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WO2014065760A1 - Méthodes de détermination de la résistance contre des molécules ciblant des protéines - Google Patents

Méthodes de détermination de la résistance contre des molécules ciblant des protéines Download PDF

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Publication number
WO2014065760A1
WO2014065760A1 PCT/SG2013/000460 SG2013000460W WO2014065760A1 WO 2014065760 A1 WO2014065760 A1 WO 2014065760A1 SG 2013000460 W SG2013000460 W SG 2013000460W WO 2014065760 A1 WO2014065760 A1 WO 2014065760A1
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WIPO (PCT)
Prior art keywords
biological molecule
molecule
hdm2
nutlin
gene encoding
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PCT/SG2013/000460
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English (en)
Inventor
Farid Ghadessy
Chandra Shekhar Verma
David Philip Lane
Jia Wei SIAU
Thomas Leonard JOSEPH
Adelene Yen Ling SIM
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Agency for Science Technology and Research Singapore
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Agency for Science Technology and Research Singapore
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Priority to US14/438,469 priority Critical patent/US20150276755A1/en
Priority to EP13849032.1A priority patent/EP2912463A4/fr
Priority to SG11201503198YA priority patent/SG11201503198YA/en
Publication of WO2014065760A1 publication Critical patent/WO2014065760A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4748—Details p53
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)

Definitions

  • the present invention generally relates to methods for determining drug resistance.
  • the present invention relates to methods for determining resistance to drug molecules that target protein-protein interactions.
  • Drug resistance is a major therapeutic bottleneck that mandates a detailed understanding of any potential molecular escape mechanisms that may arise in patients. Many patients die as a result of the disease developing resistance to the drugs that are being used to treat them.
  • Cancer is a major disease in the developed world. While significant improvements have been made in the efficacy of drugs used to treat cancer, the emergence of drug-resistance remains a significant problem in cancer treatment. Cancers are often able to adapt such that the drugs used to treat them are no longer effective. In order to address this problem, it is important to be able to predict how readily cancers can become resistant to a particular drug. Understanding drug- specific resistance mechanisms, together with the ability to screen for such resistance, may facilitate both rational therapeutic approaches and enable the development of next-generation drugs tailored to counteract the deleterious mutations in the target protein that confers the resistance.
  • target -based mutagenesis approaches have been adopted, wherein complementation by a mutated protein enables survival of an otherwise drug- sensitive cell line. These approaches, have accurately predicted drug resistance in several instances [51,52].
  • a major disadvantage of such approaches is the relatively small library of target protein variants that can be sampled due to inherent technical limitations arising from transformation efficiency ( ⁇ 10 6 ) , and the possibility of off-target drug toxicity at higher doses that limits selection pressure.
  • the viral transduction method used to stably introduce genes encoding mutant proteins introduces significant heterogeneity arising from the random nature of integration into the chromosome. This can impart significant bias due to variation in expression levels of the mutant proteins being screened. -
  • a method for determining and predicting drug resistance comprising the steps of:
  • detection of the complex in the presence of the inhibitor indicates that the biological molecule is resistant to inhibition of its interaction with the target molecule by the inhibitor
  • non-detection of the complex in the presence of the inhibitor indicates that the biological molecule is not resistant to inhibition of its interaction with the target molecule by the inhibitor.
  • the disclosed method provides a completely cell-free methodology for determining resistance of a biological molecule to its inhibitor. Being completely cell-free, the disclosed method allows the use of stringent selection pressures for selecting biological molecules with exceptionally strong resistance phenotype, as well as with enhanced accuracy.
  • the complex comprises the gene encoding the biological molecule.
  • this enables selection and isolation of biological molecules having resistance phenotype, which can be used to facilitate design of new drugs or new versions of the drug to overcome the resistance.
  • the emulsion comprises a plurality of the aqueous droplets.
  • each aqueous droplet comprises a single variant of the gene encoding the biological molecule.
  • the provision of a plurality of the aqueous droplets each comprising a single variant of the gene encoding the biological molecule allows for high- throughput screening of a large repertoire of variants.
  • a mutated HDM2 ubiquitin ligase polypeptide comprising at least one mutation selected from the group consisting of E69A, D225G, V241A, V280A, K344R, E390G, V426A, Q442R, M459T, Q24R, M62V, E124G, C461Y, T16A, P20L, L254F, N309T, G443D, H457R, L82P, and combinations thereof .
  • a DNA molecule encoding the mutated HDM2 ubiquitin ligase polypeptide as defined above.
  • a prognostic method for determining the receptiveness of a cancer patient to treatment with an anti-cancer drug capable of inhibiting the interaction of HDM2 ubiquitin ligase with p53 tumor suppressor protein comprising the step of:
  • identification of a match between the gene of the patient to at least one gene in the plurality of HDM2 ubiquitin ligase genes that have been determined to be resistant to the anti -cancer drug indicates that the cancer patient may not be receptive to treatment with the anticancer drug.
  • kits for use in a method of the first aspect comprising means to co-compartmentalize the gene encoding the biological molecule with the target molecule or the gene encoding the target molecule into aqueous droplets disposed within a water-in-oil emulsion, and means to detect the complex comprising the biological molecule and the target molecule upon expression of the gene encoding the biological molecule and the gene encoding the target molecule.
  • a method for selecting a variant form of a biological molecule that is resistant to inhibition of its interaction with a target molecule by an inhibitor of the biological molecule comprising the steps of providing a plurality of randomly mutated genes encoding the biological molecule, and determining resistance of the biological molecule to inhibition of its interaction with the target molecule by the inhibitor using the method according to the first aspect.
  • a kit for use in a method of the sixth aspect comprising means for generating the plurality of randomly mutated genes encoding the biological molecule.
  • a method of restoring the inhibitory activity of a drug on the interaction of a biological molecule with a target molecule comprising the steps of:
  • the method enables iterative improvement of the functionality and efficacy of a drug that is already in use or that is currently in development for clinical applications to reduce or avoid resistance.
  • the term "about” as used in relation to a numerical value means, for example, +50% or +30% of the numerical value, preferably +20%, more preferably +10%, more preferably still +5%, and most preferably +1%. Where necessary, the word "about” may be omitted from the definition of the invention.
  • inhibitor means “and/or” unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive.
  • inhibition refers to the act of decreasing, blocking, suppressing, or disrupting a particular activity or interaction such as the interaction between two molecules; or blocking, suppressing or disrupting a biological process, for example, an enzymatic reaction, gene expression, and the like.
  • inhibitor therefore refers to any molecule which has the ability to decrease, block, suppress, or disrupt a particular activity of another molecule, or the interaction between two other molecules.
  • resistance to inhibition is thus construed to mean the ability of a molecule to resist or withstand inhibition by an inhibitor, thereby maintaining its original activity or function, or its original interaction with other molecules in the presence of the inhibitor.
  • wild- type refers to a phenotype , genotype, or gene that predominates in a natural population of organisms or strain of organisms in contrast to that of natural or laboratory mutant forms .
  • mutant and mutant include any detectable change in genetic material, e.g. DNA, or any process, mechanism, or result of such a change. This includes gene mutations, in which the structure (e.g. DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e.g. protein or enzyme) expressed by a modified gene or DNA sequence.
  • variant may also be used to indicate a modified or altered form of a gene, DNA sequence, enzyme, cell, etc., i.e., any kind of mutant.
  • a mutant HDM2 ubiquitin ligase polypeptide comprising a L82P mutation is a variant form of the wild-type HD 2 ubiquitin ligase polypeptide.
  • a “variant” will have substantially similar polypeptide or nucleic acid sequences as the "non-variant" (or wild-type) form.
  • variants may have at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the "non-variant" polypeptide or nucleic acid.
  • Variants may be made using, for example, the methods of protein engineering and site-directed mutagenesis as is well known in the art. For example, mutations may be introduced by chemical or.
  • exogenous nucleic acid may be introduced by recombinant means employing, for example, chemical assisted cell permeation (using, for example, calcium, lithium, PEG), electroporation, microinjection, liposome-mediated transfection, microparticle bombardment (biolistics) , virus infection, or any other appropriate means as are known in the art .
  • chemical assisted cell permeation using, for example, calcium, lithium, PEG
  • electroporation using, for example, calcium, lithium, PEG
  • microinjection liposome-mediated transfection
  • microparticle bombardment biolistics
  • virus infection or any other appropriate means as are known in the art .
  • analogue refers to a chemical compound that is structurally similar to a parent compound or has chemical properties or pharmaceutical activity in common with the parent compound.
  • Analogues include, but are not limited to, homologues, i.e., where the analogue differs from the parent compound by one or more carbon atoms in series; positional isomers; compounds that differ by interchange of one or more atoms by a different atom, for example, replacement of a carbon atom with an oxygen, sulfur, or nitrogen atom; and compounds that differ in the identity of one or more functional groups, for example, the parent compound differs from its analogue by the presence or absence of one or more suitable substituents .
  • Suitable substit ents include, but are not limited to, ( Ci - C 8 ) alkyl ; ( Ci - C 8 ) alkenyl ; ( Ci-C 8 ) alkynyl ; aryl ; (C 2 -C 5 ) heteroaryl ; ( Ci - C 6 ) heterocycloaklyl ; (d-C 7 ) cycloalkyl ; O- - (d-C 8 ) alkyl ; 0--(d- C 8 ) alkenyl; 0- - (C a -C 8 ) alkynyl ; O-aryl; CN; OH; oxo; halo, C(O)0H; COhalo; 0 (CO) halo; CF 3 ; N 3 ; N0 2 ; NH 2 ; NH ( (d-C 8 ) alkyl) N((d- C e )alkyl) 2 ; NH(aryl
  • emulsion refers to a suspension of small globules of one liquid (the dispersed phase) in a second liquid (the continuous phase) with which the first will not mix.
