WO2011005219A1 - Nouveaux peptides de liaison à mdm2 et leurs utilisations - Google Patents

Nouveaux peptides de liaison à mdm2 et leurs utilisations Download PDF

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Publication number
WO2011005219A1
WO2011005219A1 PCT/SG2010/000256 SG2010000256W WO2011005219A1 WO 2011005219 A1 WO2011005219 A1 WO 2011005219A1 SG 2010000256 W SG2010000256 W SG 2010000256W WO 2011005219 A1 WO2011005219 A1 WO 2011005219A1
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peptide
mdm2
mdmx
cancer
binding
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PCT/SG2010/000256
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English (en)
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Madhumalar Arumugam
Chandra Verma
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Agency For Science, Technology And Research
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to new peptides and uses thereof in treating cancer.
  • Cancer is one of the main diseases of current times causing 13% of all deaths globally. New aspects of the genetics of cancer pathogenesis, such as DNA methylation are increasingly recognized as important. While there are several chemicals that can affect rapidly dividing cancer cells most of these are toxic with adverse side effects.
  • the P53 protein is an endogenous protein that induces apoptosis of cells. It is generally activated when cells are under stress.
  • the p53 protein is known to be inactive in many tumors. More than 50 percent of human tumors contain a mutation or deletion of the TP53 gene. Artificially elevating levels of p53 have been shown to cause premature ageing. Restoring endogenous p53 induced apoptosis in tumor cells is desirable in treating cancer but there are no such methods on the market.
  • MDM2 oncoprotein Murine Double Minute Clone 2
  • SEQ ID No. 2 The oncoprotein Murine Double Minute Clone 2
  • p53 function can be rescued through the disruption of the MDM2-p53 interactions by small molecules and peptides such as peptide 12-1(33).
  • MDM2 regulates p53 by binding to its transactivation domain inhibiting its transcriptional activity and stimulating its ubiquitination and proteasomal degradation (5).
  • MDMX SEQ ID No. 3
  • MDMX promotes the ubiquitin ligase activity of M DM2 and also stabilizes p53 (10, 11 , 15).
  • the N-terminal domain of MDMX also regulates p53 by blocking its N-terminus transactivation domain, but unlike MDM2, MDMX doesn't promote p53's degradation (15, 16).
  • MDMX has also been found to be overexpressed in several cancers (70, 71).
  • MDM2/MDMX also binds to p73 but not p63, the two homologues of p53.
  • p73 is not mutated in cancers and can induce apoptosis. This further enhances the need for an antagonist against the MDM2 protein.
  • the p53 family consists of two other homologues, p63 and p73. All three proteins share a similar architecture, which includes an N-terminal TA domain, a central DNA binding domain (DBD) and a C-terminal Oligomerization domain. Reports have shown that some isoforms of p63 and p73 can bind to p53 responsive elements, transactivate p53-responsive genes and induce apoptosis (17-20). Though p63 and p73 emulate some of the p53 functions, their main functions have been shown to be critical for development and differentiation and, are rarely found to be mutated in human cancers (21-26).
  • the present invention seeks to provide novel peptides and/or methods for the disruption of the MDM2-p53 interaction or MDMX-p53 interaction. This may be useful in treating or slowing cancer cells to ameliorate some of the difficulties with the current treatment of cancer.
  • the invention further seeks to provide in vivo and in vitro methods, for arresting or slowing cell proliferation.
  • the first aspect of the invention is a peptide comprising an amino acid sequence of 20 amino acids or less with a phenylalanine in position 3, a serine in position 4, a hydrophobic amino acid in position 6, a Tryptophan in position
  • hydrophobic amino acid in position 6 is an isoleucine.
  • the peptide comprises a cationic amino acid at position 8.
  • the cationic amino acid at position 8 is a lysine.
  • the peptide comprises the sequence set out in SEQ ID No 1.
  • the peptide of the invention has a binding affinity ( ⁇ G bmd ) to
  • MDM2 or MDMX in the range of -18 Kcal/mol to -46 Kcal/mol.
  • the peptide of the invention is suitable for use in treating cancer.
  • a further aspect of the invention comprises a method of treating a subject afflicted with cancer comprising administering to the subject a peptide of the invention.
  • the peptide is suitable for use in the preparation of a medicament for the treatment of cancer.
  • a further aspect of the invention comprises a composition comprising a therapeutically effective amount of a peptide of the invention.
