues of eukaryotic organisms

bull It can signal the degradation of the attached protein by transporting to proteasome

bull Isopeptide linkage (4)

bull Enzymes involved

ndash E1 (ub activation)

ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)

ndash E3 ( ub ligation)

Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose

NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)

bull Function

activationregulation of ubiquitin

SUMOylation (Small Ubiquitin-like Modifier)

bull SUMO proteins 100 aa 12 kDa 4 isoforms

bull Post-translational modification

bull Aktivation cleavage of 4 residues at the C-terminal

bull Attachment to target protein by using three enzymes

Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation

R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

I Experimental methods

Genetic engineering bull deletion bull chimeric proteins bull mutagenesis

ndash site directed ndash random

bull phage display libraries

Chemical synthesis bull substituted analogs bull truncatedomitted analogs bull overlapping peptides bull peptide libraries

An example Identification of epitope sequences in protein antigens

Whole virus

Viral envelope proteins (mixture)

1 Affinity chromatography 2 Gradient centrifugation

Amino acid sequence Immunodominant protein component komponens

Immuneprecipitation (Gelelectrophoresis)

Sequencing

Identification of peptid epitopes

protein

prediction of 3D structure

epitope bdquomaprdquo

Prediction of hydrophilicity

modeling

Identification of short sequences responsible for activity

bdquooverlappingrdquo strategy

bdquopredictedrdquo

Gaacutet

laacutes

TPTPTGTQ

PTGTQ

TGTQ

0

20

40

60

80

100

001 1 100 cpeptid (mmoll)

bdquoshorteningrdquo strategy

intuicioacute

bdquocombinatorialrdquo strategy

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

IITheoretical methods

Quantum chemistry bull molecular mechanics bull molecular dynamics

Predictions from the primary structure

bull Probabilistic (statistical) 1970 ndash

bull Physicochemical 1974 ndash

bull Information theory 1974 -

Approaches for the localisation of functionally relevant domains in proteins

Protein primary structure

Know Unknown

3D Structure

Know Unknown

Isolation

Fragmentation separation

Functional assay with fragments

Selection

Structure elucidation

Prediction of

bull secondary structure

bull functionally relevant domain

Fragmentation separation

Functional assay with fragments

Selection

3D Structure elucidation

Chemical modification

Genetic engineering

Chemical synthesis

Smallest functional domain

Strategies for determinations of 3D structures

Experimental methods Theoretical methods

X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)

bull crystal bull time-intensive

NMR

CD spectroscopy FT-IR spectroscopy

Quantum chemistry mechanics and dynamics

Empirical calculations

Relationship assay

Prediction of secondary structures (helix b-turn)

Prediction of hydrophobic ndash hydrophilic regions

Techniques for the detection of interactionrecognition phenomena

1 Molecular level

Detection with Separation Detection without Separation

Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis

Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation

Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy

Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)

Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy

Techniques for the detection of interactionrecognition phenomena

2 Cellular level

Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity

In vivo post-translational modifications

alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)

Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)

Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)

Ubiquitination

bull Ubiquitin protein (76 amino acids 85 kDa)

bull Almost all tissues of eukaryotic organisms

bull It can signal the degradation of the attached protein by transporting to proteasome

bull Isopeptide linkage (4)

bull Enzymes involved

ndash E1 (ub activation)

ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)

ndash E3 ( ub ligation)

Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose

NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)

bull Function

activationregulation of ubiquitin

SUMOylation (Small Ubiquitin-like Modifier)

bull SUMO proteins 100 aa 12 kDa 4 isoforms

bull Post-translational modification

bull Aktivation cleavage of 4 residues at the C-terminal

bull Attachment to target protein by using three enzymes

Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation

R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)

An example Identification of epitope sequences in protein antigens

Whole virus

Viral envelope proteins (mixture)

1 Affinity chromatography 2 Gradient centrifugation

Amino acid sequence Immunodominant protein component komponens

Immuneprecipitation (Gelelectrophoresis)