  • water-in-oil emulsion is construed to mean a suspension of small globules of water (the dispersed phase) in an oil solvent (the continuous phase) .
  • peptide refers to any compound containing two or more amino acid residues joined by an amide bond formed from the carboxyl group of one amino acid residue and the amino group of the adjacent amino acid residue.
  • peptide includes oligopeptide, peptide, polypeptide and derivatives thereof, peptide analogs and derivatives thereof, as well as pharmaceutically acceptable salts of these compounds.
  • polypeptide and “protein” are used interchangeably and refer to any polymer of amino acids (dipeptide or greater) linked through peptide bonds or modified peptide bonds, whether produced naturally or synthetically.
  • protein may refer, in addition, to a complex of two or more polypeptides.
  • proteins include an antibody, an antibody fragment and. a peptide aptamer .
  • oligopeptide refers to a peptide containing a relatively small number of amino acid residues; typically around 2 to about 20 amino acids. Examples of oligopeptides include dipeptides, tripeptides, tetrapeptides , and pentapeptides .
  • synthetic peptide or “synthetic protein” as used herein refers to man-made molecules that mimic the function and structure of natural peptides or proteins. Synthetic peptides and proteins typically have genetic sequences that are not seen in natural proteins.
  • stapled peptide refers to artificially modified peptide in which the structure is stabilized with one or more artificial molecular bridging (cross links) that connects adjacent turns of a-helices in the peptide.
  • the term “recombinant” refers to a compound or composition produced by human intervention.
  • a "recombinant" nucleic acid or protein molecule is a molecule where the nucleic acid molecule which encodes the protein has been modified in vitro, so that its sequence is not naturally occurring, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in a genome which has not been modified.
  • biological molecule refers to any molecule that are created or used by living organisms or cells, or derivatives of such molecules. These molecules may be of natural, synthetic or semisynthetic origin, and includes proteins and nucleic acids .
  • target molecule refers to any molecule with which a biological molecule interacts (e.g. binds) , and may for example be a ligand or substrate of the biological molecule.
  • a target molecule may be a peptide, a polypeptide or protein (e.g. a fusion protein), a protein or polypeptide fragment, or functional protein or polypeptide domain.
  • expression refers interchangeably to expression of a gene or gene product, including the encoded protein.
  • a gene is expressed in or by a cell to form an "expression product” such as mRNA or a protein.
  • the expression product itself e.g. the resulting mRNA or protein, may also be said to be “expressed” by the cell.
  • Expression of a gene may be determined, for example, by measuring the production of messenger RNA (mRNA) transcript levels.
  • Expression of a polypeptide gene product may be determined, for example, by immunoassay using an antibody ( ies) that bind with the polypeptide .
  • the word “substantially” does not exclude “completely” e.g. a composition which is "substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.
  • detection of the complex in the presence of the inhibitor indicates that the biological molecule is resistant to inhibition of its interaction with the target molecule by the inhibitor
  • non-detection of the complex in the presence of the inhibitor indicates that the biological molecule is not resistant to inhibition of its interaction with the target molecule by the inhibitor.
  • the complex comprises the gene encoding the biological molecule of interest.
  • the complex comprises the expressed biological molecule of interest, the expressed target molecule, and the gene encoding the biological molecule such that a protein-protein-DNA complex is formed.
  • this facilitates selection for a-nd isolation of the gene encoding the biological molecule which exhibits resistance to its inhibitor.
  • the gene encoding the biological molecule comprises at least one copy of a p53 response element (RE) fused to the gene encoding the biological molecule.
  • the gene encoding the biological molecule comprises 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 2 to 100, 5 to 100, 10 to 100, 20 to 100, 30 to 100, 50 to 100, 75 to 100, 90 to 100, 2 to 90, 2 to 75, 2 to 50, 2 to 30, 2 to 20, 2 to 10, or 2 to 5 copies of a p53 response element (RE) fused to the gene encoding the biological molecule.
  • RE p53 response element
  • the gene encoding the biological molecule comprises two (2) copies of a p53 response element (RE) fused to the gene encoding the biological molecule .
  • RE p53 response element
  • the p53 response element is a CONA response element.
  • Other exemplary response elements that may be used are known to those skilled in the art, and include, but are not limited to, p21 and puma response elements.
  • p53 can act as a linker protein to link the gene encoding the biological molecule (or a variant thereof) with the biological molecule (or a variant thereof) that is expressed in protein form from the gene, thus forming a protein- protein-DNA complex in the presence of an inhibitor of the biological molecule.
  • the p53 can act as a linker between the genotype (gene encoding the biological molecule or a variant thereof) and the phenotype of the biological molecule (or variant thereof) in formation of the complex in presence of the inhibitor.
  • the disclosed method may be applied to other protein- protein interactions (apart from interaction between p53 and HDM2) by generating a fusion protein comprising a protein of interest and p53.
  • This can be added to emulsion compartments in purified form, or expressed inside emulsion compartments from a gene construct encoding the fusion protein.
  • a gene construct comprising two or more tandem copies of a p53 response element and the biological molecule are also introduced into the emulsion compartments.
  • the emulsion comprises a plurality of the aqueous droplets.
  • each aqueous droplet comprises a single variant of the gene encoding the biological molecule of interest .
  • the biological molecule may be selected from the group consisting of an amino acid, a peptide, a protein and combinations thereof.
  • the peptide may be a polypeptide, an oligopeptide, a stapled peptide, or a synthetic peptide.
  • the protein may be a mini -protein , a recombinant protein or a protein complex.
  • polypeptides of the invention may be "free-standing", i.e. not part of or fused to other amino acids or polypeptides or they may be comprised within a larger polypeptide of which they form a part or region.
  • fusion proteins incorporating the polypeptides described herein are contemplated in the present invention.
  • additional amino acid sequences which may contain secretory or leader sequences, pro- sequences , or sequences which aid in for instance detection, expression, separation or purification of the protein or to endow the protein with additional properties as desired such as higher protein stability.
  • Examples of potential fusion partners include epitope tags (short peptide sequences for which a specific antibody is available), polyethylene glycol, beta-galactosidase , luciferase, a polyhistidine tag, glutathione S transferase (GST), a secretion signal peptide and a label, which may be, for instance, bioactive, radioactive, enzymatic or fluorescent, or an antibody.
  • a fusion protein may also be engineered to contain a cleavage site located between the sequence of a polypeptide of the invention and the sequence of a heterologous protein / polypeptide sequence so that the polypeptide may be cleaved and purified away from the heterologous protein / polypeptide sequence.
  • the biological molecule is a protein.
  • the protein may be an enzyme.
  • the protein is HDM2 ubiquitin ligase ("HD 2").
  • the protein is a homologue of HDM2 , for example HDMX or HD 4.
  • the protein may be selected from the group consisting of tumor suppressor p53 -binding proteins, such as tumor suppressor p53 -binding protein 1 (TP53BP1) and tumor suppressor p53 -binding protein 2 (TP53BP2) .
  • Other proteins that may be used as the biological protein to form a protein-protein or protein-protein-DNA complex in the methods disclosed herein can be found on publicly available databases, such as the UniProt database (http://www.uniprot.org/).
  • the target molecule may also be selected from the group consisting of an amino acid, a peptide,, a protein and combinations thereof.
  • the peptide may be a polypeptide, an oligopeptide, a stapled peptide and a synthetic peptide.
  • the protein may be a mini-protein, a recombinant protein and a protein complex.
  • the target molecule is a protein.
  • the protein is a p53 tumor suppressor protein ("p53") .
  • the biological molecule of interest whose resistance to its inhibitor is to be determined is HDM2
  • the target molecule to which HDM2 binds is its substrate, p53.
  • the target molecule may comprise a fusion protein.
  • the fusion protein may comprise a p53 fused to a protein (e.g. protein Y) with which the biological molecule interacts.
  • the protein-protein-DNA complex that is formed comprises the biological molecule, the fusion protein (e.g. comprising p53 and protein Y) , and the gene encoding the biological molecule.
  • the fusion protein e.g. comprising p53 and protein Y
  • the fusion protein may be included in the compartments within the emulsion in purified form, or expressed inside the compartments from a gene construct encoding the fusion protein.
  • a gene construct comprising two or more tandem copies of a p53 response element and the biological molecule may also be included in the compartments within the emulsion .
  • the inhibitor may be a small organic molecule, a peptide or a protein.
  • the peptide may be selected from the group consisting of a polypeptide, an oligopeptide, a stapled peptide and a synthetic peptide.
  • the protein may be selected from the group consisting of a mini-protein, a recombinant protein and a protein complex.
  • An inhibitor may also be a ribozyme or an antibody. Analogues of the inhibitor are also included herein.
  • a "small molecule” is an organic (having at least one carbon atom) or inorganic (having no carbon atoms) compound that has a molecular weight that is sufficiently low (typically ⁇ 900 Daltons) to allow the small molecule to rapidly diffuse across cell membranes so that they can reach intracellular sites of action.
  • the inhibitor is a small organic molecule.