  • the composition comprises a chemotherapeutic agent.
  • composition is suitable for use in treating cancer.
  • composition is suitable for use in the preparation of a medicament for the treatment of cancer.
  • a further aspect of the invention comprises a method of disrupting MDM2 interaction with p53 comprising the steps of introducing a peptide of the invention that preferentially binds to MDM2. [00023].
  • a further aspect of the invention comprises a method of disrupting MDMX interaction with p53 comprising the steps of introducing a peptide of the invention that preferentially binds to MDMX.
  • Figure 1 The structure of the p53-MDM2 complex taken from the crystal structure 1YCR solved at a resolution of 2.6A. MDM2 and p53 are shown in grey and black respectively. The three critical residues, Phe19, Trp23 and Leu26 are shown in sticks.
  • Root Mean square Deviations of the backbone atoms as a function of time with respect to the starting structures during the MD simulations are shown for MDM2 apo (black), p53 (orange), p63(red),p73(cyan),12-1 (yellow) and pmad(green).
  • the regions of higher fluctuations of MDM2 are shown in ribbon model (ii).
  • FIG. 2B Temporal evolution of the secondary structure profiles peptides over simulations of 20ns (i for the free peptides) and 10ns (ii for the bound peptides), (a) p53 (b) p63 (c) p73 (d) 12-1 (e) pmad.
  • the secondary structure is colored as follows: purple- ⁇ -helix; blue- 3-10 helix; green-turn; white-random coil.
  • Figure 3 Residue wise contributions of van der Waals energies of the peptides taken from the simulations of the complexes.
  • FIG. 4 Location of Lys24 of p53 peptide with respect to the electrostatic surface of MDM2.
  • the residues Asp21 , Lys24, Leu 26, Pro27 and Glu28 of p53 are shown in sticks.
  • MDM2 is represented as a electrostatic potential mapped on to the solvent accessible surface. The potentials are colour coded from dark grey (+1 kcal e-1 mol- 1) to light grey (-1 kcal e-1 mol-1).
  • the p53 peptide is shown in black ribbon.
  • Figure 5 The packing of Pro27 (spheres) of the p53 peptide (shown in black ribbon) against the surface of MDM2.
  • FIG. 6 Three different conformations of Glu17 of pmad peptide. MDM2 is shown in grey ribbon and pmad peptide is shown in black ribbon. The residues Glu17, Lys94 and Gln59 are shown in sticks (A) pmad interacting with Lys94 of ⁇ 2' (B) pmad is exposed to solvent (C) pmad interacting with Gln59.
  • Figure 7 The packing of Pro27 (spheres) of pmad peptide against the MDM2 surface. Three critical residues Phe19, Trp23 and Leu26 are shown in sticks.
  • A Packing of Pro27 against the surface of MDM2
  • B the interaction of Pro27 residue with the backbone of Thr26 of MDM2.
  • FIG 8 The packing against the MDM2 surface of (A) 12-1 peptide (shown in black ribbon) (B) pmad peptide (shown in black ribbon). Three critical residues Phe19, Trp23, Leu26 are shown in sticks. MDM2 surface is shown in grey. Figure 9 Residues of MDMX that contribute to the differential binding of pmad. MDM2 and pmad peptide are shown in ribbon. (A) Glu17 of pmad peptide is not able to make interactions at the N-terminus due the presence of Gln66.
  • Pro95 provides nicer packing near the C-terminus and the presence of Ty ⁇ 99 and Arg 103 makes favorable interaction with the peptide backbone and carboxy terminal (B) pmad peptide (shown in black ribbon) packed against the MDMX surface.
  • B pmad peptide
  • Three critical residues Phe19, Trp23, Leu26 along with the N-terminus Glu17 and C-terminus Pro29 are shown in sticks.
  • M DM2 surface is shown in light grey.
  • peptide are short polymers formed from linking, amino acids with amide bonds.
  • the peptide is in the range of a 8mer to 20mer, preferably a 13mer or a 12mer having the features that allow the peptide to have high affinity bonding with MDM2 or MDMX.
  • the peptide has the sequence set out in SEQ ID No 4: GIu, X, Phe, Ser, X, Ne, Trp, X, X, Leu, X, X, X.
  • the peptide in one embodiment preferably comprising the sequence set out in SEQ ID No 1 : GIu, VaI, Phe, Ser, Asp, Me, Trp, Lys, Leu, Leu, GIu, GIn, Pro.
