EP1290004A1 - Procede d'identification des points critiques dans des interactions proteines-proteines - Google Patents

Procede d'identification des points critiques dans des interactions proteines-proteines

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Publication number
EP1290004A1
EP1290004A1 EP01935592A EP01935592A EP1290004A1 EP 1290004 A1 EP1290004 A1 EP 1290004A1 EP 01935592 A EP01935592 A EP 01935592A EP 01935592 A EP01935592 A EP 01935592A EP 1290004 A1 EP1290004 A1 EP 1290004A1
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European Patent Office
Prior art keywords
prp
protein
compound
gene
amino acid
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EP01935592A
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German (de)
English (en)
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Stanley B. Prusiner
Fred E. Cohen
Thomas L. James
Kiyotoshi Kaneko
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University of California
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University of California
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease

Definitions

  • This invention relates generally to a method of analyzing protein-protein interactions and more specifically to using the results of the analysis to determine molecules which effect those interactions.
  • Each protein of biological significance is likely to be involved in a large number of protein- protein interactions.
  • Each of these interactions provides a site at which a drug might either (a) inhibit the interactions, i.e. act as an antagonist, or (b) enhance the response obtained, i.e. act as an agonist.
  • the interactions may have other effects such as allowing for or preventing a protein from undergoing a conformational change.
  • PrP proteins can exist in a normal PrP c configuration or in a disease related PrP Sc configuration known as a prion. The following provides a description of prions and diseases caused by and/or related to conformationally altered proteins from which the importance of inhibiting protein-protein interactions which result in prion formation will be appreciated.
  • Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in humans and animals. Prions are distinct from bacteria, viruses and viroids. The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein. Further, a prion which infects one species of animal (e.g., a human) will not efficiently infect another (e.g., a mouse).
  • the prion diseases represent a variety of neurodegenerative states characterized at the neuropathologic level by the presence of spongiform degeneration and astrocytic gliosis in the central nervous system (DeArmond & Prusiner (1996) Current Topics in MicroBiology and Immunology, 207: 125-146). Frequently, protein aggregates and amyloid plaques are seen that are often resistant to proteolytic degradation. The neuroanatomic distribution of the lesions varies with the specific types of prion disease. In humans, sporadic Creutzfeldt- Jakob Disease (CJD) accounts for 85% of all cases. The disease presents in the sixth decade of life with dementia and ataxia.
  • CJD Creutzfeldt- Jakob Disease
  • Familial disease carries a variety of monikers such as Gertsmann-Straussler-Scheinker disease (GSS), familial CJD (fCJD) and Fatal Familial Insomnia (FFI) that relate the precise mutation in the PrP gene to a clinical syndrome (Prusiner & Hsaio (1994) Annals of Neurology, 35:385-395; Parchi, et al. (1996) Annals of Neurology, 39:767-778; Montagna, et al. (1998) Brain Pathology, 8:515-520). Disease typically presents in the fourth decade of life with an autosomal dominant pedigree.
  • GSS Gertsmann-Straussler-Scheinker disease
  • fCJD familial CJD
  • FFI Fatal Familial Insomnia
  • the gene contains a single open reading frame and codes for a protein that is proteolytically processed and glycosylated to form a macromolecule with 219 amino acids, a disulfide bridge, two N-linked sugars and a glycophosphotidyl inositol anchor that is exported to the cell surface and concentrated in an endocytic compartment known as the caveolar space ( Endo, et al. Biochemistry, 28:8380-8 1989); Stahl, et al. Biochemistry 29:8879-84 (1990); Yost, et al. Nature, 343:669-72 (1990); DeFea, et al. J. Biol. Chem., 269:16810-16820 (1994); Hegde, et al.
  • the disease causing form of the prion protein is a multimeric assembly substantially enriched in ⁇ -sheet structure (40% ⁇ -sheet, 30% ⁇ -helices as judged by FTIR spectroscopy) (Pan, et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 10962-10966).
  • PrP c is a kinetically trapped intermediate in the folding of PrP Sc (Cohen & Prusiner (1998) Annual Review of Biochemistry, 67:793-819). This kinetic barrier can be reduced by exogenous administration of the PrP Sc template, mutations to the wild type (wt) PrP sequence, or stochastic processes resulting in infectious, inherited, or sporadic prion diseases. Epitope mapping and peptide studies suggest that much of this conformational plasticity is localized to the middle third of this 231 residue GPI anchored glycoprotein with a 22 amino acid signal sequence (Peretz, et al. (1991) J. Mol. Biol, 273:614-622).