  • Exemplary small organic molecules include, but are not limited to, pharmaceuticals (i.e. drugs, such as anti-cancer drugs), sugars, fatty acids, steroids, saccharides, purines, pyrimidines, derivatives, structural analogs, or combinations thereof .
  • An inhibitor of the HDM2 ubiquitin ligase includes any molecule capable of decreasing the activity of the ligase, for example by interfering with interaction of the ligase with another molecule, such as its substrate, e.g. p53.
  • One such inhibitor is the small organic molecule Nutlin or analogues thereof.
  • Analogues of Nutlin may be "Nutlin-like" molecules that have the same mode of action as Nutlin. Examples of Nutlin include but are not limited to Nutlin 1, Nutlin 2, and Nutlin 3.
  • the Nutlin 3 is Nutlin 3A.
  • Nutlin-like molecules include but are not limited to RG7112, MI-219, AM-8553 and BZD-17.
  • Other small organic molecules that may inhibit HDM2 ubiquitin ligase include but are not limited to MI-5, MI -17, I- 63, MI-219, MI-888, N, -dibenzylcinnamoyl amide, N,N- dibensylbenzamide , 1 , 4 -benzodiazepine- 2 , 5-dione (EZD) , WK298 and WK23.
  • the disclosed method may be used for determining the resistance of HDM2 (or a variant thereof) to inhibition of its interaction with its target molecule, p53, by the inhibitor Nutlin.
  • the disclosed method may be used for determining the resistance of steroid receptors (e.g. estrogen receptors) to inhibition of its interaction with its target proteins (e.g. p300, CBP, SP1 etc.) by an inhibitor.
  • the inhibitor may be a small molecule inhibitor, such as tamoxifen.
  • the inhibitor may be present at a concentration that is capable of inhibiting the interaction of a wild-type form of the biological molecule with the target molecule.
  • concentration of the inhibitor may be, for example, at least about 1 ⁇ , at least about 2 ⁇ , at least about 3 ⁇ , at least about 4 ⁇ , at least about 5 ⁇ , at least about 6 ⁇ , at least about 7 ⁇ , at least about 8 ⁇ , at least about 9 ⁇ , at least about 10 ⁇ , at least about 20 ⁇ , at least about 30 ⁇ , at least about 40 ⁇ , at least about 50 ⁇ , at least about 60 ⁇ , at least about 70 ⁇ , at least about 80 ⁇ , at least about 90 ⁇ , at least about 100 ⁇ , at least about 110 ⁇ , at least about 120 ⁇ , at least about 130 ⁇ , at least about 140 ⁇ , or at least about 150 ⁇ .
  • concentration will vary from one biological molecule- target molecule pair to another. For any given case, an appropriate concentration may be determined by one of ordinary skill
  • the co-compartmentalization of a gene encoding the biological molecule of interest with a gene encoding its target molecule and its inhibitor in step a) of the disclosed method may be carried out using methods known in the art for forming emulsions.
  • the aqueous phase and the oil phase are mixed in the presence of an emulsifying agent of the water-in- oil type.
  • Any conventional water- in-oil emulsifying agent can be used, such as mineral oil, hexadecyl sodium phthalate, sorbitan monooleate (e.g. Span 80), sorbitan monostearate , polysorbitan (e.g. Tween 80), cetyl or stearyl sodium phthalate, metal soaps, and the like.
  • step a) of the disclosed method comprises co-compartmentalizing the gene encoding the biological molecule and the target molecule into an aqueous droplet disposed within a water- in-oil emulsion.
  • the target molecule is in the form of an expressed product, i.e. a protein, and may be in purified form. In such an embodiment, it may not be necessary to include the gene encoding the target molecule in the aqueous droplet .
  • step a) of the disclosed method comprises co-compartmentalizing the gene encoding the biological molecule and the gene encoding the target molecule into an aqueous droplet disposed within a water- in-oil emulsion.
  • any complex between the biological molecule and the target molecule may be formed upon expression of the gene encoding the biological molecule and the gene encoding the target molecule.
  • step a) comprises co-compartmentalizing an inhibitor with the gene encoding the biological molecule, and the target molecule or the gene encoding the target molecule into an aqueous droplet disposed within a water- in-oil emulsion.
  • step b) comprises assaying for the complex comprising the biological molecule and the target molecule upon expression of the gene encoding the biological molecule and/or the gene encoding the target molecule after rupturing the aqueous droplet and contacting the contents thereof with the inhibitor.
  • step b) comprises assaying for the complex by "off-rate" selection. Methods for performing off-rate selection have been described, for example in Ylera F et al. (2013). Anal Biochem. 2013 Oct 15; 441 (2 ) : 208 - 13.
  • Step b) of the disclosed method may also comprise rupturing the aqueous droplets and contacting the contents thereof with a detectable label capable of binding to a complex comprising the expressed biological molecule of interest, its expressed target molecule, and the gene encoding the biological molecule, to assay for any such complex that may have been formed.
  • the "detectable label” may be a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or non-covalently joined to a polynucleotide or polypeptide.
  • Exemplary detectable labels include, but are not limited to, magnetic bead labels, antibodies, radioisotope labels, luminescent labels, fluorescent labels, enzyme labels, colloidal metal labels, colored glass bead labels, colored latex bead labels, carbon black labels, or combinations thereof.
  • the label may be a "direct" label that is coupled (i.e. physically linked) to a component of the complex to be detected, or an "indirect” label of the complex by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end- labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin .
  • the disclosed method further comprises step c) of amplifying the gene encoding the biological molecule in the complex that has been detected and detecting the amplified gene.
  • amplification refers to the production of additional copies of a nucleic acid. Amplification may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid amplification technologies well known in the art.
  • the disclosed method comprises identifying a mutation/ (s) in the gene encoding the biological molecule in the complex that has been detected.
  • Methods for identification of a mutation/ (s) present in a gene are well known in the art, and include for example, sequence analysis.
  • the disclosed method comprises analyzing in silico the interaction between the biological molecule and/or the target molecule and/or the inhibitor to determine the mechanism of resistance of the biological molecule to inhibition of its interaction with the target molecule by the inhibitor .
  • Steps a) , b) and c) of the disclosed method may be repeated at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least 10 times, more than 10 times, more than 15 times, more than 20 times , more than 25 times , more than 30 times , more than 35 times , more than 40 times , more than 45 times , more than 50 times , more than 55 times , more than 60 times , more than 55 times , more than 70 times , more . than 75 times , more than 80 times , more than 80 times , more than 90 times , more than 90 times, or more than 100 times.
  • the disclosed method is conducted in vitro.
  • conducting the disclosed method in vitro enables the application of stringent selection pressures, thus enabling identification of mutant/variant forms of the biological molecule with exceptionally strong phenotypes.
  • kits for use in a method as described above comprising means to co- compartmentalize the gene encoding the biological molecule with the target molecule, or the gene encoding the biological molecule with a gene encoding the target molecule, and optionally the inhibitor, into aqueous droplets disposed within a water-in-oil emulsion, and means to detect the complex comprising the biological molecule and the target molecule upon expression of the gene encoding the biological molecule and/or the gene encoding said target molecule.
  • the means for co- compartmentalizing the gene encoding the biological molecule with the gene encoding the target molecule, and optionally the inhibitor, into aqueous droplets may include buffers, emulsifying agents etc.
  • the means for detecting any complex that may have been formed between the biological molecule and the target molecule upon expression of the gene encoding the biological molecule and/or the gene encoding said target molecule include, for example, a detectable label as described above.
  • the kit comprises means to detect the gene encoding the biological molecule also present in the complex.
  • the reagents that are suitable for detecting the complex may include reagents that may incorporate a detectable label, such as a fluorophore, radioactive moiety, enzyme, biotin/avidin label, chromophore , chemiluminescent label, or the like, or the kits may include reagents for labeling the nucleic acids for detecting the presence or absence of the gene encoding the biological molecule as described herein.
  • the kit may further comprise reagents including, but are not limited to reagents for isolating peptides/proteins from samples, reagents for positive or negative controls and reagents for assays as described herein.
  • the kits may include reagents used in the Experimental section below.
  • the kit may further comprise instructions that may be provided in paper form or in computer- readable form, such as a disc, CD, DVD or the like.
  • the kits may optionally include quality control reagents, such as sensitivity panels, calibrators, and positive controls.
  • kits can optionally include other reagents required to conduct a diagnostic or prognostic assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co- factors, substrates, detection reagents, and the like.
  • Other components such as buffers and solutions for the isolation and/or treatment of a test sample (e.g. pretreatment reagents), may also be included in the kit.
  • the kit may additionally include one or more other controls.
  • One or more of the components of the kit may be lyophilized and the kit may further comprise reagents suitable for the reconstitution of the lyophilized components .
  • the various components of the kit optionally are provided in suitable containers.
  • the kit further can include .containers for holding or storing a sample (e.g. a container or cartridge for a blood or urine sample) .
  • a sample e.g. a container or cartridge for a blood or urine sample
  • the kit may also optionally contain reaction vessels, mixing vessels and other components that facilitate the preparation of reagents or the test sample.
  • the kit may also include one or more instruments for assisting with obtaining a test sample, such as a syringe, pipette, forceps, measured spoon, or the like.
  • mutated ubiquitin ligase polypeptides comprising at least one mutation.