  • the new peptide of SEQ ID No 1. has been named pmad.
  • pmad preferably GIuI is attracted by Lys94 of MDM2 which also stabilizes an hydrogen bond between the Glu1 backbone and Gln72 of MDM2; the 2nd state is an intermediate state where Glu1 of pmad is solvated and doesn't make any specific interactions.
  • the 3rd conformation involves a stable packing between the N-terminus of pmad and Met62 of MDM2 and is associated with an increase in the length of the peptide helix by a half turn.
  • the backbone of GIuI forms a hydrogen bond with Ser4 side chain, thus stabilizing the pmad helix.
  • Asp5 is involved in interactions with Lys8 of pmad.
  • Glu11 is involved in a salt bridge with Arg97 of MDM2 and prevents TyMOO of MDM2 from flipping in.
  • Gln12 may form a hydrogen bond largely with the backbone of Lys8 and Leu10 and with Lys51 of MDM2; this gives the C-terminal region of pmad a stable turn-like fold.
  • Pro13 nestles against the aV side of MDM2 in a manner that enables the carboxy terminus to make a charge- charge interaction with Thr27 of MDM2 (Figs 7A 1 B).
  • Ile6 provides a local disruption to the packing of the peptide with MDM2. Indeed, although the N-terminal 3 residues and the C-terminal 4 residues of p63 and pmad are the same, the introduction of lie in place of Leu and 3 other changes (ie only a 30% transformation) appears to yield a transformation from a non-binding p63 to the highest affinity binding peptide, pmad to MDM2 or MDMX protein.
  • the new peptide pmad appear to offers a novel approach to peptide design.
  • the hydrophobic collapse is introduced via a bulkier hydrophobic residue (Ile6) that nucleates the aggregation of the other hydrophobic residues in the new peptide.
  • the frustration in the conformational landscape is generated by a cation (Lys8) toggling between two anionic fields (Asp5 and Glu11) on either side.
  • the new peptide design results in a dynamic peptide that can exist in different states.
  • Peptides of the invention may be synthesized chemically using known techniques such as liquid phase synthesis or solid phase synthesis such as t-BOC or Fmoc, or BOP SPPS or any chemical synthesis method known to those in the art.
  • the peptide may be made biologically within a cell or vector designed to use the machinery of translation and/or transcription for peptide synthesis.
  • cancer refers to malignant neoplasm, or a group of cells that display uncontrolled division and growth beyond the normal limits, ie: abnormal proliferation of cells invasion, intrusion on and destruction of adjacent tissues, and sometimes metastasis where the cancer cells have spread to other locations in the body via lymph system or blood. Most cancers form a tumor but some, like leukemia, do not. For the purpose of the invention cancer refers to cells where MDM2 has been upregulated.
  • the present invention provides a method for treating a patient with cancer, which comprises the step of: contacting the cells within and around a cancer with a peptide as described above.
  • the peptide is provided in a therapeutically effective amount.
  • An alternative form of the present invention resides in the use of the peptide in the manufacture of a medicament for treating a patient with cancer preferably a medicament used in treatment to affect cells over expressing MDM2.
  • Treatment and “treat” and synonyms thereof refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a cancer condition.
  • Those in need of such treatment include those already diagnosed with cancer or having cells over expressing MDM2.
  • a "therapeutically effective amount" of a compound will be an amount of active peptide that is capable of preventing or at least slowing down (lessening) a cancer condition, in particular increasing the average 5 year survival rate of cancer patients.
  • Dosages and administration of an antagonist of the invention in a pharmaceutical composition may be determined by one of ordinary skill in the art of clinical pharmacology or pharmacokinetics.
  • An effective amount of the peptide to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the mammal. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect.
  • a typical daily dosage might range from about 10 ng/kg to up to 100 mg/kg of the mammal's body weight or more per day, preferably about 1 ⁇ g/kg/day to 10 mg/kg/day.
  • Peptides produced according to the invention can be administered for the treatment of cancer in the form of pharmaceutical compositions.
  • compositions including pharmaceutical compositions comprising a therapeutically effective amount of a peptide that binds to MDM2 with high affinity.
  • a compound will be therapeutically effective if it is able to affect cancer growth either in vitro or in vivo.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions and or one or more carrier.
  • injectable solutions may be delivered encapsulated in liposomes to assist their transport across cell membrane.