  • Peptide fragments derived from regions of the PrP sequence have been studied extensively (Gasset, et al. (1992) Proc. Natl. Acad. Sci. USA, 89:10940-10944; Tagliavini, et al. (1993) Proc. Natl Acad. Sci. USA, 90:9678-9682; Forloni, etal (1993) Nature, 362:543-546; Come, etal. (1993) Proc. Natl. Acad. Sci. USA, 90:5959-5963; Zhang, et al. (1995) J. Mol. Biol, 250:514-526; Nguyen, et al.
  • peptides chosen from the region 90-145 are compatible with ⁇ -helical, irregularly coiled, and ⁇ -sheet rich conformations when characterized under different conditions (Zhang, et al. (1995) J. Mol. Biol, 250:514-526).
  • catalytic amounts of ⁇ -sheet rich peptides can facilitate the conformational conversion of peptides with distinct structures into ⁇ -sheet rich isoforms (Gasset, et al. (1992) Proc. Natl. Acad. Sci. USA, 89:10940-10944; ; Nguyen, et al. (1995) Biochemistry, 34:4186-4192).
  • BSE bovine spongiform encephalopathy
  • the method can be carried out in a variety of ways and preferably results in identifying one or more positions in an amino acid sequence which are critical to protein-protein interactions. After determining the critical positions, protein variants can be produced which are altered at the critical positions or small molecules are designed to interact with those positions thereby obtaining useful pharmaceuticals.
  • the method preferably involves identifying the amino acid sequence of a biologically significant protein and/or the amino acid sequence of its receptor.
  • analysis (such as the use of X-ray crystallography) is carried out on the protein to obtain as much information as possible and develop a pharmacophore which defines the three-dimensional structure and electrochemical characteristics of the protein.
  • a pharmacophore which defines the three-dimensional structure and electrochemical characteristics of the protein.
  • an initial determination can be made of the amino acid positions or segments of the proteins which might be most important in interacting with another protein.
  • DNA sequences encoding either or both of the interacting protein sequences are then produced and cloned.
  • the cloned sequences of DNA are subjected to site specific mutagenesis.
  • site specific mutagenesis sites specific mutagenesis.
  • large numbers of mutated sequences are produced which are expressed to produce large numbers of protein variants.
  • the variants are assayed to determine the ability of any to affect protein-protein interactions.
  • the results of such an assay will provide further information on the point mutations and/or areas of the protein which are most significant in terms of enhancing or inhibiting the protein-protein interaction of interest. This makes it possible to identify specific amino acids or segments in the protein which most influence the protein-protein interaction.
  • mutagenesis can thus be carried out so as to specifically affect those amino acids or segments of the protein and the process steps repeated until the resulting proteins variant created can be used to obtain any desired degree of inhibition or enhancement of the protein-protein interaction of interest.
  • the process can be supplemented by (1) creating transgenic animals which express or test variants; and/or (2) using a computer program to determine small molecules which would be the best candidates for having a desired effect on the protein-protein interaction of interest. Molecules are disclosed that interact with the cellular components involved in conversion of
  • PrP c to PrP Sc The molecules disclosed can be small molecules, peptides or protein analogs, e.g. analogs of PrP c . In one embodiment, these molecules interfere with prion formation and/or replication, e.g. by preventing interactions of proteins involved in a prion complex or by interfering with ⁇ -sheet formation. In another embodiment, the molecules of the invention promote PrP c conversion to PrP So , e.g.
  • the molecules of the invention may be designed to bind to PrP c or PPMF of a genetically diverse species, i.e. the molecules will not be limited by the "species barrier" that normally limits prion infectivity.
  • Binding to PrP c or PPMF can present the interaction needed to bring about the conversion of PrP c to PrP So .
  • the invention features a compound defined by a pharmacophore.
  • the compound corresponds to a geometric and chemical description of a molecular structure or collection of molecular structures.
  • a preferred compound is characterized by an ability to modulate conversion of PrP to PrP Sc in vivo.
  • a preferred compound defined by a pharmacophore can be a peptide or a small molecule with the ability to bind to PPMF and/or PrP c .