  • the mutation may be selected from the group consisting of E69A, D225G, V241A, V280A, K344R, E390G, V426A, Q442R, M459T, Q24R, M62V, E124G, C461Y, T16A, P20L, L254F, N309T, G443D, H457R, L82P, and combinations thereof.
  • the mutated ubiquitin ligase polypeptide comprises the mutations E69A, D225G, V241A, V280A, K344R, E390G, V426A, Q442R and M459T.
  • the mutated HDM2 ubiquitin ligase polypeptide comprises the mutations Q24R, M62V, E124G and C461Y.
  • the mutated HD 2 ubiquitin ligase polypeptide comprises the mutations T16A, P20L, L254F, V280A, N309T, G443D and H457R.
  • the mutated HDM2 ubiquitin ligase polypeptide comprises the mutation L82P.
  • the disclosed method may be used to assess the receptiveness of a cancer patient to treatment with an anticancer drug.
  • a prognostic method for determining the receptiveness of a cancer patient to treatment with an anti-cancer drug capable of inhibiting the interaction of HDM2 ubiquitin ligase with p53 tumor suppressor protein comprising the step of:
  • the anti-cancer drug is a Nutlin or analogues thereof (e.g. Nutlin-like molecules) .
  • the Nutlin may be selected from the group consisting of Nutlin 1, Nutlin 2 and Nutlin 3.
  • the Nutlin 3 is Nutlin 3A.
  • Nutlin- like molecules may be selected from the group consisting of RG7112, MI-219, AM-8553 and BZD-17.
  • the Nutlin analogue is RG7112.
  • the anti -cancer drug is a stapled peptide.
  • the stapled peptide targets the same interaction and/or the same site on the HD 2 as Nutlin.
  • the sample used in the disclosed prognostic method may be a biological sample such as tissues, cells, whole blood, blood fluids (e.g. serum and plasma), lymph and cystic fluids, sputum, stool, tears, mucus, hair, skin, ascitic fluid, cystic fluid, urine, nipple exudates, nipple aspirates, sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, archival samples, and explants, primary and transformed cell cultures derived from patient tissues.
  • blood fluids e.g. serum and plasma
  • the sample may be untreated, treated, diluted or concentrated from a patient, and may comprise an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; etc.
  • the cancer of the patient who may be subjected to the disclosed prognostic method includes but is not limited to retinoblastoma, blood malignancies (e.g. leukaemia), biliary tract cancer; brain cancer; breast cancer; cervical cancer ; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; intraepithelial neoplasms; lymphomas; liver cancer; lung cancer (e.g.
  • the cancer is leukaemia or melanoma.
  • the disclosed method may also be used to select for a variant form of a biological molecule of interest that is resistant to inhibition of its interaction with a target molecule by an inhibitor of the biological molecule.
  • a method for selecting a variant form of a biological molecule that is resistant to inhibition of its interaction with a target molecule by an inhibitor of the biological molecule comprising the steps of providing a plurality of randomly mutated genes encoding the biological molecule, and determining resistance of the biological molecule to inhibition of its interaction with the target molecule by the inhibitor . using the method as described above.
  • the plurality of randomly mutated genes encoding the biological molecule may be generated using methods known in the art, for example by passing cloned genes through mutator strains, by "error-prone" PCR mutagenesis, by rolling circle error-prone PCR, or by saturation mutagenesis .
  • the plurality of randomly mutated genes encoding the biological molecule may comprise at least 10 4 randomly mutated genes encoding the biological molecule, at least 10 5 randomly mutated genes encoding the biological molecule, at least 10 6 randomly mutated genes encoding the biological molecule, at least 10 7 randomly mutated genes encoding the biological molecule, at least 10 s randomly mutated genes encoding the biological molecule, at least 10 9 randomly mutated genes encoding the biological molecule, at least 10 10 randomly mutated genes encoding the biological molecule, at least 10 11 randomly mutated genes encoding the biological molecule, at least 10 12 randomly mutated genes encoding the biological molecule, at least 10 13 randomly mutated genes encoding the biological molecule, at least 10 randomly mutated genes encoding the biological molecule, or at least 10 15 randomly mutated genes encoding the biological molecule.
  • the plurality of randomly mutated genes encoding the biological molecule comprises at least 10 7 randomly mutated genes encoding the biological molecule, at least 10 s randomly mutated genes encoding the biological molecule, at least 10 9 randomly mutated genes encoding the biological molecule, or at least 10 10 randomly mutated genes encoding the biological molecule.
  • kits for use in the method of selecting a variant form as described above comprising means for generating the plurality of randomly mutated genes encoding the biological molecule.
  • the kit may further comprise means for co-compartmentalizing a randomly mutated gene encoding the biological molecule, a gene encoding a target molecule of the biological molecule, and an inhibitor of the interaction of the biological molecule with the target molecule into an aqueous droplet disposed within a water-in-oil emulsion as described above, as well as means for detecting a complex comprising the biological molecule and the target molecule upon expression of the randomly mutated gene encoding the biological molecule and the gene encoding the target molecule as described above.
  • Step i) may comprise determining the mechanism of resistance of the biological molecule to inhibition of its interaction with its target molecule by the drug.
  • the method may also comprise analyzing the structure of the biological molecule determined to be resistant to inhibition of its interaction with the target molecule by the inhibitor.
  • the structural analysis may be carried out using methods well known in the art, such as nuclear magnetic resonance (NMR) and X-ray crystallography. Determination of the mechanism of resistance and analysis of the structure of the biological molecule having the resistant phenotype provides information to facilitate iterative refinement of the drug so as to improve their functionality and/or restore their inhibitory effect on the biological molecule.
  • the information may also be. useful for assessing alternative therapeutic candidates for overcoming or circumventing the drug resistance, and for predicting future resistant phenotypes that may arise in a clinical setting.
  • the modification of the drug in step b) may comprise modifying the drug to overcome the mechanism of resistance such that the inhibitory activity of the drug on the interaction of the biological molecule with its target molecule can be restored.
  • Fig. 1 shows a schematic depicting a complex formed between HA- tagged HDM2 , p53 and DNA captured on beads coated with anti-HA antibody. Arrows represent PCR primers for quantifying the captured DNA or amplifying HDM2 genes during selection.
  • C shows a Western blot of p53 captured by immobilised HDM2 and the effect of Nutlin (at a concentration of 10 ⁇ ) .
  • Fig. 2 shows a schematic depicting the selection of Nutlin- resistant HDM2 by in vitro compartmentalization (I C) .
  • HDM2 expression constructs appended with 2CONA p53 response element ("RE") and HA- tag coding sequence ("HA") and p53 expression construct- - (“p53”) are segregated into aqueous emulsion compartments along with Nutlin ("N” orbs) . Protein expression occurs within the compartments .
  • Nutlin inhibition of HDM2 results in no HDM2-p53-DNA complex formation (left bubble) , whereas resistant HD 2 can form the complex (right bubble) .
  • the emulsion is broken and complexes captured with anti-HA antibody.
  • DNA encoding resistant HDM2 variants is amplified by PCR. 4. Selectants are further evaluated by secondary pulldown assay or subjected to further rounds of selection.
  • Fig. 3 depicts the in vitro pulldown assay showing reduced inhibition by Nutlin (10 ⁇ ) to binding of p53 for indicated parental HDM2 variants.
  • Fig . 4 depicts the results of inhibition of selected variants in p53-null H1299 cells.
  • A shows the results on H1299 cells co- transfected with either p53 alone or p53 and the indicated HDM2 variants.
  • p53 function was measured by reporter gene activity in presence of Nutlin (at concentrations of 0, 2, or 5 ⁇ ) . The values represent mean +/- SD .
  • B As in A, with Nutlin- induced increases plotted relative to the base line value of inhibition in the absence of drug treatment (set to 1) . The values represent mean +/- SD .
  • C Western blot showing expression levels of HDM2 variants and p53 in H1299 cells.
  • Fig . 5 shows the results of inhibition of selected HDM2 variants in p53/HDM2-null DKO cells.
  • A Wild-type and indicated N-terminal domain mutants were co- transfected with p53 and p53 reporter gene activity was measured in the presence of Nutlin (at concentrations of 0, 2, 5, or 10 ⁇ ) . Representative data from one experiment are shown.
  • B Wild- type and indicated acidic (V280A) , zinc finger (C308Y, N309T, C322R) and RING (G443D) domain mutants were co- transfected with p53 and p53 reporter gene activity was measured in the presence of Nutlin (at concentrations of 0, 2, 5, or 10 ⁇ ) . Representative data from one experiment are shown.
  • C Representative Western Blots showing expression levels of HDM2 variants and p53 DKO cells.
  • Fig . 6 depicts a bar graph showing the results of Fluorescent 2-Hybrid (F2H) assay of p53 with wild-type (WT) HDM22 , mutant Q24R and M62A.
  • the F2H assay investigates the interaction of p53 (bait) with wild-type (WT) HDM2 , mutant Q24R and M62A (preys) .
  • the F2H assay measures the interaction between two proteins as ratio of cells showing co- localization of bait and prey at the nuclear F2H interaction platform, to cells not showing this co- localization .
  • Fig. 7 shows images of the Q24R mutation in the HDM2 lid region.
  • the Q24R mutation in the HDM2 lid region was predicted to enhance affinity for p53 but not Nutlin.
  • A Molecular simulations indicate mutation of Q24 to arginine (right structure) leads to repulsion of proximal K51 and stabilization of E28 in p53 (circled, right structure) through charge-charge interaction. This additional stabilization is not seen in wild- type HDM2 bound to p53 (circled, left structure) .