  • preparations may contain constituents of self-assembling pore structures to facilitate transport across the cellular membrane. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating/destructive action of microorganisms such as, for example, bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as, for example, lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Preventing the action of microorganisms in the compositions of the invention is achieved by adding antibacterial and/or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active peptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying, to yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • the active ingredients in particular small peptides contemplated within the scope of the invention, are suitably protected they may be orally administered, for example, with an inert diluent or with an edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound.
  • compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
  • the amount of active peptide in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared so that a dosage unit form contains between about 0.1 ⁇ g and 20 g of active compound.
  • the tablets, troches, pills, capsules and the like may also contain binding agents, such as, for example, gum, acacia, corn starch or gelatin. They may also contain an excipient, such as, for example, dicalcium phosphate. They may also contain a disintegrating agent such as, for example, corn starch, potato starch, alginic acid and the like. They may also contain a lubricant such as, for example, magnesium stearate. They may also contain a sweetening agent such a sucrose, lactose or saccharin. They may also contain a flavouring agent such as, for example, peppermint, oil of wintergreen, or cherry flavouring.
  • binding agents such as, for example, gum, acacia, corn starch or gelatin. They may also contain an excipient, such as, for example, dicalcium phosphate. They may also contain a disintegrating agent such as, for example, corn starch, potato starch, alginic acid and the like. They may also contain a
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. [00044]. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparaben as preservatives, a dye and flavouring such as, for example, cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound(s) may be incorporated into sustained-release preparations and formulations.
  • Pharmaceutically acceptable carriers and/or diluents may also include any and all solvents, dispersion media, coatings, antibacterials and/or antifungals, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • those supplementary active ingredients are anticancer agents such as chemotherapy agents like, for example; cisplatin, platinum, carboplatin, gemcitabine, paclitaxel, docetaxel, etoposide, vinorelbine, topotecan, or irinotecan; tyrosine kinase inhibitors (e.g., Axitinib, Bosutinib, Cediranib, Dasatinib, Erlotinib, Gefitinib, Imatinib, Lapatinib, Lastaurtinib, Nilotinib, semaxanib, sunitinib, vandetanib, vatalanib or any other suitable tyrosine kinase inhibitor); apoptosis inducing enzymes, for example TNF polypeptides, TRAIL (TRAIL R1 , TRAIL R2) or FasL, Exis
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
  • the principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form.
  • a unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 ⁇ g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 pg to about 2000 mg/ml of carrier.
  • the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • compositions for administration by inhalation may take the form of inhalable powder compositions or liquid or powder sprays, and can be administrated in standard form using powder inhaler devices or aerosol dispensing devices. Such devices are well known.
  • the powdered formulations typically comprise the active compound together with an inert solid powdered diluents such as lactose or starch.
  • Inhalable dry powder compositions may be presented in capsules and cartridges of gelatin or a like material, or blisters of laminated aluminum foil for use in an inhaler or insufflators. Each capsule or cartridge may generally contain between 20 pg-10 mg of the active compound.
  • the compound of the invention may be presented without excipients.
  • the inhalable compositions may be packaged for unit dose or multi-dose delivery.
  • the compositions can be packaged for multi-dose delivery in a manner analogous to that described in GB 2242134, US6632666, US5860419, US5873360 and US5590 645 (all illustrating the "Diskus” device), or GB2178965, GB2129691 , GB2169265, US4778 054, US4811731 and US5035237 (which illustrate the "Diskhaler” device), or EP 69715 (“Turbuhaler” device), or GB 2064336 and US4353656 ("Rotahaler” device).
  • Spray compositions for topical delivery to the lung by inhalation may be formulated as aqueous solutions or suspensions or as aerosols delivered from pressurised packs, such as a metered dose inhaler (MDI), with the use of a suitable liquefied propellant.
  • MDI metered dose inhaler
  • the medication in pressurized MDI is most commonly stored in solution in a pressurized canister that contains a propellant, although it may also be a suspension.
  • Aerosol compositions suitable for inhalation can be presented either as suspensions or as solutions and typically contain the active compound and a suitable propellant such as a fluorocarbon or hydrogen-containing chlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanes such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and especially 1 ,1 , 1, 2- tetrafluoroethane, 1 ,1 , 1,2, 3,3, 3-heptafluoro-n-propane and mixtures thereof.