  • a preferred structure of a compound defined by the pharmacophore is defined by a tertiary surface reflecting the negative image of PPMF at its PrP binding domain and/or a tertiary surface defined by the positive image of a specific discontinuous epitope of PrP protein that includes a small subset of residues.
  • the compound defined by the pharmacophore structure reflects geometric and chemical positions defined by the relative positions of specific amino acid side chains corresponding to the positions of residues 90-231 of the human PrP protein, and in particular residues 168, 172, 215 and 219 corresponding to the human PrP protein.
  • the compound defined by the pharmacophore can also contain an epitope from PPMF that binds to PrP.
  • An object of the invention is to provide an ex vivo system for studying the structural events occurring in conversion, where the system is a cell line treated with a small organic molecule or a peptide that is able to mimic the chemical and geometric features of proteins involved in prion complexing.
  • the pharmacophore defines (specifies at a molecular level) a collection of molecules that repress prion infectivity and or progression of prion-mediated disease.
  • Any compound defined by a pharmacophore of the invention may inhibit initial infectivity, conversion of PrP c to PrP Sc and/or progression of neurodegeneration by any number of mechanisms, including but not limited to binding a molecule involved in prion complexing, e.g. PrP c or PPMF or inhibiting ⁇ -sheet formation or elongation
  • Yet another aspect of the invention features a method of repressing conversion of PrP c to PrP Sc , comprising administering an inhibitor that meets the criteria specified by the pharmacophore model.
  • This may be administered prophylactically to a subject at risk of developing a prion-mediated disorder, e.g. a mammal exposed to infectious prions, or to treat a subject that is exhibiting signs of prion-mediated neurodegeneration.
  • a feature of the invention is that the inhibitors can be used to treat subjects suffering from prion-mediated disorders.
  • Yet another aspect of the invention features an assay to identify a PrP pharmacophore, a geometric and chemical specification of a collection of small molecules that could inhibit PrP Sc formation.
  • the assay utilizes the steps of determining functional residues of the PrP protein involved in prion complex interactions, developing three dimensional structures based on these functional residues, comparing the three dimensional structures with a series of compounds having known or calculated tertiary structures, and identifying compounds having a spatial orientation consistent with binding to components of the PrP Sc replication complex (PrP c , PrP Sc , PPMF) at these functional residues.
  • Figure 1 is a ribbon drawing of the NMR of rSHa PrP(90-231). Residues 90-115 are not shown, ⁇ -helical regions are shown in mauve and ⁇ -strands are shown in cyan.
  • Figure 2 is a model of the putative structure of PrP Sc highlighting the dramatic increase in the ⁇ -sheet structure that has been localized to the region between residues 90-145 by immunologic studies.
  • Figure 3 illustrates mutations causing inherited human prion disease and polymorphism's in human, mouse and sheep.
  • the x-axis represents the human PrP sequence, with the five octarepeats, the three ⁇ -helices A, B and C and the two ⁇ -strands S 1 and S2.
  • Above the line of the human sequence are mutations that cause prion disease.
  • polymorphisms some but not all of which are known to influence the onset as well as the phenotpye of disease.
  • FIG 4 is an illustration of the distinction between thermodynamic and kinetic models for the energetics of the conversion of PrP c carrying the wild-type (WT) and mutant (MUT) sequences into PrP Sc .
  • ⁇ G is the free energy difference between the PrP c and PrP So states and ⁇ G* is the activation energy barrier separation these two states.
  • ⁇ G is the difference between ⁇ G and
  • Figure 5 is an approach to small molecules in computer screening for inhibitors of PrP So replication based on blocking the PrP c : PPMF interaction.
  • FIG 6 shows dependence of Cp-60 inhibition on (MHM2)PrP Sc concentration using the Time Resolved Fluorescence (TRF) technique of Safar et al (1998).
  • TRF Time Resolved Fluorescence
  • Different concentrations of Cp- 60 were applied on transiently transfected ScN2a cells.
  • Samples were digested by proteinase K and the remaining proteins were quantified by i munoassay using the TRF technique.
  • An Eu-mAb3F4 is used to detect (MHM2)PrP proteins. Data represent average + s.e.m. from three independent experiments measured in duplicate.
  • Figure 7 illustrates the chemical composition of compound 60.