Sequencing

Identification of peptid epitopes

protein

prediction of 3D structure

epitope bdquomaprdquo

Prediction of hydrophilicity

modeling

Identification of short sequences responsible for activity

bdquooverlappingrdquo strategy

bdquopredictedrdquo

Gaacutet

laacutes

TPTPTGTQ

PTGTQ

TGTQ

0

20

40

60

80

100

001 1 100 cpeptid (mmoll)

bdquoshorteningrdquo strategy

intuicioacute

bdquocombinatorialrdquo strategy

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

IITheoretical methods

Quantum chemistry bull molecular mechanics bull molecular dynamics

Predictions from the primary structure

bull Probabilistic (statistical) 1970 ndash

bull Physicochemical 1974 ndash

bull Information theory 1974 -

Approaches for the localisation of functionally relevant domains in proteins

Protein primary structure

Know Unknown

3D Structure

Know Unknown

Isolation

Fragmentation separation

Functional assay with fragments

Selection

Structure elucidation

Prediction of

bull secondary structure

bull functionally relevant domain

Fragmentation separation

Functional assay with fragments

Selection

3D Structure elucidation

Chemical modification

Genetic engineering

Chemical synthesis

Smallest functional domain

Strategies for determinations of 3D structures

Experimental methods Theoretical methods

X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)

bull crystal bull time-intensive

NMR

CD spectroscopy FT-IR spectroscopy

Quantum chemistry mechanics and dynamics

Empirical calculations

Relationship assay

Prediction of secondary structures (helix b-turn)

Prediction of hydrophobic ndash hydrophilic regions

Techniques for the detection of interactionrecognition phenomena

1 Molecular level

Detection with Separation Detection without Separation

Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis

Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation

Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy

Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)

Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy

Techniques for the detection of interactionrecognition phenomena

2 Cellular level

Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity

In vivo post-translational modifications

alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)

Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)

Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)

Ubiquitination

bull Ubiquitin protein (76 amino acids 85 kDa)

bull Almost all tissues of eukaryotic organisms

bull It can signal the degradation of the attached protein by transporting to proteasome

bull Isopeptide linkage (4)

bull Enzymes involved

ndash E1 (ub activation)

ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)

ndash E3 ( ub ligation)

Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose

NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)

bull Function

activationregulation of ubiquitin

SUMOylation (Small Ubiquitin-like Modifier)

bull SUMO proteins 100 aa 12 kDa 4 isoforms

bull Post-translational modification

bull Aktivation cleavage of 4 residues at the C-terminal

bull Attachment to target protein by using three enzymes

Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation

R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)

Identification of peptid epitopes

protein

prediction of 3D structure

epitope bdquomaprdquo

Prediction of hydrophilicity

modeling

Identification of short sequences responsible for activity

bdquooverlappingrdquo strategy

bdquopredictedrdquo

Gaacutet

laacutes

TPTPTGTQ

PTGTQ

TGTQ

0

20

40

60

80

100

001 1 100 cpeptid (mmoll)

bdquoshorteningrdquo strategy

intuicioacute

bdquocombinatorialrdquo strategy

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

IITheoretical methods

Quantum chemistry bull molecular mechanics bull molecular dynamics

Predictions from the primary structure

bull Probabilistic (statistical) 1970 ndash

bull Physicochemical 1974 ndash

bull Information theory 1974 -

Approaches for the localisation of functionally relevant domains in proteins

Protein primary structure

Know Unknown

3D Structure

Know Unknown

Isolation

Fragmentation separation

Functional assay with fragments

Selection

Structure elucidation

Prediction of

bull secondary structure

bull functionally relevant domain

Fragmentation separation

Functional assay with fragments

Selection

3D Structure elucidation

Chemical modification

Genetic engineering

Chemical synthesis

Smallest functional domain

Strategies for determinations of 3D structures

Experimental methods Theoretical methods

X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)

bull crystal bull time-intensive

NMR

CD spectroscopy FT-IR spectroscopy

Quantum chemistry mechanics and dynamics

Empirical calculations

Relationship assay

Prediction of secondary structures (helix b-turn)

Prediction of hydrophobic ndash hydrophilic regions

Techniques for the detection of interactionrecognition phenomena

1 Molecular level

Detection with Separation Detection without Separation

Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis

Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation

Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy

Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)

Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy

Techniques for the detection of interactionrecognition phenomena

2 Cellular level

Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity

In vivo post-translational modifications

alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)

Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)

Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)

Ubiquitination

bull Ubiquitin protein (76 amino acids 85 kDa)

bull Almost all tissues of eukaryotic organisms

bull It can signal the degradation of the attached protein by transporting to proteasome

bull Isopeptide linkage (4)

bull Enzymes involved

ndash E1 (ub activation)

ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)

ndash E3 ( ub ligation)

Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose

NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)

bull Function

activationregulation of ubiquitin

SUMOylation (Small Ubiquitin-like Modifier)

bull SUMO proteins 100 aa 12 kDa 4 isoforms

bull Post-translational modification

bull Aktivation cleavage of 4 residues at the C-terminal

bull Attachment to target protein by using three enzymes

Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation

R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)

