Conditions

1. Three components, three lecturers.

2. Lectures 1-4 Prof. F. HudeczLectures 5-8 Prof. Á. PálovicsLectures 8-12 Prof. J. Borvendég

3. Examination: three parts determined by the lecturersand one mark.- option A: written test- option B: presentation based on literature

4. Participation at lectures > 70 %

fhudecz@elte.hu

First generatioin Second generation New generation

Oxytocin (L) Carbetocin (S) Abarelix (GnRH) (L)ACTH (1-24) & (1-39) (L,S) Terlipressin (L,S) Cetrorelix (GnRH) (L)Vasopressin (L,S) Felypressin (L,S) Ganirelix (GnRH) (L)Insulin (E,SS, R) Buserelin (L,S) EptifibatideGlucagon (E,S,R) Deslorelin (L,S) Bivalirudin (L)Calcitonins (L,S,R) Goserelin (L) Copaxone (L)TRH (L) Histrelin (L) Techtide P-289(S)Gonadorelin (L,S) Leuprolide (L,S) Cubicin (F)Somatostatin (L,S) Nafarelin (S) Fuzeon (antiHIV (H)GHRH (1-29) & (1-44) (S) Tryptorelin (L,S) Ziconotide (pain) (S)CRF (Human & Ovine) (S) Lecirelin (S) Pramlintide (diabetes) (S)Cyclosporin (F) Lanreotide (S) Exenatide (diabetes) (S)Thymopentin (L) Octreotide (L,S) Icatibant (brady-rec)Thymosin Alpha-1 (S) Atosiban (L) Romiplostim (hormon)Secretins (Human & Porcine) (E,S) Desmopressin (L,S) Degarelix (GnRH)Parathyroid Hormone (1-34) & (1-84)(S) Lypressin (L) Mifamurtide (rák, adj.)Vasoactive Intestinal Polypeptide (S) Ornipressin Ecallantide (ödéma)Brain Natriuretic Peptide (R) Pitressin (L) Liraglutide (diabetes)Cholecystokinin (L) ACE Inhibitors (Enalapril, Lisinopril) (L) TesamorelinTetragastrin (L) HIV Protease Inhibitors (L) SurfaxinPentagastrin (L) PeginesatideEledoisin (L) Carfilzomib

Linaclotide (enz.inh)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

Peptide *

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

40’s 50’s 60’s 70’s 80’s 90’s 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 Cushing’s disease 6 Phase III

Albiglutide Type 2 diabetes -- Phase III

E75 Breast cancer (therapeutic vaccine) -- Phase II/III

Pexiganan Diabetic foot infections 22 Phase II/III

Cilengitide Glioblastoma 5 Phase III

KAI-4169 Seconday hyperparathyroidism -- Phase II

TRV120027 Acute heart failure -- Phase II

MIM-D3 Dry eye -- Phase II

AIDS Gynecological Disorders Allergies Hypertension Analgesia IBD/IBSArthritis Immune Deficiencies Birth Control Infections (anti-viral, anti-microbial) Cardiovascular Diseases InflammationCNS Disorders Lung Surfactant Cystic Fibrosis Obesity Diabetes OncologyEpilepsy Ophthalmology Gastrointestinal Disorders OsteoporosisGrowth Deficiencies Vaccines

Main fields of applications

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- 1000/year

Three-dimensional (3D) structure (J. Kendrew, 1962)

1962-1985 (20 years) 200

1986-1991 ( 5 years) 480

1992- 100/years

CLASSIFICATION OF PROTEINSACCORDING TO THEIR FUNCTION

1. Enzymatic catalysis (e.g. Ser proteases)

2. Transport (e.g. transferrin for iron, serum albumin for fatty acids)

3. Storage (e.g. ferrin for iron in liver, casein in milk)

4. Protection

• toxins (e.g. ricin [plant], diphteria [bacteria])

• self and non-self discrimination, immune protection

(e.g. antibodies, antigenes)

5. Signal transduction (e.g. hormones, receptors)

• nerve impulses

• growth

• differentiation

6. Cell to cell communication (e.g. adhesion, molecules; factors, acceptors)

7. Coordinated motion (e.g. muscle proteins)

8. Mechanical support

• at cellular level (e.g. Membrane proteins)

• at tissue level (e.g. structural proteins, e.g. collagen in skin, bone)

RECOGNITION PHENOMENA

Interaction Kd[M]

1. Enzyme – substrate 10-3 – 10-5

2. Transporter – ligand 10-6 – 10-8

3. Hormone – receptor 10-9

4. Antibody – antigen 10-7 – 10-11

5. Storage protein – ligand

6. Toxin – receptor

7. Protein – protein (in a contractile superassembly)

8. Lectin – carbohydrate 10-4 – 10-7

9. Avidin – biotin 10-15

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

I. Experimental methods

Chemical modification

• side chain modification

• conjugation

Fragmentation

• enzymatic (e.g. trysin)

