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 %
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)