  • a suitable propellant such as a fluorocarbon or hydrogen-containing chlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanes such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and especially 1 ,1 , 1, 2- tetrafluoroethane, 1 ,1 , 1,2,
  • the aerosol composition may optionally contain additional excipients typically associated with such compositions, for example surfactants such as oleic acid or lecithin and co-solvents such as ethanol.
  • Pressurized formulations will generally be contained within a canister (for example an aluminum canister) closed with a metering valve and fitted into an actuator provided with a mouthpiece.
  • Peptides can also be delivered by protein delivery methods known in the art such as transfection, macromolecule delivery vehicles and other methods known to those skilled in the art.
  • compositions may be for use in treating cancer.
  • Use includes use of a composition of the invention for the preparation of a medicament or a pharmaceutically acceptable composition for the treatment of cancer.
  • the preparation may further comprise a chemotherapeutic agent for the preparation of a medicament for the treatment of cancer.
  • MDM2 The N- and C- termini of MDM2 were capped with acetyl (ACE) and N-methyl (NME) respectively to keep them neutral; the N-terminus of the p53 peptides was capped with ACE (53).
  • MD simulations were performed using the SANDER module of the AMBER8 (54) package employing the all-atom Cornell force field (55) Simulations were carried out for the complexes of p53-MDM2, p63-MDM2, p73-MDM2, pmad-MDM2,12-1-MDM2 and 20 nano seconds (ns) simulations for the uncomplexed protein (MDM2 apo and p53, p63, p73, pmad, 12-1).
  • Each system was solvated with TIP3P water (56) box, which extended at least 12 A in each direction from the solute.
  • a cut off of 10 A was used for the non-bonded van der Waals interaction. All bonds involving hydrogen atoms were constrained using SHAKE (57). Particle mesh Ewald (PME) (58) was used for the long range electrostatic interactions. After initial minimizations, the systems were gradually heated to 300 K, equilibrated for 100 (pico seconds) (ps) and production run was extended up to 10 ns.
  • the binding free energy ( ⁇ G b in d ) of the p53 peptide (residues 16-27) to MDM2 has been reported to be -6.6 to -7.8 kcal/mol (37) and is similar to that of longer peptide (residues 15-29) (38)
  • Our methods are robust in that they are close to other computationally derived data.
  • Vibrational entropy was estimated using normal mode analysis (Nmode module of Amber) (64) and averaged over 50 frames of complex and 100 frames of free peptides and MDM2. Secondary structures were computed using VMD, which has DSSP assignments (65), PyMOL (66) and Visual Molecular Dynamics (VMD) were used for visualizations (67).
  • Peptides of the invention have a binding affinity ( ⁇ G b in d ) to MDM2 or MDMX in the range of -18 Kcal/mol to -46 Kcal/mol.
  • peptides of the invention have a binding affinity ( ⁇ G b i nd ) to MDM2 or MDMX in the range of -20 Kcal/mol to -46 Kcal/mol.
  • the pmad peptide has a binding affinity ( ⁇ G b ind) to MDM2 in the range of -30 Kcal/mol to -36 Kcal/mol and a binding affinity ( ⁇ G b in d ) to MDMX in the range of -30 Kcal/mol to -46 Kcal/mol.
  • the van der Waals energy of p73 is the smallest and originates in the p73 peptide being rich in smaller amino acids, Thr/Ser. Further, the p63 peptide has a very unfavorable internal energy term. This strained conformation arises from a hydrophobic collapse that appears to be caused by the clustering of Phe19, Ile22, Trp23, Leu26 and Pro29, which in turn leads to higher van der Waals interactions. The packing against MDM2 in 12-1 and pmad is the most favorable amongst all the peptides.
  • the first half of the simulation is characterized by a hydrophobic collapse as seen in p63 above; this is expected because the residues that are part of this cluster- Phe19, Ile22, Trp23, Leu26 and Pro29 - are conserved between the p63 and pmad (movies of wtp53,pmad at http://web.bii.a- star.edu.sg/ ⁇ madhumalar/movies/).
  • this collapse is reversed during the course of the simulation because of the long range attraction between Asp21 and Lys24 which straightens the helix and releases the strain in pmad.