  • Figure 8 is a graphic representation of an open reading frame of the MoPrP and synthetic peptides 89-143 and 89-143, P101L of the MoPrP (MoPrP(89-143)) and MoPrP(89-143, P101L)). The residue corresponding to the mutation is underlined. Confirming that the N-terminal half of the PrP open reading frame is dispensable in prion propagation, PrP27-30 containing G89-S230 can induce prion propagation. HA, HB and HC represent Helices A, B and C, respectively. GPI represents the glycophosphatidyl inositol anchor.
  • Figure 9 illustrates the chemical structure of analogs based on a substructure search using compound 60.
  • Figure 8 shows the results of a substructure search of the available chemicals directory with compound 60 as a probe. Nine commercially available compounds have been identified and six have been screened.
  • Figures 10A, 10B and IOC are images of gels run to demonstrate the presence or lack of protease resistant PrP Sc .
  • Figures 11A and 1 IB are images of gels run to show the presence of or a lack of a protease resistant PrP So .
  • Figures 12A-12L are images of brains showing the presence of a plaque due to the presence of a PrP
  • Figures 13A-13F are cross-sectional images of mice brains.
  • Figure 14A and 14B are graphs of data points demonstrating the presence of insoluble PrP Sc in different transgenic mice brains.
  • Figure 15 A and 15B are each images of gels run showing the presence or lack thereof of PrP Sc in different transgenic mice brains.
  • PPMF Prion Protein Modulator Factor
  • Prion Protein Modulator Factor is a protein which can be glycosylated and is characterized by binding to PrP c and facilitating a conformational change from PrP c to PrP Sc .
  • the term encompasses any PPMF from any animal allowing for specific differences between different species of animals.
  • the PPMF compounds of the present invention are more particularly characterized herein.
  • pharmacophore means a geometric and/or chemical description of a class or collection of compounds. Compounds defined by the pharmacophore have a biochemical activity which activity is obtained by the 3 -dimensional physical shape of the compound and the electrochemical properties of the atoms making up the compound.
  • the term "pharmacophore” is a description of a collection of compounds which have defined characteristics. Specifically, the "pharmacophore” defines a compound with those 3 -dimensional physical and electrochemical characteristics. More specifically, pharmacophores of the invention may, for example, define a class of compounds which mimic or inhibit PrP Sc activity by interaction with (1) the discontinuous epitope on PrP c to which PPMF binds or (2) the surface of PPMF which binds to PrP c . Thus, a pharmacophore defines compounds of the invention which have a given shape (i.e., the geometric specifications) and given electrochemical characteristics.
  • Examples of such are compounds with a shape and characteristic as defined by PrP Sc , PPMF, PrP c , or other proteins involved in the prion complex that facilitate the conversion of PrP c to PrP Sc .
  • the term pharmacophore defines properties of peptides, peptide analogs and small molecules.
  • small molecule refers to small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons, which preferably are not comprised of DNA or RNA.
  • treatment means obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of completely or partially preventing a prion disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a prion disease or adverse effect attributable to the disease.
  • treatment covers any treatment of a disease in a mammal, particularly a cow, pig, sheep, mouse or human, and includes:
  • isolated shall mean separated away from its natural environment.
  • An isolated protein is not necessarily separated away from all materials it is normally present with and may remain glycosylated.
  • corresponding position means the position of an amino acid in a peptide or the position of a codon in a nucleotide sequence corresponding to the same position in the sequence of a different species.
  • amino acid sequence of PPMF also has corresponding positions from one species to another and corresponding positions for four different positions on the discontinuous epitope of PrP c (for five different proteins) are shown in Table 1.
  • FVB refers to a mouse strain commonly used in the production of transgenic mice.
  • PrP mouse prion protein
  • Prnp 0/0 or "Prnp-Abl” refers to a transgenic animal which has its PrP gene ablated with the " 0/0 " indicating that both alleles are ablated whereas " 0/+ " indicates only one is ablated.
  • the animal being referred to is generally a transgenic mouse which has its PrP gene ablated i.e., a PrP knockout mouse. In that the PrP gene is disrupted no mouse PrP protein is expressed.
  • CJD sporadic CJD
  • CJD Creutzfeldt- Jakob Disease
  • Iatrogenic CJD refers to disease resulting from accidental infection of people with human prions. The most noted example of such is the accidental infection of children with human prions from contaminated preparations of human growth hormone.