Identification of short sequences responsible for activity

bdquooverlappingrdquo strategy

bdquopredictedrdquo

Gaacutet

laacutes

TPTPTGTQ

PTGTQ

TGTQ

0

20

40

60

80

100

001 1 100 cpeptid (mmoll)

bdquoshorteningrdquo strategy

intuicioacute

bdquocombinatorialrdquo strategy

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

IITheoretical methods

Quantum chemistry bull molecular mechanics bull molecular dynamics

Predictions from the primary structure

bull Probabilistic (statistical) 1970 ndash

bull Physicochemical 1974 ndash

bull Information theory 1974 -

Approaches for the localisation of functionally relevant domains in proteins

Protein primary structure

Know Unknown

3D Structure

Know Unknown

Isolation

Fragmentation separation

Functional assay with fragments

Selection

Structure elucidation

Prediction of

bull secondary structure

bull functionally relevant domain

Fragmentation separation

Functional assay with fragments

Selection

3D Structure elucidation

Chemical modification

Genetic engineering

Chemical synthesis

Smallest functional domain

Strategies for determinations of 3D structures

Experimental methods Theoretical methods

X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)

bull crystal bull time-intensive

NMR

CD spectroscopy FT-IR spectroscopy

Quantum chemistry mechanics and dynamics

Empirical calculations

Relationship assay

Prediction of secondary structures (helix b-turn)

Prediction of hydrophobic ndash hydrophilic regions

Techniques for the detection of interactionrecognition phenomena

1 Molecular level

Detection with Separation Detection without Separation

Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis

Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation

Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy

Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)

Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy

Techniques for the detection of interactionrecognition phenomena

2 Cellular level

Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity

In vivo post-translational modifications

alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)

Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)

Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)

Ubiquitination

bull Ubiquitin protein (76 amino acids 85 kDa)

bull Almost all tissues of eukaryotic organisms

bull It can signal the degradation of the attached protein by transporting to proteasome

bull Isopeptide linkage (4)

bull Enzymes involved

ndash E1 (ub activation)

ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)

ndash E3 ( ub ligation)

Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose

NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)

bull Function

activationregulation of ubiquitin

SUMOylation (Small Ubiquitin-like Modifier)

bull SUMO proteins 100 aa 12 kDa 4 isoforms

bull Post-translational modification

bull Aktivation cleavage of 4 residues at the C-terminal

bull Attachment to target protein by using three enzymes

Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation

R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

IITheoretical methods

Quantum chemistry bull molecular mechanics bull molecular dynamics

Predictions from the primary structure

bull Probabilistic (statistical) 1970 ndash

bull Physicochemical 1974 ndash

bull Information theory 1974 -

Approaches for the localisation of functionally relevant domains in proteins

Protein primary structure

Know Unknown

3D Structure

Know Unknown

Isolation

Fragmentation separation

Functional assay with fragments

Selection

Structure elucidation

Prediction of

bull secondary structure

bull functionally relevant domain

Fragmentation separation

Functional assay with fragments

Selection

3D Structure elucidation

Chemical modification

Genetic engineering

Chemical synthesis

Smallest functional domain

Strategies for determinations of 3D structures

Experimental methods Theoretical methods

X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)

bull crystal bull time-intensive

NMR

CD spectroscopy FT-IR spectroscopy

Quantum chemistry mechanics and dynamics

Empirical calculations

Relationship assay

Prediction of secondary structures (helix b-turn)

Prediction of hydrophobic ndash hydrophilic regions

Techniques for the detection of interactionrecognition phenomena

1 Molecular level

Detection with Separation Detection without Separation

Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis

Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation

Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy

Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)

Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy

Techniques for the detection of interactionrecognition phenomena

2 Cellular level

Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity

In vivo post-translational modifications

alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)

Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)

Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)

Ubiquitination

bull Ubiquitin protein (76 amino acids 85 kDa)

bull Almost all tissues of eukaryotic organisms

bull It can signal the degradation of the attached protein by transporting to proteasome

bull Isopeptide linkage (4)

bull Enzymes involved

ndash E1 (ub activation)

ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)

ndash E3 ( ub ligation)

Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose

NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)

bull Function

activationregulation of ubiquitin

SUMOylation (Small Ubiquitin-like Modifier)

bull SUMO proteins 100 aa 12 kDa 4 isoforms

bull Post-translational modification

bull Aktivation cleavage of 4 residues at the C-terminal

bull Attachment to target protein by using three enzymes

Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation

R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)