  • DNA encoding residues Gln18 to Asn125 of MDM2 was cloned as a Ndel/BamHI fragment into pET19b (Novagen). Recombinant clones carrying the protein insert were identified by DNA sequencing. Plasmid from these clones was introduced into E. coli BL21(DE3) and protein production in LB medium was induced with IPTG(I mM) when the cell density reached an OD600 between 0.4 and 0.6. Induced cells were grown at room temperature for 5 h, harvested, and then resuspended in buffer A [50 mM Tris pH 8.0, 10 % sucrose].
  • NTA Ni-nitrilotriacetic acid
  • MDM2 was eluted with a 1 M imidazole linear gradient. MDM2 was then dialysed into 1OmM Sodium phosphate and 0.05M NaCI.
  • the protein was further purified by cation-exchange chromatography [Pharmacia Mono S 5/5, 1 ml/min, Sodium phospsphate (10 mM), pH 7.0] with a 1M NaCI gradient. Protein concentration was determined using A280 with an extinction
  • F t is the fluorescence intensity at temperature T ;
  • T m is the midpoint temperature of the protein-unfolding transition,
  • F pre and F post are the pre-transitional and post-transitional fluorescence intensities,
  • R is the gas constant,
  • ⁇ H is the enthalpy of protein unfolding, and
  • ⁇ C is the heat capacity change on protein
  • K L(T) can be calculated from K L(Tm) using Eq. (3A):
  • K is the ligand association constant at temperature T
  • ⁇ H is the van't Hoff enthalpy of binding at temperature T.
  • the value of ⁇ H was taken to be - 10kcal/mol.
  • pmad peptide has an affinity somewhat higher than that of 12-1 (data not shown).
  • the binding affinity of peptides of the invention to MDM2 or MDMX can be easily tested in this way by a person skilled in the art in addition to any of the other in-silico methods mentioned throughout the specification. In can be quickly and easily established whether a peptide comprising a amino acid sequence of 20 amino acids or less with a phenylalanine in position 3, a serine in position 4, a hydrophobic amino acid in position 6, a Tryptophane in position 7 and Leucine in position 10 is capable of binding to a MDM2 protein or a MDMX protein using the methods described.
  • GIu in both p53/p63 can interact with Lys70/Lys94, while Thr in p73 is not long enough to interact with either of them; but it gets oriented towards Lys94 and or Gln71.
  • Thr18 in both p53/p73 makes favorable interactions with Asp21/His21 of the peptide as well as with the side chain of Gln72; the equivalent Va118 in p63 is not involved in any interactions. Indeed these accounts for the greater disorder in the N-terminus of p63.
  • the well conserved Phe19 fits well in the hydrophobic cleft in all the three cases.
  • p53 has Ser whose side chain remains solvated while equivalent GIn and GIu in p63 and p73 are stabilized by Gln59. Asp21 of p53 is stabilized by Lys 24 for extended periods; in p63 and p73, the equivalent residue is His which interact with the N-terminus of the peptide leading to some loss of the packing between MDM2 and this end of the peptide. In p73 interactions between this His and ThM 8 stabilize the system a little.
  • Trp23 packs well in the hydrophobic cleft in all the complexes, including the hydrogen bond between its side chain and the backbone of Leu54.
  • Lys 24 of p53 points largely towards Asp21 , this is not- possible in p63 as it has anionic Asp at position 24.
  • the dynamics in this region are quite correlated.
  • the Lys24 in p53 can only either be solvated or point towards the anionic field of Asp21 ; it is pushed towards this by the cationic field of Lys51 of MDM2 which in turn is stabilized by the anionic field created across the backbone carbonyls of Lys24, Leu26 and Pro27 and the side chain of Glu28 occasionally (Fig. 4).
  • Asp 24 points in the direction of Lys51 (doesn't reach it as it is short) and adds to the anionic field. This anionic field now pulls Lys51 side chain as well as the side chain of Gln28 of p63.
  • Ser24 is only involved in intra peptide interactions that stabilize the helix.
  • Glu28 in p53 interacts with Lys51 of MDM2 while GIn of p63 is involved with the anionic field of the region of the peptide mentioned above.
  • Pro28 in p73 is nestled against the surface of MDM2 (this is the surface opposite to where the Pro27 in p53 nestles).
  • the side chain of Gln29 in p53 is largely exposed even as the carboxy terminus interacts briefly with the side chains of TyMOO and Tyr104 and for long periods with Arg97; indeed the orientation of Arg97 is such that its hydrophobic side-chain forms the surface against which Pro27 nestles.
  • Pro29 only creates a curved peptide but doesn't really interact with anything on MDM2, lending this part high disorder.