  • Familial CJD refers to a form of CJD which occurs rarely in families and is inevitably caused by mutations of the human prion protein gene. The disease results from an autosomal dominant disorder. Family members who inherit the mutations succumb to CJD.
  • Gerstmann-Strassler-Scheinker Disease refers to a form of inherited human prion disease. The disease occurs from an autosomal dominant disorder. Family members who inherit the mutant gene succumb to GS S .
  • prion shall mean an infectious particle known to cause diseases (spongiform encephalopathies) in humans and animals.
  • the term “prion” is a contraction of the words “protein” and “infection” and the particles are comprised largely if not exclusively of PrP So molecules encoded by a PrP gene which expresses PrP c which changes conformation to become PrP Sc .
  • Prions are distinct from bacteria, viruses and viroids. Known prions include those which infect animals to cause scrapie, a transmissible, degenerative disease of the nervous system of sheep and goats as well as bovine spongiform encephalopathies (BSE) or mad cow disease and feline spongiform encephalopathies of cats.
  • BSE bovine spongiform encephalopathies
  • prion diseases known to affect humans are (1) kuru, (2) Creutzfeldt- Jakob Disease (CJD), (3) Gerstmann-Straussler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI).
  • CJD Creutzfeldt- Jakob Disease
  • GSS Gerstmann-Straussler-Scheinker Disease
  • FFI fatal familial insomnia
  • prion includes all forms of prions causing all or any of these diseases or others in any animals used — and in particular in humans and in domesticated farm animals.
  • PrP gene and "prion protein gene” are used interchangeably herein to describe genetic material which expresses PrP proteins, including proteins with polymorphisms and mutations such as those listed herein under the subheading "Pathogenic Mutations and Polymorphisms.” Unless stated otherwise the term refers to the native wild-type gene and not to an artificially altered gene.
  • the PrP gene can be from any animal including the “host” and “test” animals described herein and any and all polymorphisms and mutations thereof, it being recognized that the terms include other such PrP genes that are yet to be discovered.
  • PrP gene refers generally to any gene of any species which encodes any form of a PrP amino acid sequences including any prion protein.
  • PrP sequences are described in Gabriel et al., Proc. Natl. Acad. Sci. USA S :9097-9101 (1992) and Wopfner et al., JMol Biol 289: 1163-78 (1999), which is incorporated herein by reference to disclose and describe such sequences.
  • the term “genetic material related to prions” is intended to cover any genetic material which affects the ability of an animal to become infected with prions.
  • the term encompasses any "PPMF gene,” “PrP gene,” “artificial PrP gene,” “chimeric PrP gene” or “ablated PrP gene” which terms are defined herein as well as mutations and modifications of such which affect the ability of an animal to become infected with prions.
  • Standardized prion preparations are produced using animals which all have substantially the same genetic material related to prion so that all of the animals will become infected with the same type of prions and will exhibit signs of infection at about the same time.
  • host animal and "host mammal” are used to describe animals which will have their genome genetically and artificially manipulated so as to include genetic material which is not naturally present within the animal.
  • host animals include mice, hamsters and rats which have their endogenous PrP gene altered by the insertion of an artificial gene or by the insertion of a native PrP gene of a genetically diverse test animal.
  • test animal and “test mammal” are used to describe the animal which is genetically diverse from the host animal in terms of differences between the PrP gene of the host animal and the PrP gene of the test animal.
  • the test animal may be any animal for which one wishes to run an assay test to determine whether a given sample contains prions to which the test animal would generally be susceptible to infection.
  • the test animal may be a human, cow, sheep, pig, horse, cat, dog or chicken, and one may wish to determine whether a particular sample includes prions which would normally only infect the test animal. This is done by including PrP gene sequences of the test animal into the host animal, administering PPMF and inoculating the host animal with prions which would normally only infect the test animal.
  • a mouse PrP gene is genetically diverse with respect to the PrP gene of a human, cow or sheep, but is not genetically diverse with respect to the PrP gene of a hamster.
  • prions of a given animal will not infect a genetically diverse animal and PPMF of a given animal will not bind to PrP c of a genetically diverse animal.
  • ablated prion protein gene means an endogenous prion protein gene which has been altered (e.g., add and/or remove nucleotides) in a manner so as to render the gene inoperative.