Approaches for the localisation of functionally relevant domains in proteins

Protein primary structure

Know Unknown

3D Structure

Know Unknown

Isolation

Fragmentation separation

Functional assay with fragments

Selection

Structure elucidation

Prediction of

bull secondary structure

bull functionally relevant domain

Fragmentation separation

Functional assay with fragments

Selection

3D Structure elucidation

Chemical modification

Genetic engineering

Chemical synthesis

Smallest functional domain

Strategies for determinations of 3D structures

Experimental methods Theoretical methods

X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)

bull crystal bull time-intensive

NMR

CD spectroscopy FT-IR spectroscopy

Quantum chemistry mechanics and dynamics

Empirical calculations

Relationship assay

Prediction of secondary structures (helix b-turn)

Prediction of hydrophobic ndash hydrophilic regions

Techniques for the detection of interactionrecognition phenomena

1 Molecular level

Detection with Separation Detection without Separation

Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis

Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation

Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy

Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)

Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy

Techniques for the detection of interactionrecognition phenomena

2 Cellular level

Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity

In vivo post-translational modifications

alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)

Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)

Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)

Ubiquitination

bull Ubiquitin protein (76 amino acids 85 kDa)

bull Almost all tissues of eukaryotic organisms

bull It can signal the degradation of the attached protein by transporting to proteasome

bull Isopeptide linkage (4)

bull Enzymes involved

ndash E1 (ub activation)

ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)

ndash E3 ( ub ligation)

Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose

NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)

bull Function

activationregulation of ubiquitin

SUMOylation (Small Ubiquitin-like Modifier)

bull SUMO proteins 100 aa 12 kDa 4 isoforms

bull Post-translational modification

bull Aktivation cleavage of 4 residues at the C-terminal

bull Attachment to target protein by using three enzymes

Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation

R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)

Strategies for determinations of 3D structures

Experimental methods Theoretical methods

X-ray diffraction (Mioglobin hemoglobin Kendrew Perutz 1960)

bull crystal bull time-intensive

NMR

CD spectroscopy FT-IR spectroscopy

Quantum chemistry mechanics and dynamics

Empirical calculations

Relationship assay

Prediction of secondary structures (helix b-turn)

Prediction of hydrophobic ndash hydrophilic regions

Techniques for the detection of interactionrecognition phenomena

1 Molecular level

Detection with Separation Detection without Separation

Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis

Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation

Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy

Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)

Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy

Techniques for the detection of interactionrecognition phenomena

2 Cellular level

Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity

In vivo post-translational modifications

alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)

Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)

Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)

Ubiquitination

bull Ubiquitin protein (76 amino acids 85 kDa)

bull Almost all tissues of eukaryotic organisms

bull It can signal the degradation of the attached protein by transporting to proteasome

bull Isopeptide linkage (4)

bull Enzymes involved

ndash E1 (ub activation)

ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)

ndash E3 ( ub ligation)

Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose

NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)

bull Function

activationregulation of ubiquitin

SUMOylation (Small Ubiquitin-like Modifier)

bull SUMO proteins 100 aa 12 kDa 4 isoforms

bull Post-translational modification

bull Aktivation cleavage of 4 residues at the C-terminal

bull Attachment to target protein by using three enzymes

Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation

R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)

Techniques for the detection of interactionrecognition phenomena

1 Molecular level

Detection with Separation Detection without Separation

Separation techniques bull equilibrium dialysis bull chromatography - gel filtration - affinity bull electrophoresis

Detection techniqes bull spectroscopic bull radiochemical (125I 35S 3H 14C) bull imunochemical - RIAELISA - blotting - immunprecipitation

Optical techniques bull absorption spectroscopy bull CD bull fluorescence spectroscopy bull IR and Raman spectroscopy

Resonance techniques bull NMR bull electron paramagnetic resonance (EPR)

Scattering and Diffraction techniques bull X-ray crystallography bull neutron scattering bull electron microscopy

Techniques for the detection of interactionrecognition phenomena

2 Cellular level

Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity

In vivo post-translational modifications

alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)

Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)

Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)

Ubiquitination

bull Ubiquitin protein (76 amino acids 85 kDa)

bull Almost all tissues of eukaryotic organisms

bull It can signal the degradation of the attached protein by transporting to proteasome

bull Isopeptide linkage (4)

bull Enzymes involved

ndash E1 (ub activation)

ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)

ndash E3 ( ub ligation)

Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose

NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)

bull Function

activationregulation of ubiquitin

SUMOylation (Small Ubiquitin-like Modifier)

bull SUMO proteins 100 aa 12 kDa 4 isoforms

bull Post-translational modification

bull Aktivation cleavage of 4 residues at the C-terminal

bull Attachment to target protein by using three enzymes

Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation

R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)

Techniques for the detection of interactionrecognition phenomena

2 Cellular level

Bioassay (in vitro) bull binding to cell bull hemolysis bull antibacterial effect bull cytotoxicity

In vivo post-translational modifications

alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)

Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)

Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)

Ubiquitination

bull Ubiquitin protein (76 amino acids 85 kDa)

bull Almost all tissues of eukaryotic organisms

bull It can signal the degradation of the attached protein by transporting to proteasome

bull Isopeptide linkage (4)

bull Enzymes involved

ndash E1 (ub activation)

ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)

ndash E3 ( ub ligation)

Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose

NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)

bull Function

activationregulation of ubiquitin

SUMOylation (Small Ubiquitin-like Modifier)

bull SUMO proteins 100 aa 12 kDa 4 isoforms

bull Post-translational modification

bull Aktivation cleavage of 4 residues at the C-terminal

bull Attachment to target protein by using three enzymes

Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation

R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)

In vivo post-translational modifications

alkyl acyl (O-N-S-) N-terminal Lys Ser Thr amide C-terminal phosphoric acid ester (Ser Thr Tyr) sulphonic acid ester glycosylation O- in Golgi (Ser Thr) N- in RER (Asn) nitrosation desamidation decarboxylation Arg desamination citrullination (Arg -gt citrullin) hydroxylation (Pro Lys) oxidation gamma-carboxylation (eg Glu ) beta-elimination (eg Thr -gtalkene)

Cleavage of peptide bond N-terminals Met or fMet signalpeptide precursor activation (proinzuline rarr inzuline) Disulphid bond formation Isomerisation (Pro) Coupling of nucleotide (eg flavine)

Coupling of proteinpeptide sumoylation (SUMO protein) ubiquitination (ubiquitin) neddylation (Nedd)

Ubiquitination

bull Ubiquitin protein (76 amino acids 85 kDa)

bull Almost all tissues of eukaryotic organisms

bull It can signal the degradation of the attached protein by transporting to proteasome

bull Isopeptide linkage (4)

bull Enzymes involved

ndash E1 (ub activation)

ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)

ndash E3 ( ub ligation)

Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose

NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)

bull Function

activationregulation of ubiquitin

SUMOylation (Small Ubiquitin-like Modifier)

bull SUMO proteins 100 aa 12 kDa 4 isoforms

bull Post-translational modification

bull Aktivation cleavage of 4 residues at the C-terminal

bull Attachment to target protein by using three enzymes

Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation

R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)

Ubiquitination

bull Ubiquitin protein (76 amino acids 85 kDa)

bull Almost all tissues of eukaryotic organisms

bull It can signal the degradation of the attached protein by transporting to proteasome

bull Isopeptide linkage (4)

bull Enzymes involved

ndash E1 (ub activation)

ndash E2 (ub conjugation to e-amino group of Lys to thiol of Cys by thioester to OH of ThrSer by ester)

ndash E3 ( ub ligation)

Nobel Prize in Chemistry 6 October 2004 A Ciechanover A Hershko I Rose

NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)

bull Function

activationregulation of ubiquitin

SUMOylation (Small Ubiquitin-like Modifier)

bull SUMO proteins 100 aa 12 kDa 4 isoforms

bull Post-translational modification

bull Aktivation cleavage of 4 residues at the C-terminal

bull Attachment to target protein by using three enzymes

Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation

R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)

NEDDylation (Neural-precursor-cell-expressed developmentally down-regulated 8)

bull Function

activationregulation of ubiquitin

SUMOylation (Small Ubiquitin-like Modifier)

bull SUMO proteins 100 aa 12 kDa 4 isoforms

bull Post-translational modification

bull Aktivation cleavage of 4 residues at the C-terminal

bull Attachment to target protein by using three enzymes

Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation

R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)

SUMOylation (Small Ubiquitin-like Modifier)

bull SUMO proteins 100 aa 12 kDa 4 isoforms

bull Post-translational modification

bull Aktivation cleavage of 4 residues at the C-terminal

bull Attachment to target protein by using three enzymes

Involved in nuclear-cytosolic transport transcriptional regulation apoptosis protein stability but not in degradation

R Geiss-Friedlander amp F Melchior Nature Rev Mol Cell Biol 8 947-956 (2007)