  • Phe19, Trp23 and Leu26 are packed in a manner similar to that seen in the other systems while Met20 adds stability to the complex by packing against the ⁇ 1 helix (including Met62).
  • Glu24 is largely exposed although it appears to be involved in long range stabilization with Arg18.
  • Gln28 and this side chain together with the carboxy terminus are stabilized by Lys51 thus giving the peptide a final helical turn.
  • TyMOO and TyM 04
  • the N-terminus occupies three different states ( Figures 6A-C) that are separated by 3-4 kcal/mol (data not shown): in one state, Glu17 is attracted by Lys94 which also stabilizes an h-bond between the Glu17 backbone and Gln72; the 2nd state is an intermediate state where Glu17 is solvated and doesn't make any specific interactions.
  • the 3rd conformation involves a stable packing between the N-terminus and Met62 and is associated with an increase in the length of the peptide helix by a half turn. In this conformation, the backbone of Glu17 h-bonds with Ser20 side chain, thus stabilizing the helix.
  • Asp21 is involved in interactions with Lys24, analogous to p53.
  • Glu27 is involved in a salt bridge with Arg97 and prevents TyMOO from flipping in.
  • Gln28 h-bonds largely with the backbone of Lys24 and Leu26 and with Lys51 ; this gives the C-terminal region a stable turn-like fold.
  • Pro29 nestles against the aV side of MDM2 in a manner that enables the carboxy terminus to make a charge-charge interaction with Thr27 of MDM2 (Figs 7A,B).
  • Ne22 provides a local disruption to the packing of the peptide with MDM2 (as seen in p63).
  • MDM2 MDM2
  • MDMX MDM2
  • the invention described herein may include one or more range of values (eg size, concentration etc).
  • a range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.
  • MDMX a novel p53-binding protein with some functional properties of MDM2.
  • Vassilev LT Small-Molecule Antagonists of P53-Mdm2 Binding: Research Tools and Potential Therapeutics. Cell Cycle 2004; 3:419-21.
  • MDM2 Uldrijan S, Pannekoek WJ, Vousden KH.
  • An essential function of the extreme C- terminus of MDM2 can be provided by MDMX. EMBO J. 2007 Jan 10;26(1 ):102-12.
  • Bottger V Bottger A
  • Garcia-Echeverria C Ramos YF
  • van der Eb AJ van der Eb AJ

Abstract

Nous avons mis au point un peptide qui est intrinsèquement moins hélicoïdal que p53 et possède néanmoins une affinité plus élevée pour MDM2 et MDMX, qui peut être capable de secourir la fonction de p53 en apoptose par l'interruption de l'interaction MDM2-p53 ou de l'interaction MDMX-p53. Le peptide peut être utilisé pour traiter le cancer.
PCT/SG2010/000256 2009-07-07 2010-07-07 Nouveaux peptides de liaison à mdm2 et leurs utilisations WO2011005219A1 (fr)

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EP2639240A2 (fr) 2012-02-21 2013-09-18 Consiglio Nazionale Delle Ricerche Peptides capables de neutraliser l'activité d'inhibition d'une hétérodimère MDM2/MDM4 vers p53 et leur utilisation pour le traitement du cancer
US8859723B2 (en) 2010-08-13 2014-10-14 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US8889632B2 (en) 2007-01-31 2014-11-18 Dana-Farber Cancer Institute, Inc. Stabilized p53 peptides and uses thereof
US8927500B2 (en) 2012-02-15 2015-01-06 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US8987414B2 (en) 2012-02-15 2015-03-24 Aileron Therapeutics, Inc. Triazole-crosslinked and thioether-crosslinked peptidomimetic macrocycles
US9096684B2 (en) 2011-10-18 2015-08-04 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US9408885B2 (en) 2011-12-01 2016-08-09 Vib Vzw Combinations of therapeutic agents for treating melanoma
US9604919B2 (en) 2012-11-01 2017-03-28 Aileron Therapeutics, Inc. Disubstituted amino acids and methods of preparation and use thereof
WO2017201449A1 (fr) 2016-05-20 2017-11-23 Genentech, Inc. Conjugués anticorps-protac et procédés d'utilisation
US10023613B2 (en) 2015-09-10 2018-07-17 Aileron Therapeutics, Inc. Peptidomimetic macrocycles as modulators of MCL-1
US10164661B2 (en) 2014-03-05 2018-12-25 Saturn Licensing Llc Data processing device and data processing method
US10253067B2 (en) 2015-03-20 2019-04-09 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