  • Q24 is seen to make no significant contact with Nutlin (left structure) and mutation to arginine (right structure) did not result in any additional differences.
  • Fig. 8 depicts images of the packing interactions between the HDM2 p53 -binding domain and Nutlin.
  • the M62A mutant in the HDM2 p53 -binding domain selectively resulted in loss of Nutlin binding.
  • Fig. 9 shows that the mutation V280A in acidic domain resulted in reduced interaction with p53 DNA binding domain.
  • A shows models of the native (left) and V280A mutant (right) peptides docked to the p53 DNA binding domain. Residues set as "active" in the Haddock server run (details in Materials and Methods) are shown as sticks (red and yellow for p53 and blue for peptide) . Residues of p53 known to make direct contact with DNA are colored in yellow. V280 and A280 are shown in green. In the native case, V280 appears to make more contacts with p53 than A280 in the mutated peptide.
  • Fig. 10 depicts a table indicating the mutations present in HDM2 selectants displaying in vitro Nutlin resistance.
  • the upper schematic shows the domain architechture of the HDM2 gene.
  • Fig. 11 shows the results of the pulldown assay investigating the resistance of the HDM2 variants.
  • Nutlin (10 ⁇ ) no significant increase in p53 binding was observed for mutant HD 2 compared to wild-type.
  • Fig. 12 shows a graph depicting the results of inhibition of HDM2 variants in p53/HDM2-null DKO cells.
  • DKO cells were co- transfected with either p53 alone or p53 and the indicated HDM2 variants.
  • p53 function was measured by reporter gene activity in the presence of the indicated amounts of Nutlin. Activity was expressed as percentage of reporter gene transact ivation seen with wild-type HDM2 (set to 100, indicated by dotted line) .
  • the values represent mean + SD from two to three independent experiments, *p ⁇ 0.05, **p ⁇ 0.05, ***p ⁇ 0.005.
  • Fig. 13 depicts time lapse images which indicate persistence of mutant HDM2-p53 complex in presence of Nutlin using F2H assay.
  • Green dot shows p53 bound to DNA.
  • Co-localised red dot shows HDM2 in complex with p53. Arrows indicate last time point where HDM2 (wild-type or indicated mutant) was present in complex. Time is indicated in minutes.
  • Fig. 14 depicts the distribution of energies of interactions (enthalpies) of p53 (top) and Nutlin (bottom) with wild-type and the mutants Q24R and M62A.
  • the enthalpies were computed using standard protocols as outlined in [16] .
  • Fig. 15 shows that Nutlin-resistant HDM2 variants are inhibited by stapled peptides.
  • A In vitro pull-down assay shows that Nutlin (10 ⁇ ) inhibits p53-H D2 interaction but was less effective for the Q24R and M62A HD 2 variants (top row) . Stapled peptides PM2 and 011 (10 ⁇ ) showed inhibition of p53 binding to both T and mutant HD 2 (second row) . 10% of respective HDM2 inputs loaded. PM2CO was used as negative control stapled peptide.
  • B Sequences of stapled peptides PM2 and M011. The residues at positions 3, 7, 9 of PM2 were mutated to alanine in PM2C0N. "X" denotes staple tethering sites. A chlorine atom is added to the C6 position of W7 in Oll.
  • Fig. 16 shows that stapled peptides inhibit wild- type and mutant HDM2 function in p53/MD 2 -null DKO cells.
  • Fig. 19 shows that the direct interaction between p53 and HDM2 N- terminal domain was inhibited by stapled peptides.
  • F2H assay was carried out to investigate the interaction of p53 (bait) with wild-type HDM2 (WT) , mutant Q24R and M62A (preys) .
  • the F2H assay measures the interaction between two proteins as ratio of cells showing co- localization of bait and prey at the nuclear F2H interaction platform, to cells not showing this co- localization.
  • Fig. 20 shows that the M62A mutation in HDM2 does not perturb binding of the stapled peptide PM2.
  • Left Simulations (see Materials and Methods) indicate packing interactions between M62 (yellow) and hydrophobic staple (cyan) of PM2.
  • the peptidic component of PM2 is depicted in magenta.
  • Fig. 21 depicts molecular simulations showing the negative impact of the P20L and Q24R mutations on the docking of Nutlin to the HDM2 N-terminal domain.
  • A Space- filling (left) and ribbon (right) depictions of Nutlin binding the main (arrowed) and secondary (circled) binding sites of HDM2. The p53 -peptide (cyan) binding to the main site is overlaid.
  • B Space- filling model of the HDM2 N-terminal domain showing migration of the lid region when P20 (left) was mutated to leucine (right, L20 sphere) . The arrow depicts the main Nutlin binding site.
  • C Mutation of Q24 (left) to arginine (right) resulted in extensive hydrogen bond network with E23 and Y100 and likely occlusion of the secondary Nutlin binding site.
  • Fig. 23 shows a table on the energetic contribution to the differences in the binding free energies (AG B inding) between wild type HDM2 and alanine mutant variants for binding to the indicated ligands. Residues contributing >2 kcal/mol are italicized and underlined, and are further depicted in Venn diagram below the table.
  • Fig. 24 shows the expression levels of HA-tagged wild-type (WT) and indicated HDM2 mutants transfected into HCT116 p53 +/+ cells and treated with either Nutlin or stapled peptides PM2CON and PM2 as indicated.
  • WT HA-tagged wild-type
  • indicated HDM2 mutants transfected into HCT116 p53 +/+ cells and treated with either Nutlin or stapled peptides PM2CON and PM2 as indicated.
  • Fig. 25 shows the energetic, contribution (kcal/mol) of indicated HDM2 residues to binding of p53 peptide, Nutlin, and stapled peptide PM2 as determined by computational alanine scanning (see Materials and Methods) .
  • Non- limiting examples of the invention, including the best mode, and a comparative example will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention .
  • the p53 tumor suppressor functions as a master regulator of cell fate [1,2] and is commonly mutated in cancer [3,4,5,6] . Its pro-apoptotic activity is negatively regulated by HDM2 , the ubiquitin- ligase that binds to p53 and targets it for proteosomal degradation [7,8,9,10] . Approximately 50% of cancers harbor wild-type p53, and elevation of p53 levels in these cancers by targeted disruption of the HDM2-p53 complex represents an attractive therapeutic modality [89,38] .
  • Nutlin-3A The small molecule Nutlin-3A (hereinafter referred to as "Nutlin"), presently in clinical trials, competitively binds to HDM2 , a key negative regulator of p53 and blocks its activity. Nutlin binds to the p53-binding pocket in the N-terminal domain of HD 2 by mimicking core interactions of residues in the p53 transactivation domain that interact with the pocket [93] . It is presently in advanced preclinical development and clinical trials for the treatment of retinoblastoma and blood malignancies with wild- type p53 status [94,95] .
  • HDM2 has been previously identified in some tumour samples [11,22] . Furthermore, HDM2 gene amplification and overproduction in cancer [13,14], and correlation with poor response to therapy [15] , suggest that HDM2 mutation could render cells recalcitrant to Nutlin therapy. To investigate this possibility in a targeted manner, it would therefore be desirable to interrogate large numbers of mutated HDM2 variants for a Nutlin- resistance phenotype, wherein the interaction with p53 is not attenuated by the drug [16] .
  • oligonucleotides used in this work were from 1st Base (Singapore) , restriction enzymes from ' NEB and chemical reagents from Sigma. Nutlin-3A was from Calbiochem .
  • Hdm2-Ndel 5' - CACAACATATGTGCAATACCAACATGTCTGTACC- 3' (SEQ ID NO: 1
  • INF-Hdm2 - cmvF 5' -CGAACCTAAAAACAAATGTGCAATACCAACATGTCTGTAC
  • Error-prone PCR [54] was carried out on HDM2 - PET22b using primers petF2 and petR and mutant genes re-amplified with Hdm2- Ndel and Hdm2-HA-BamHl .
  • the library was ligated into 2ConA- PET22b via Ndel/BamHl sites and re-amplified with petF2 and petR to make library amplicons with T7 promoter and ribosome binding site required for in vitro transcription- translation (IVT) , as well as the 2ConA RE site located before the T7 promoter site.
  • IVTT in vitro transcription- translation
  • 2ConA-HD 2-PET22b, HD 2 - PET22b and p53-PET22b were also amplified with petF2 and petR for IVT of wild-type HDM2 and p53.
  • Nutlin-resistant parental clones obtained from the selection were amplified with petF2 and petR to create amplicons for secondary assays.
  • Three parental clones (5-3, 5-9 and 5-14) were also amplified with INF-Hdm2 - cmvF and INF-HA-cmvRcor for cloning by infusion (Clontech) into the pCMV vector expression in cells.