  • transgenic or hybrid ariimal such as a transgenic mouse Tg(MHu2M) which, without the chimeric PrP gene, would not be susceptible to infection with a human prion (less than 20% chance of infection) but with the chimeric gene is susceptible to infection with human prions (80% to 100% chance of infection).
  • resistant to infection means the animal includes a PrP gene which renders the animal resistant to prion disease when inoculated with an amount and type of prion which would be expected to cause prion disease in the animal.
  • the resistant animals PrP gene includes non-native codons which express amino acids different from those of the native PrP gene which effect the PrP c /PPMF binding site.
  • incubation time shall mean the time from inoculation of an animal with a prion until the time when the animal first develops detectable symptoms of disease resulting from the infection.
  • a reduced incubation time is six months or less, preferable about 100 days ⁇ 25 days or less, more preferably about 30 days ⁇ 10 days or less.
  • CNS central nervous system
  • FFI fatal familial insomnia
  • HuPPMF for human Prion Protein Modulator Factor
  • HuPrP for a human PrP protein
  • MHu2M for a chimeric mouse/human PrP gene wherein a region of the mouse PrP gene is replaced by a corresponding human sequence which differs from mouse PrP at 9 codons;
  • Mo for mouse
  • MoPPMF for mouse Prion Protein Modulator Factor
  • MoPrP Sc for the scrapie isoform of the mouse PrP protein
  • PPMF for Prion Protein Modulator factor in general, i.e., that protein as in any species
  • Prnp 0/0 for ablation of both alleles of an endogenous PrP protein gene, e.g., the Mo PrP gene; PrP CJD for the CJD isoform of a PrP gene;
  • PrP Sc for the scrapie isoform of the PrP protein
  • SHa for a Syrian hamster SHa PrP for a Syrian hamster PrP protein
  • Tg(MHu2M) mice are transgenic mice of the invention which include the chimeric MHu2M gene;
  • Tg(MHu2M)/Prnp 0/0 for a hybrid mouse obtained by crossing a mouse with a chimeric PrP protein gene (MHu2M) with a mouse with both alleles of the endogenous PrP protein gene disrupted; Tg(SHa PrP) for a transgenic mouse containing the PrP gene of a Syrian hamster;
  • Tg(SHa PrP) for transgenic mice containing the complete sheep PrP gene Tg(SHa PrP) for transgenic mice containing the complete sheep PrP gene.
  • GENERAL ASPECTS OF THE INVENTION The present invention is based at least in part on understanding aspects of dominant negative mutations of PrP genes resulting in the discovery that synthetic or isolated molecules (e.g. small molecules, peptides, and the like) with the appropriate tertiary structure have the ability to interact with members of the prion replication complex and to inhibit the formation of de novo protease resistant forms of PrP in appropriate animals.
  • An aspect of the invention involves identifying compounds which effect protein-protein interactions.
  • the invention can identify all types of compounds including peptides and small molecules and can identify those compounds which effect all types of protein-protein interactions.
  • the invention is particularly focused on protein-protein interactions wherein one protein being acted on is changed from a first conformational shape which is not pathogenic to a second conformational shape which is pathogenic.
  • the method is carried out by providing a DNA sequence which encodes at least a portion of one of the two proteins involved in the protein-protein interaction of interest. After identifying the DNA sequence the sequence is copied or cloned in order to produce a large number of copies of the sequence of interest.
  • the copies or clones are then subjected to mutagenesis using standard procedures thereby producing a plurality of DNA sequence variants. These variants may include additions, deletions or changes in the sequence.
  • the variants are then operatively inserted into expression vectors and expressed in order to produce a large number of amino acid sequence variants.
  • the variants produced are then tested. The test may involve providing the variants in groups or individually into a situation where the protein-protein interaction of interest would be expected to take place and observing the effect of the variants, if any, on the protein-protein interaction. If an effect is observed it is desirable to isolate the amino acid sequence variant which has the greatest effect on the protein-protein interaction of interest.
  • the isolated amino acid sequence variant of interest is sequenced and a determination is made of the differences between the sequence variant and at least a portion of the protein of the protein-protein interaction of interest.
