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Protein-protein interaction
 

Protein-protein interaction

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    Protein-protein interaction Protein-protein interaction Presentation Transcript

    • Protein-protein interaction: Is this a “druggable”target? CAD - R&D Dr.ssa G.Gallo 20-12-2006
    • Protein-protein interaction (PPI)
      • G.Gallo: strategies and examples
      • S.Vincenti: bioinformatic approaches
      • D.Vignola: from peptide to peptidomimetic
      • L.Mastrofrancesco: fragment-based approaches- tools for lead discovery
    • Why
      • PPI are critical for almost every important cellular pathway
        • S. cerevisiae 6200 proteins, estimated 30.000 PPI
      • Structural and biophysical methods to clarify the binding mechanism
        • Analytical ultracentrifugation (AUC)
        • Surface plasmon resonance (SPR)
        • Nuclear magnetic resonance (NMR)
        • X-ray crystallography
      • Antagonists antibody-based
        • The fastest growing segment of prescription-drug market (+42% over previous 5 years)
      Arkin M.R.&Wells J.A. Nature reviews 2004, 3, 301
    • Antibodies
      • Advantages
      • Highly specific
      • Fewer adverse effects
      • Quicker and less costly development
      • Higher success rates
      • Very stable in serum
      • Drawbacks
      • Difficulties in manifacture
      • High costs of goods
      • Lack of oral bioavailability
      • Not cell-permeable
      Small molecules inhibitors
      • Temporary and stable complexes
      • Low affinity
        • Large and flat sites
        • Wide distribution of weak interactions
        • Shallow surface
      • Low selectivity
        • Anti binding elements
      • Regulation via phosphorilation
      • Functional restoration of interaction as therapeutic strategy
      Why not Fry D.C. Biopolymers (2006) 84, 535-552
    • Proposed solutions
      • Protein
        • Hot spots (Clackson T.&Wells J.A. Science 1995, 267, 383)
        • Deep subpocket (McDowell R.S. et al. JACS 1994, 116, 5077)
        • Temporary instead of stable complexes (TooGood P.L. J.Med.Chem. 2001, 45, 1543)
        • Both proteins can be targeted
      • Ligand
        • High end of MW range
        • Rigid conformation (rigid core or multiple ring in succession)
        • No exact matches in key protein elements
    • Strategies
      • Random HTS screening
      • Peptide to peptidomimetics
        • Small continuous peptide epitopes
      • Computational approaches
        • Structure-based design
        • In silico screening
      • Fragment-based approaches
      • Covalent modificators
      • Antibody as template
      • Allosteric interaction
      • Protein stabilization
    • Therapeutic target
      • Cancer
        • Integrin
        • MDM2
        • XIAP
        • BCL-2/BCL-XL
        • SH2 domain
        • HSP90
        • ErBB
        • PTX3
      • Antibiotic, antiviral
        • ZipA
        • HIV enzymes
      • Analgesic
        • NGF
      • Inflammatory, autoimmune disease
        • Interleukin-1
        • Interleukin-2
        • iNOS
        • TNF- 
        • C5a receptor
        • MyD88/IRAK1
      • Diabetes
        • Somatostatin
        • Ped
      • Heart disease
        • Integrin
        • Trombopoietin
    • From peptide to peptidomimetic
      • Integrin
      • Grb2-SH2 domain
        • pTyr-Ile- Asn (~ 1  M) -> 19 (10 nM) (Yao Z-J. Et al. J.Med.Chem. 1999, 42, 25)
      • C5a Receptor
        • N-Me-FKPdChaWr (0.1  M) -> 20 (20 nM) ( Finch A. et al. J.Med.Chem. 1999, 42, 1965)
      • HDM2
        • 12-mer p 53 (8.7  M) -> 21 (5 nM) (Garcia-Echeverria C. et al.
        • J.Med.Chem. 2000, 43, 3205)
    • Integrins
      • 18  chain 8  chain -> 24 different families
        • RGD mimetics (  IIb  3,  V  3 )
        • LDV mimetics (  4  1 )
        • Allosteric inhibitors (  L  2 )
      Xiao T. et al. Nature 2004, 32, 59 a : low affinity in a bent conformation b : intermediate affinity in an extended form and closed headpiece c : High affinity in an extended form and open headpiece with exposure of ligand induced binding sites (LIBS)
    • Antitrombotic agents
      • End points
        • Affinity on  IIb  3
        • Selectivity versus  V  3
        • Parenteral versus oral administration
      • Parenteral antagonists
        • Monoclonal antibodies (abciximab; 1995 centocor)
        • Natural substances (snake venom peptides: barbourin KGDW peptide)
        • Linear peptides
        • Cyclic peptides (eptifibatide; 1998 COR therapeutics)
        • Screening compounds (tirofiban; 1998 Merck)
      Scarborough R. M.&Gretler D.D. J.Med. Chem. 2000, 3, 353