US10301351B2 (en) 2007-03-28 2019-05-28 President And Fellows Of Harvard College Stitched polypeptides
US10471120B2 (en) 2014-09-24 2019-11-12 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
US10905739B2 (en) 2014-09-24 2021-02-02 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and formulations thereof
WO2023056069A1 (fr) 2021-09-30 2023-04-06 Angiex, Inc. Conjugués agent de dégradation-anticorps et leurs procédés d'utilisation

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WO2003105880A1 (fr) * 2002-03-12 2003-12-24 The Research Foundation Of The State University Of New York Peptides selectivement letaux pour des cellules de mammiferes transformees et malignes

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WO2003105880A1 (fr) * 2002-03-12 2003-12-24 The Research Foundation Of The State University Of New York Peptides selectivement letaux pour des cellules de mammiferes transformees et malignes

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US9527896B2 (en) 2007-01-31 2016-12-27 Dana-Farber Cancer Institute, Inc. Stabilized p53 peptides and uses thereof
US8889632B2 (en) 2007-01-31 2014-11-18 Dana-Farber Cancer Institute, Inc. Stabilized p53 peptides and uses thereof
US10301351B2 (en) 2007-03-28 2019-05-28 President And Fellows Of Harvard College Stitched polypeptides
US8859723B2 (en) 2010-08-13 2014-10-14 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10703780B2 (en) 2010-08-13 2020-07-07 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US11008366B2 (en) 2010-08-13 2021-05-18 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US9957299B2 (en) 2010-08-13 2018-05-01 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US9522947B2 (en) 2011-10-18 2016-12-20 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10308699B2 (en) 2011-10-18 2019-06-04 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US9096684B2 (en) 2011-10-18 2015-08-04 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US9408885B2 (en) 2011-12-01 2016-08-09 Vib Vzw Combinations of therapeutic agents for treating melanoma
US10227380B2 (en) 2012-02-15 2019-03-12 Aileron Therapeutics, Inc. Triazole-crosslinked and thioether-crosslinked peptidomimetic macrocycles
US9505804B2 (en) 2012-02-15 2016-11-29 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US8987414B2 (en) 2012-02-15 2015-03-24 Aileron Therapeutics, Inc. Triazole-crosslinked and thioether-crosslinked peptidomimetic macrocycles
US8927500B2 (en) 2012-02-15 2015-01-06 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10213477B2 (en) 2012-02-15 2019-02-26 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
EP2639240A2 (fr) 2012-02-21 2013-09-18 Consiglio Nazionale Delle Ricerche Peptides capables de neutraliser l'activité d'inhibition d'une hétérodimère MDM2/MDM4 vers p53 et leur utilisation pour le traitement du cancer
US9604919B2 (en) 2012-11-01 2017-03-28 Aileron Therapeutics, Inc. Disubstituted amino acids and methods of preparation and use thereof
US9845287B2 (en) 2012-11-01 2017-12-19 Aileron Therapeutics, Inc. Disubstituted amino acids and methods of preparation and use thereof
US10669230B2 (en) 2012-11-01 2020-06-02 Aileron Therapeutics, Inc. Disubstituted amino acids and methods of preparation and use thereof
US10164661B2 (en) 2014-03-05 2018-12-25 Saturn Licensing Llc Data processing device and data processing method
US11271595B2 (en) 2014-03-05 2022-03-08 Saturn Licensing Llc Data processing device and data processing method
US10707903B2 (en) 2014-03-05 2020-07-07 Saturn Licensing Llc Data processing device and data processing method
US10471120B2 (en) 2014-09-24 2019-11-12 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
US10905739B2 (en) 2014-09-24 2021-02-02 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and formulations thereof
US10253067B2 (en) 2015-03-20 2019-04-09 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
US10023613B2 (en) 2015-09-10 2018-07-17 Aileron Therapeutics, Inc. Peptidomimetic macrocycles as modulators of MCL-1
WO2017201449A1 (fr) 2016-05-20 2017-11-23 Genentech, Inc. Conjugués anticorps-protac et procédés d'utilisation
WO2023056069A1 (fr) 2021-09-30 2023-04-06 Angiex, Inc. Conjugués agent de dégradation-anticorps et leurs procédés d'utilisation

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