  • Single mutant HDM2 clones were generated by Quickchange mutagenesis (Stratagene) of 2ConA-HDM2 - PET22b using primers mdm2-T16A-QCl and mdm2 -T16A-QC2 , mdm2-P20L-QCl and mdm2-P20L- QC2, mdm2-Q24R-QCl and mdm2 -Q24R-QC2 , HDM 62A- 1 and HDMM62A-2, mdm2-M62V-QCl and mdm2 -M62V-QC2 , mdm2 -V280A-QC1 and mdm2-V280A- QC2, mdm2-G443D-QCl and mdm2 -G443D-QC2 to create 2ConA-HDM2- T16A-pET22b, 2ConA-HDM2 - P20L-pET22b , 2ConA-HDM
  • IVC reactions consisting of 0.5 ⁇ ZnCl 2 , 8ng p53 (1.6ng and 0.8ng in subsequent rounds), 5ng library amplicons (lng and 0.5ng in subsequent rounds) in a total volume of 50uL PURExpress ® in vitro protein synthesis solution (New England Biolabs) were assembled on ice and emulsified in 450 ⁇ i in vitro compartmentalization (IVC) oil comprising 95% (v/v) mineral oil, 4.5% (v/v) Span-80 and 0.5% (v/v) Tween-80 as previously described [17] . After incubation at 37°C, the reactions were centrifuged at 8000rpm for 10 mins to separate the aqueous and oil phase.
  • IVC in vitro compartmentalization
  • TNTB buffer 0.1M Tris pH 7.4, 0.15M NaCl, 0.05% Tween-20, 0.5% BSA
  • 50uL TNTB buffer 0.1M Tris pH 7.4, 0.15M NaCl, 0.05% Tween-20, 0.5% BSA
  • the compartments were disrupted by six rounds of hexane extraction and the aqueous phase incubated with anti-HA antibody-coated protein G beads (Invitrogen) at 4°C with rotation.
  • the beads were washed thrice with PBST-0.1%BSA, and thrice with PBST.
  • the beads were resuspended in 20 ⁇ 1 water and the protein-protein-DNA complexes eluted by incubation at 95°C for 5 mins .
  • the eluates were amplified with Hdm2-Ndel and Hdm2 - HA-BamHl and products cloned back into 2ConA-PET22b via Ndel/BamHl sites and re-amplified with petF2 and petR for the next round of selection.
  • Protein G beads were incubated with anti-HA (l g per lOpL beads) for 1 hour in PBST-3%BSA and subsequently washed twice in PBST-0.1%BSA. IVT-expressed protein was incubated with the beads on a rotator for 30 mins. Nut1in was added at required concentrations and incubation carried out for 30 mins. IVT- containing secondary protein was added to the mixture and incubation allowed for 1 hour. Beads were finally washed thrice in PBST-0.1%BSA and thrice with PBS, and bound proteins eluted by resuspension in 20 ⁇ 1, SDS-PAGE loading buffer and incubation at 95°C for 5 minutes.
  • blank IVT extract (no template DNA added) was used as control.
  • the eluates were subjected to electrophoresis, transferred to nitrocellulose membranes and probed for p53 with horseradish peroxidise conjugated D01 antibody (Santa Cruz) or for HDM2 with anti-HA antibody followed by rabbit anti-mouse (Dakocytomation) .
  • Protein G beads were incubated with anti-HA (lyg per 5yL beads) for 1 hour in PBST-3.%BSA and subsequently washed twice in PBST-0.1%BSA.
  • IVT-expressed HDM2 (with either HDM2 or HDM2 2ConA as template DNA) was incubated with the beads on a rotator for 30 mins. Nutlin was added at required concentrations and incubation carried out for 1 hour. IVT-expressed p53 was added to the mixture and incubation allowed for 1 hour.
  • DKO (p53/HDM2 null) cells were maintained in Dulbecco' s modified -Eagle' s medium (D E ) with 10% foetal calf serum (FCS) and 1% penicillin/streptomycin. The cells were seeded at 1.0 x 10 5 cells/well in 6-well plates, 24 hours prior to transfection . Cells were co-transfected with parental or individual Nutlin- resistant HDM2 plasmid, p53-pcDNA plasmid, LacZ reporter plasmid and luciferase transfection efficiency plasmid using TurboFect transfection reagent (Thermo Scientific) according to the manufacturer's instructions.
  • DKO cells were harvested 24 hours after transfection and ⁇ - galactosidase activities were assessed using the Dual-light System (Applied Biosystems) according to the manufacturer's protocol.
  • the ⁇ -galactosidase activity was normalized with luciferase activity for each sample.
  • the Fluorescent 2-Hybrid (F2H) assay is an intracellular, direct, fully reversible protein-protein interaction assay.
  • This microscopy-assisted assay consists of two components, a bait and a prey protein.
  • the bait is a fusion of p53 (amino acid 1- 81) with a lac repressor binding domain (Lacl) and GFP.
  • the prey is a fusion of FP with either N-terminal domain of wild-type HDM2, mutants Q24R or M62A (amino acids 7-134) .
  • These two plasmids are co-transfected in a specific transgenic BHK cell line containing an array of lac operator repeats, the F2H interaction platform.
  • the bait protein is then captured at this interaction platform and forms a bright green spot in the cell nucleus [27] .
  • the prey protein co-localizes to the same nuclear spot.
  • Compounds, which disrupt the protein- protein interaction here Nutlin
  • the declined percentage of co-localization is measured using imaging techniques .
  • BHK cells were co-transfected with the bait p53 and different prey HD 2 plasmids overnight in 96 multiwell plates (yClear Greiner Bio-One, Germany) using the Lipofectamine 2000 (Life Technologies) reverse transfection protocol according to the manufacturer's instructions with 0.2 g DNA * and 0.4 ⁇ Lipofectamine 2000 per well. Cells were incubated with a dilution series of 50 ⁇ , 10 ⁇ , 2 ⁇ , 1 ⁇ , 0.5 ⁇ , 0.25 ⁇ and 0.13 ⁇ Nutlin for 1 hour at 37°C, 5% C0 2 .
  • Interaction was determined as the ratio of cells showing co-localization of fluorescent signals at the nuclear spot to the total number of evaluated cells.
  • an INCell Analyzer 1000 with a 20X objective (GE Healthcare) was used. Automated image segmentation and analysis was performed with the corresponding INCell Workstation 3.6 software. At least 100 co- transfected cells were analyzed per well. Titrations were carried out independently three to five times .
  • the acidic domain of MDM2 is known to be unstructured [37] , and hence molecular dynamics in implicit solvent (AMBER molecular modeling package) [55] was used to model a 2 -residue peptide in the acidic domain (residues 259-282 of MDM2) starting from an extended chain.
  • AMBER molecular modeling package molecular modeling package
  • HADDOCK HADDOCK
  • Fig. 1A DNA template encoding HDM2 did not have the 2CONA RE appended.
  • Fig. IB shows that DNA is only captured on the beads when the 2CONA RE is present, indicating the formation of an HDM2-p53-DNA complex.
  • addition of Nutlin resulted in a clear dose-dependent reduction in the amount of 2CONA- appended DNA pulled down (-383 fold reduction at 100 ⁇ ) , indicating disruption of the p53-HDM2 interaction in vitro. Disruption was also observed in a pull -down assay measuring p53 bound to immobilised HDM2 by Western blot (Fig. 1C) .
  • a library of randomly mutated HDM2 genes was created and a selection for Nutlin-resistance by IVC was carried out.
  • Fig. 2 depicts the selection protocol, wherein variant HDM2 expression constructs tagged with the CONA RE, along with p53 expression construct and Nutlin are dispersed into the aqueous compartments of the water- in-oil emulsion. Within each compartment protein expression occurs, and in the presence of Nutlin, the HDM2-p53-DNA complex is not expected to form if Nutlin binds HDM2 (left bubble) , but will form if the variant HDM2 is resistant to Nutlin inhibition (right bubble) .
  • the emulsion is broken and complexes are captured using anti-HA coated magnetic beads.
  • the genes encoding Nutlin-resistant HDM2 variants are then amplified by PCR prior to further rounds of selection and/or secondary characterisation.
  • the parental selectants and the single HDM2 mutants Q24R and M62V were next analysed in a functional assay measuring p53 activity in the H1299 cell line (Fig. 4A) .
  • Plasmids encoding HDM2 (wild-type or selectants) and p53 were transfected along with a p53 transactivation reporter construct.
  • Nutlin inhibition by HDM2 was attenuated, with p53 activity being restored up to 76% of that observed in the absence of HDM2 co- transfection (5 ⁇ Nutlin) .
  • HDM2 Q24R showed an appreciable Nutlin-resistant phenotype, with p53 activity only being restored to 47% (5 ⁇ Nutlin) .
  • the N309T mutant in the zinc finger domain showed slight resistance (-88% activity of wild-type). However, its proximity to C308, shown to be mutated in non-Nutlin treated cancer [12] led us to test the clinically observed C308Y mutant and this showed moderate resistance (-70% activity of wild-type) .
  • the C322R mutation, also in the zinc finger domain also showed moderate resistance (-73% activity of wild-type) .
  • the G443D mutant in the RING domain showed slight resistance (-85% restoration) .
  • the direct cellular binding of the HDM2 wild- type, Q24R and M62A N-terminal domains to p53 were further characterized in the Fluorescent 2-Hybrid (F2H) assay [27] .
  • the F2H assay differs from the DKO reporter assay in that it does not measure reactivation of a reporter gene but the precise interaction to be disrupted.
  • the assay visualizes the interaction of RFP-tagged HDM2 (amino acids 7-134) with GFP-tagged p53 (amino acids 1-81) at a defined nuclear F2H interaction platform, in specific BHK cells.
  • the addition of Nutlin results in a dissociation of the complex, which can be imaged and quantified.
  • time- lapse analysis indicated enhanced persistence of mutant HDM2-p53 complexes compared to wild-type after Nutlin challenge (1 ⁇ ) .