• chemical (e.g. BrCN)

Separation

• centrifugation

• chromatography

• electrophoresis

Identification

• amino acid analysis

• sequencing

• mass spectrometry

Genetic engineering

• deletion

• chimeric proteins

• mutagenesis

– site directed

– random

• phage display libraries

Chemical synthesis

• substituted analogs

• truncated/omitted analogs

• overlapping peptides

• peptide libraries

An example: Identification of epitope sequences in protein antigens

Whole virus

Viral envelope proteins (mixture)

1. Affinity chromatography2. Gradient centrifugation

Amino acid sequence Immunodominant protein component komponens

Immuneprecipitation(Gelelectrophoresis)

Sequencing

Identification of peptid epitopes

protein

prediction of 3D structure

epitope „map”

Prediction of hydrophilicity

modeling

Identification of short sequences responsible for activity

„overlapping” strategy

„predicted”

Gátlás %

TPTPTGTQ

PTGTQ

TGTQ

0

20

40

60

80

100

0.01 1 100 cpeptid (mmol/l)

„shortening” strategy

intuició

„combinatorial” strategy

Identification of linear antibody epitopes by monoclonal antibodies

Identification of linear T cell epitopes by T cell interaction

cleavage+ T cells

IL4 / IFγγγγ

METHODS FOR THE LOCALISATION OF FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS

II. Theoretical methods

Quantum chemistry• molecular mechanics

• molecular dynamics

Predictions from the primary structure

• Probabilistic (statistical) 1970 –

• Physicochemical 1974 –

• Information theory 1974 -

Approaches for the localiation of functionally relevant domains in proteins

Protein primary structure

Know Unknown

3D Structure

Know Unknown

IsolationFragmentation, separation

Functional assay with fragments

SelectionStructure elucidation

Prediction of• secondary structure• functionally relevant domain

Fragmentation, separationFunctional 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)

• crystal• time-intensive

NMR

CD spectroscopyFT-IR spectroscopy

Quantum chemistry:mechanics and dynamics

Empirical calculations

Relationship assay

Prediction of secondary structures(helix, β-turn)

Prediction of hydrophobic – hydrophilic regions

Techniques for the detection of interaction/recognition phenomena

1. Molecular level

Detection with Separation Detection without Separation

Separation techniques• equilibrium dialysis• chromatography

- gel filtration- affinity

• electrophoresis

Detection techniqes• spectroscopic• radiochemical (125I, 35S, 3H, 14C)

• imunochemical- RIA/ELISA

- blotting- immunprecipitation

Optical techniques• absorption spectroscopy• CD

• fluorescence spectroscopy• IR and Raman spectroscopy

Resonance techniques• NMR• electron paramagnetic resonance (EPR)

Scattering and Diffraction techniques• X-ray crystallography• neutron scattering

• electron microscopy

Techniques for the detection of interaction/recognition phenomena

2. Cellular level

Bioassay (in vitro)• binding to cell• hemolysis

• antibacterial effect• cytotoxicity

In vivo post-translational modifications

alkyl acyl (O-,N-,S-)

N-terminal, Lys,Ser, Thr

amid C-terminal

phosphoric acid ester (Ser, Thr, Tyr)sulphonic acid esterglycosilation

O- in Golgi (Ser, Thr)N- in RER (Asn)

nitrolizációdezamidationdecarboxilationArg dezamination,

citrullination (Arg -> citrullin)hidroxilation (Pro, Lys) oxidationgamma-carboxilation (pl. Glu )béta-elimination (eg. Thr ->alkene)

Cleavage of peptide bondN-terminals Met or fMetsignalpeptideprecursor activation (proinzuline → inzuline)

Disulphid bond formation

Isomerisation (Pro)

Coupling of nucleotide (e.g. flavine)

Coupling of protein/peptide :szumoiláció (SUMO fehérje)ubikvitinálás (ubiquitin)neddiláció (Nedd)

Ubiquitináció

• Ubiquitin: fehérje (76 aminosav)• Lebontásra váró fehérje

proteoszómába juttatása• Izopeptid kötés (4 db)• Enzim:

– E1 (ub aktiváló)– E2 (ub konjugáló)– E3 ( ub ligáz)

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

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

• Function: activation/regulation of ubiquitin

SUMOylation(Small Ubiquitin-like Modifier)

• SUMO proteins: 100 aa. , 12 kDa, 4 isoforms • Aktivation: cleavage of 4 residues at the C-

terminal• Attachment to target protein by using three

enzymes .

Involved in nuclear-cytosolic transport, transcriptional regulation, apoptosisprotein stability,but, not in degradation

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