    • Linear peptides (RGD >300  M; RGDS 26  M) Cyclic peptides Platelet aggregation test IC 50 =32  M IC 50 =72 nM IC 50 = 97 nM selective 1985- 1999 Searle, SK, Roche, Fujisawa, Rhone-Poulenc , Merck, Genentech, COR ther. IC 50 =26 nM selective IC 50 = 46 nM Selective Intravenous and oral administration
      • INTEGRILIN (Eptifibatide) This injectable agent is approved for use in patients with acute coronary syndrome (unstable angina and non Q-wave myocardial infarction. As with Aggrastat, it inhibits platelet aggregation however by a different mechanism, by preventing the binding of fibrinogen to platelet. That is a necessary step to clot formation. The incidence the angina progressing to either death or myocardial infarction was significantly reduced from 7.6% to 5.9%.
      Platelet aggregation test IC 50 =140 nM; selective Fibrinogen: KQAGDV
    • Subset of a corporate library (Merck) containing amine and carboxylic acid functionalities at distance 10-20 Å IC 50 = 11 nM Selective N-  -sulfonamide “ exosite” binding Platelet aggregation test IC 50 = 27  M Hit compound Hartman G.D. et al. J.Med. Chem. 1992, 35, 4640 IC 50 = 260 nM
      • AGGRASTAT (Tirofiban) This injectable drug is the first of a new class of drugs called glycoprotein inhibitors which block the ability of platelets to aggregate thus preventing or reducing clotting. When combined with intravenous heparin and aspirin for the treatment of acute coronary syndrome and unstable angina, it reduces the risk of death and the probability of a heart attack by 32%.
    • Magenta  subunit Cyano  subunit Xiao T. et al. Nature 2004, 32, 59
    • Table 1. Parenteral GPIIb-IIIa Antagonists Table 2. Clinical Status of Oral GPIIb-IIIa Antagonists
    •  
      • Interleukin-2 (IL-2)
      • Interact with heterotrimeric receptor (IL-2R  ,  ,  )
      • X-ray cristallography (IL-2 Brandhuber B.J. Science 1987, 238, 1707, IL-2/IL-2R  Rickert M. et al Science 2005, 308, 1477)
      • Mutagenesis
        • critical residues for binding IL-2 to IL-2R  (Sauve K. Et al. PNAS 1991, 88, 4636)
        • non contiguous aa located in various segments (R38, F42)
      • Antagonists
        • Monoclonal antibodies (daclizimab; 1997 PDL biopharma and basiliximab; 1998 novartis)
        • Structure-based design (Roche 24)
        • Fragment-based approach (SP-4206; preclinical Sunesis)
      Fragment-based approach
    • Tilley J.W. Et al JACS 1997, 119, 7589 Arkin M.R. et al. PNAS 2003, 100, 1603 Roche 24 Mimicking of R36 and E29 on IL-2R  and bindind of E62 and K43 on IL-2 Biaryl alchene group in a deep groove
    • Raimundo B.C. et al. J.Med.Chem. 2004, 47, 3111 Sunesis – Tethering approach
    • 60 nM
      • Structure-based design
      • IL-1R antagonists binding epitopes: R4, F46 and K93
      Computational approaches Core 2,4-dihydroxybenzene not interacting IC 50 = 770  M Sarabu R et al. Drug. Des. Discovery 1998, 15, 191 2,4-dihydroxybenzene mimicking F46 IC 50 < 10  M
    • Computational approaches
      • In silico screening on Bcl-2 inhibitors:
        • Homology model based on NMR structure of BCL- XL
        • 193,383 docked compounds in a hydrophobic pocket
        • 1000 best solutions optimized
        • 53 best compounds criteria: binding energy, shape complementarity, hydrogen bond forming
        • 28 commercially available compounds
      Wang J-L. et al. PNAS 2000, 97, 7124 HA14-1: IC 50 = 9  M
    • Covalent modificators
      • HIV-1 Protease
        • Catalytic site and substrate binding pocket formed in the dimeric state
        • Small tethered peptides have IC 50 in the submicromolar range
        • Epoxide and  ,  unsaturated ketone deriv. directed against D25 in the active site
        • Dimerization interface C95 as target of irreversible inhibitors
      Zutshi R.&Chmielewski J. Bioorg.Med.Chem.Lett. 2000, 10, 1901 K inact = 3.7  M
    • Antibody as template
      • (D) EGFR/cetuximab
      • (E) HER2/trastuzumab
      • (F)HER2/pertuzumab
      Rationally designed anti-HER2 peptide mimetic Trastuzumab CDR3 mimetic AHNP: FCDGFYACYMDV Park B.W. et al. Nat. Biotechnol. 2000, vol. 18 pag. 194
    • Allosteric modulation
      • iNOS
      • Screening of a combinatorial library
      • A potent and selective compound IC 50 = 0.6 nM
      • Bound to iNOS monomers and prevented their dimerization
      • Binding location in the active site as the natural substrate arginine
      • Conformational change propagate to the
      • dimerization interface
      • X-ray structure validate the mechanism
      McMillan K. et al. PNAS 2000, 97, 1506
    • Protein stabilization Microtubule stabilizing drugs: Taxol Downing K.H. Emerging Ther. Targets 2000, 4, 219 Stabilized mutant p53 in DNA binding domain: Pfizer: CP-31398 Foster B.A. et al. Science 1999, 286, 2507