  • the wild- type complex was not visible after 20 minutes, whilst the Q24R complex lasted for 40 minutes.
  • the M62A complex was still visible after one hour (Fig. 13).
  • IVC was used to select for Nutlin-resistant variants from a large repertoire ( ⁇ 10 9 ) of randomly mutated HDM2 genes. It is desirable to increase selection pressure during rounds of directed evolution. However, the present study was restricted to some extent by the low solubility limit of Nutlin [28] and its strong hydrophobicity, which most likely led to much of it partitioning into the oil phase of the emulsion. Despite this, enough selection pressure was applied to yield several clones harbouring multiple mutations which showed the desired phenotype . As acquired drug resistance can arise through point mutation [29,30,31], the mutations were analysed in isolation, and several of these displayed the Nutlin-resistant phenotype both in vitro and ex vivo.
  • HDM2-5.3 showed appreciable binding to p53 in the presence of Nutlin in the in vitro pull-down assay, but displayed a mild phenotype in the ex vivo functional assay.
  • the first assay measures binding of HDM2 to p53, whilst the second is the aggregate readout for inhibition of p53 activity arising from the binding, inhibition of transactivation, .and E3 ligase activites of HDM2.
  • mutations ancillary - to V280A in selectant 5.3 (which confers Nutlin resistance in isolation) likely impact negatively on the latter two activities in the ex vivo assay.
  • the V280A mutation is also present in selectant 5.14 which shows essentially the same phenotype in both assays, indicating context-dependency. Overall, the assumption that in vitro binding can be used as a proxy to measure HDM2 function in the cell-based assay is validated through selection of HDM2 variants 5.9 and 5.14 which behave similarly in both assays.
  • Residues 16-24 in the p53 binding domain of apo HDM2 comprise a flexible lid region shown to behave as a weak pseudo- substrate in the absence of p53 binding [32,33] .
  • NMR studies indicate that whilst the lid predominantly adopts the "open” conformation when p53 is bound, Nutlin-binding is compatible with both the “open” and “closed” lid-binding states [34] .
  • the mutations T16A, P20L and Q24R may further weaken this intra-molecular interaction to selectively increase the interaction with p53.
  • biochemical studies have shown the phosphomimetic mutation S17D in the lid to stabilize the HDM2-p53 interaction [35,36] .
  • M62V mutation was of particular interest as it was previously shown that M62A confers Nutlin resistance in vitro [26] .
  • the amino acid M62 is an essential part of the subpocket accommodating F19 of p53 in the HDM2-p53 interaction
  • the central acidic domain of HDM2 contains a secondary binding site that interacts with the p53 DNA binding domain
  • the present data indicates that resistance to Nutlin can arise through several mechanisms.
  • these are predicted to either selectively reduce affinity for Nutlin (M62A, M62V, L82P) , increase affinity for p53 (P20L, Q24R) , or influence lid dynamics (T16A, P20L, Q24R) .
  • These effects can possibly be overcome by designing small molecule derivatives capable of forming additional contacts with the HDM2 binding pocket.
  • Recently described examples include a series of piperidinones , which in addition to the three core p53-mimetic interactions, form additional Van der Waals, pie- stacking and electrostatic interactions with HDM2 [46] .
  • stapled-peptide derivatives of the p53 motif that interact with HD 2 may prove more recalcitrant to mutation by virtue of the increased interaction footprint [47,48] .
  • the absence of structural data for full-length HDM2 makes it difficult to understand probable allosteric effects of mutations outside the N- terminal domain that impact on Nutlin binding (V280A, C308Y, N309T, C322R, G443D).
  • C-terminal RING domain mutants have been described which increase the affinity of the HD 2-p53 interaction [40] . Therefore, as with N-terminal domain mutations that increase p53 -binding, the use of small molecules and stapled peptides with increased binding footprints may offset allosterically induced structural variation and compete more efficiently with p53 for binding.
  • IVC readily enables interrogation of up to 10 10 variants [53] and generally allows for application of stringent selection pressures (although in this particular case the physicochemical properties of Nutlin were limiting) .
  • stringent selection pressures although in this particular case the physicochemical properties of Nutlin were limiting.
  • IVC may not be suitable where the function of target proteins requires post- translational modification, and for certain targets it may not be trivial to devise a selection strategy.
  • a robust approach to modeling drug resistance could entail primary use of IVC to sample a large pool of diversity for mutation hotspots. Smaller, focused libraries covering these regions could then be generated, and these further analysed in cell-based complementation assays.
  • Stapled peptides are a relatively new class of macrocyclic compounds with promising drug- like properties [60] .
  • the introduction of a covalent linkage bridging adjacent turns of an alpha helical peptide (the "staple") can pre-stabilize the conformer(s) preferentially adopted when it binds a target protein.
  • Stapling increases affinity by reducing the entropic cost of binding, imparts proteolytic stability / increased in vivo half-life, and in certain cases permits adjunct-free cellular uptake [61-63] .
  • Stapled peptide analogues of Nutlin that target the N- terminal domain of HDM2 have been described
  • oligonucleotides used in this work were from 1st Base (Singapore) , restriction enzymes from NEB and chemical reagents from Sigma. Nutlin-3A was from Calbiochem. The stapled peptides PM2 , P 2CON and M011 (>90% purity) were from AnaSpec (USA) .
  • HDM2 - P20L-QC1 5 ' - CCACCTCACAGATTCTAGCTTCGGAACAAGA- 3 ' (SEQ ID NO: 1) HDM2 - P20L-QC1 : 5 ' - CCACCTCACAGATTCTAGCTTCGGAACAAGA- 3 ' (SEQ ID NO: 1) HDM2 - P20L-QC1 : 5 ' - CCACCTCACAGATTCTAGCTTCGGAACAAGA- 3 ' (SEQ ID
  • HDM2 - P20L-QC2 5 ' -TCTTGTTCCGAAGCTAGAATCTGTGAGGTGG- 3 ' (SEQ ID NO: 1)
  • HDM2 -Q24R-QC1 5' -TTCCAGCTTCGGAACGAGAGACCCTGGTTAG- 3' (SEQ ID NO: 3) HDM2 -Q24R-QC1 : 5' -TTCCAGCTTCGGAACGAGAGACCCTGGTTAG- 3' (SEQ ID NO: 3) HDM2 -Q24R-QC1 : 5' -TTCCAGCTTCGGAACGAGAGACCCTGGTTAG- 3' (SEQ ID
  • HDM2 -Q24R-QC2 5' -CTAACCAGGGTCTCTCGTTCCGAAGCTGGAA- 3' (SEQ ID NO: 4)
  • HDM2-M62A-1 5' -CTTGGCCAGTATATTGCGACTAAACGATTATATG- 3'
  • HDM2-M62A-2 5' -CATATAATCGTTTAGTCGCAATATACTGGCCAAG- 3' (SEQ ID NO: 6)
  • petF2 5 ' -CATCGGTGATGTCGGCGAT- 3 ' (SEQ ID NO: 3)
  • petR 5 ' -CGGATATAGTTCCTCCTTTCAGCA- 3'(SEQ ID NO: 4)
  • h_p53_forward 5' -CCCCTCCTGGCCCCTGTCATCTTC-3' (SEQ ID NO:
  • h_b-actin_forward 5' -TCACCCACACTGTGCCCATCTACGA- 3'
  • Single mutant HDM2 clones were generated by Quickchange mutagenesis (Stratagene) of parental HDM2 -PET22b using appropriate primers 1-6.
  • the constructs were amplified with primers petF2 and petR to make HDM2 amplicons with T7 promoter and ribosome binding site required for in vitro transcription- translation (IVT) of wild-type or mutant HDM2.
  • Primers 1-6 were used to introduce mutations into the parental pCMV-HDM2 mammalian expression construct by Quickchange mutagenesis.
  • Both the HDM2 - PET22b and pCMV-HDM2 constructs additionally encode a C-terminal HA tag.
  • the plasmid p53-PET22b was also amplified with petF2 and petR to make template for IVT of wild-type p53.
  • Protein G beads (Invitrogen) were incubated with anti-HA (1ig per 10 yL beads) for 1 hour in PBST-3%BSA and subsequently washed twice in PBST- 0.1%BSA.
  • IVT expressed wild- type or mutant HDM2 was incubated with the beads on a rotator for 30 mins .
  • Nutlin or stapled peptides were added at required concentrations and incubation carried out for 30 mins.
  • IVT-expressed p53 was added to the mixture and incubation allowed for 1 hour.
  • DKO Mouse embryonic fibroblast p53/ dm2 double -knockout (DKO) cells (a kind gift from Guillermina Lozano) [68] and H1299 p53 _/ ⁇ cells [69] were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) foetal calf serum (FCS) and 1% (v/v) penicillin/streptomycin . The cells were seeded at 1.0 x 10 s cells/well in 6-well plates, 24 hours prior to transfection .
  • DMEM Dulbecco's modified Eagle's medium
  • FCS foetal calf serum
  • penicillin/streptomycin penicillin/streptomycin
  • HCT116 p53 + + cells [70] were maintained in McCoy' s 5A medium with 10% (v/v) foetal calf serum (FCS) and 1% (v/v) penicillin/streptomycin. The cells were seeded at 3.5 x 10 s cells/well in 6-well plates, 24 hours prior to transfection.
  • DKO cells were harvested 24 hours after transfection and ⁇ - galactosidase activities were assessed using the Dual-light System (Applied Biosystems) according to the manufacturer's protocol.
  • the ⁇ -galactosidase activity was normalized with luciferase activity for each sample.
  • 5 pg of the cell lysates were probed for p53 with horseradish peroxidise conjugated DOl antibody, for HDM2 and actin with anti-HA antibody and AC15 antibody respectively followed by rabbit anti- mouse .
  • HDM2 DNA encoding HDM2 (amino acids 1-125) was ligated into the GST fusion expression vector pGEX- 6P-1 (GE Lifesciences) via BamHl and Ndel double digest. Mutants of HDM2 (P20L, Q24R and M62A) were made using the QuickChange site-directed mutagenesis kit (Strategene) and appropriate primers 1-6. BL21 DE3 competent bacteria were then transformed with the GST tagged HDM2 (1-125) constructs. Cells harbouring the GST fusion constructs were grown in LB medium at 37°C to an OD600 of -0.6 and induction was carried out with 1 mM IPTG at room temperature.
  • RNA quanti f ication Total RNA was prepared from appropriately treated HCT116 p53 +/+ cells using the R easy Mini Kit (QIAGEN) .
  • Reverse transcription was performed using SuperscriptTM First-Strand Synthesis System (Invitrogen) with random hexamers.
  • Realtime PCR assays (with appropriate primers 9-18) were carried out using the iQ SYBR Green Supermix (Bio-Rad) on the Bio-Rad CFX384 realtime PCR detection system.
  • Experimental Ct values were normalized to ⁇ -actin and relative mRNA expression was calculated versus a reference sample . Data is shown as fold change in gene expression by RT-qPCR (A'Ct method) .
  • Transgenic BHK cells [27] were co- transfected with plasmids encoding the bait p53 (amino acids 1-81) fusion protein and different prey HDM2 (amino acids 7-134) fusion proteins overnight in 96 multiwell plates (pClear Greiner Bio-One, Germany) using the Lipofectamine 2000 (Life Technologies) reverse transfection protocol according to manufacturer' s instructions with 0.2 pg DNA and 0.4 ⁇ i Lipofectamine 2000 per well. Cells were incubated with a dilution series of Nutlin or stapled peptides for 1 hour at 37°C, 5% C0 2 .
  • Interaction was determined as the ratio of cells showing co- localization of fluorescent signals at the nuclear spot to the total number of evaluated cells.
  • an INCell Analyzer 1000 with a 20X objective (GE Healthcare) was used.
  • Automated image segmentation and analysis was performed with the corresponding INCell Workstation 3.6 software. At least 100 co- transfected cells were analyzed per well. Titrations were carried out independently three to five times.
  • the mutations P20L and Q24R lie in the flexible lid region of HDM2 and are missing from the crystal structures of HDM2 (1YCR, 1 RV1) [19,38] .
  • 11 conformations of the lid from the ensemble of NMR structures (1Z1M) [33] were grafted onto 1YCR (residues 25-109) .
  • the 11 structures were chosen visually to represent the 3 major states: open, closed and partially open.
  • HDM2 (residues 6-109) that has become available (in complex with a small molecule; PDB code 4HBM, resolved at 1.9A) [72] with an ordered lid and so a 12 th structure of 1YCR was also created where this lid (only from residues 6-24) was grafted.
  • the 12 structures generated (wild- type) and the P20L and Q24R mutants generated were all subject to 20 ns molecular dynamics simulations each, in 3 states: apo, complex with p53 peptide and complex with Nutlin, totaling a simulation time of 720 ns for the wild type and for each mutant.
  • a shorter HDM2 was used for HDM2-stapled peptide complexes.
  • the computational alanine-scanning methodology [74] is based on the assumption that replacing the original residue with an alanine will only introduce local changes and not cause a large conformational change to alter the binding mode. Trajectories were sampled every 100 ps for computational alanine scanning using the MM-PBSA post-processing module in amberll. Alanine mutant structures were generated by modifying each residue of the receptor at the C Y atom and by replacing the C Y atom with a hydrogen atom with appropriate distance at the C y -C (3 ⁇ 4 bond. PyMOL
  • Fig. 15 [24] . These have been designed to target the same hydrophobic cleft of HDM2 to which Nutlin binds.
  • Either wild- type or mutant (M62A and Q24R) HDM2 was captured on beads followed by incubation with either Nutlin or stapled peptide.
  • p53 was subsequently added, and interaction with HDM2 determined by Western blot.
  • the results in Fig. 15 indicate strong repression of the HDM2-p53 interaction by both Nutlin and the stapled peptides.
  • the M62A and Q24R mutants showed resistance to Nutlin, with increased p53 being pulled down compared to wildtype HDM2 [77] .
  • HCT116 p53 +/+ cells HCT116 p53 +/+ cells.
  • Wild-type or variant HDM2 was transfected and cells treated with either Nutlin or stapled peptide PM2.
  • p53 activation of p21 , gadd45a and 14 - 3 -3 ⁇ transcript levels [78] [79,80] was measured by qPCR.
  • Nutlin treatment (10 ⁇ ) significant reduction of p53 transcriptional activity was observed for the M62A and Q24R mutants compared to wild- type, consistent with results obtained in DKO cells.
  • the stapled peptide PM2 (40 ⁇ ) did not discriminate significantly between inhibition of wild-type and mutant HDM2 with regards to up- regulation of the p21 and Gadd 5a genes.
  • some resistance to PM2 was observed for the mutants, although this was not as pronounced when compared to Nutlin treatment.
  • No significant differences in expression of the HDM2 mutants were observed compared to wild-type in this cell line (Fig. 24) .
  • the Q24R and P20L mutants also displayed reduced affinity for Nutlin compared to wild-type (respectively 5282.67 ⁇ 1335.47 and 3041.67 ⁇ 879.71 versus 784.15 ⁇ 11.45 nM) .
  • the trend in Nutlin binding affinity for the mutants (M62A ⁇ Q24R ⁇ P20L) mirrors the resistance phenotypes observed for these mutants in cell-based assays (Figs. 16, 17) [77] .
  • Binding to the stapled peptide MOll was not perturbed by the Q24R and P20L mutations (respectively 16.94 ⁇ 3.20 and 16.46 ⁇ 4.61 versus 12.94 ⁇ 3.02 nM for wild-type) .
  • no significant differences were observed for p53 peptide binding to these mutants (10.39 ⁇ 1.30 and 17.22 ⁇ 4.10 versus 13.9 + 4.4 nM for wild-type) .
  • the direct cellular binding of the HDM2 wild-type, Q24R and M62A N-terminal domains to p53 was further characterized in the Fluorescent 2-Hybrid (F2H) assay [27] .
  • the F2H assay visualizes the interaction of RFP-tagged HDM2 (amino acids 7-134) with GFP- tagged p53 (amino acids 1-81) at a defined nuclear F2H interaction platform, in specific BHK cells. Dissociation of the complex due to interaction with N tlin or stapled peptide can be imaged and quantified.
  • Simulations of the P20L mutation shows that L20 together with 119 packs against the ridge of the p53 binding pocket leading to a partial occlusion of the main binding site (Fig. 21B) , notably the region where L26 of p53 embeds.
  • Cluster analysis on the apo P20L MD data shows that 84% of the conformations sampled place the I19-L20 in this position within HDM2. This places a barrier for Nutlin migration from the secondary to the main site and may account for the resistance of this mutant to Nutlin binding.
  • Binding to p53 is retained as the lid only occludes the L26 site; it has previously been shown that p53 likely binds with F19 docking first and enabling a crack to propagate [84] . This suggests that in these mutants, p53 and stapled peptides can dock into the open F19 docking site and then slowly edge the lid out .
  • Wild- type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci U S A 89: 7491-7495.
  • Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53.
  • Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53. J Biol Chem 275: 8945-8951.

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Abstract

La présente invention concerne une méthode de détermination de la résistance d'une molécule biologique à l'inhibition de son interaction avec une molécule cible par un inhibiteur de la molécule biologique, la méthode comprenant les étapes suivantes : a) co-compartimentaliser un gène codant pour la molécule biologique avec la molécule cible, ou un gène codant pour la molécule biologique avec un gène codant pour la molécule cible dans une gouttelette aqueuse située dans une émulsion d'eau dans l'huile, et b) rechercher un complexe comprenant la molécule biologique et la molécule cible lors de l'expression du gène codant pour la molécule biologique et du gène codant pour la molécule cible; la détection du complexe en présence de l'inhibiteur indiquant que la molécule biologique est résistante à l'inhibition de son interaction avec la molécule cible par l'inhibiteur. L'invention concerne aussi des polypeptides ubiquitines ligases mutés HDM2 présentant une résistance à l'inhibition par la nutline de la liaison p53.
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WO2022081827A1 (fr) 2020-10-14 2022-04-21 Dana-Farber Cancer Institute, Inc. Conjugués chimériques destinés à la dégradation de protéines virales et de protéines hôtes et méthodes d'utilisation

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016056673A1 (fr) * 2014-10-09 2016-04-14 Daiichi Sankyo Company, Limited Algorithmes pour prédicteur basé sur des signatures géniques prédisant la sensibilité aux inhibiteurs de mdm2
WO2022081827A1 (fr) 2020-10-14 2022-04-21 Dana-Farber Cancer Institute, Inc. Conjugués chimériques destinés à la dégradation de protéines virales et de protéines hôtes et méthodes d'utilisation

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