  • DNA sequence encoding amino acid sequence variants which are determined to effect the protein-protein interaction of interest can be operatively inserted into the genome of a transgenic animal and the transgenic animal can be observed in order to determine differences between the transgenic animal and an animal without the inserted DNA sequence.
  • the three-dimensional shape of amino acid sequence variants determines the three-dimensional shape of amino acid sequence variants and analyze the shape relative to at least a portion of a protein-protein interaction of interest.
  • the program selects molecules based on their three-dimensional shape and determines three-dimensional amino acid variants and creates pharmacophores.
  • the computer program molecules which the computer indicates would be expected to effect the protein-protein interaction of interest are assayed for their ability to inhibit prion formation.
  • analytical methodology selected of the group consisting of NMR spectroscopy and X-ray crystallography.
  • the invention further includes compounds which are defined by a pharmacophore characterized by its ability to effect a protein-protein interaction of interest which pharmacophore is identified by the methodology disclosed herein.
  • the invention includes compounds which are characterized by the ability to modulate the conversion of PrP c to PrP Sc in vivo.
  • Such molecules include the small molecules characterized by binding PPMF and molecules which bind to PrP c
  • an assay is used to identify a compound defined by a PrP pharmacophore.
  • the assay comprises determining the functional residues of the PrP protein involved in prion complex interactions. Thereafter a plurality of three-dimensional structures based on these functional residues are developed. These structures are compared with a series of compounds having calculable tertiary structures. Then, compounds are identified which have a spatial orientation consistent with binding PrP c at the determined functional residues.
  • dominant negative mutations are made in sequences which encode proteins such as the PrP protein which protein is subject to a conformational change to a pathogenic conformation.
  • the resulting PrP protein can prevent the formation of the pathogenic form of the PrP protein i.e. prevent prion formation.
  • the invention includes making the mutations in a PrP in both alleles or in a single allele and inserting the mutated gene into a transgenic animal in order to determine if the mutation is sufficient to effect expected conformational changes in the protein. This can, for example, be carried out by subjecting one or both alleles of the PrP gene to mutagenesis and thereafter inserting the mutated sequence into a transgenic mouse.
  • the mouse is then innoculated with a composition containing prions which would be expected to cause the mouse to develop a prion disease. If the mouse does not develop a prion disease then the dominant negative mutation inserted into the mouse has somehow sequestered the transformation of the non-pathogenic form of the protein to the pathogenic form.
  • the methodology of the present invention makes it possible to determine the sequences which are necessary for important dominant negative mutations in all types of animals including humans, cows, sheep, pigs, horses, etc. which mutations prevent the occurrence of undesirable conformational changes in proteins from a non-pathogenic conformation to a pathogenic conformation associated with the disease. Specifically, the dominant negative mutations can result in preventing the conformational change from PrP c to PrP Sc .
  • human cell lines can be genetically engineered. Human cell lines can be used to produce an array of different useful pharmaceutical compounds including human antibodies. By genetically engineering cell lines to include the dominant negative mutation which prevents the conformational change to an undesirable pathogenic form of a protein the genetically engineered cell line can be used in a cell culture more safely to produce pharmaceuticals such as antibodies which could then be used as pharmaceuticals for humans.
  • PrP gene mutated at any one, two, three or all four specific positions.
  • the gene is preferably mutated at one and preferably only one position and the resulting in a variant protein with an amino acid substituted at one position, i.e. an amino acid at one position and preferably only one position is different from the amino acid present at that position in the wild-type PrP protein.
  • an amino acid substituted at one position i.e. an amino acid at one position and preferably only one position is different from the amino acid present at that position in the wild-type PrP protein.
  • the four positions of interests are Q168, Q172, Q215 and Q219.
  • PrP genes there are differences between PrP genes and as such the position of interest will be different positions for different species of animals.
  • polymorphisms at positions equivalent to 168, 172, 215 and 219 in a hamster as follows:
  • a gene of the invention is mutated at any or all of the relevant positions and a resulting position has an amino acid at that position resulting from the mutated codon.
  • An important aspect of the invention is a PrP gene which has had a codon which encodes an amino acid at one of the relevant positions with a codon which encodes an amino acid different from the wild-type at that position.
  • a bovine PrP protein may have five or six octa-repeats at the N-terminus. Thus, the bovine has two different proteins referred to as bovine (5) and (6).
  • the codon present at codon 179 in a bovine (6) codes for Q i.e. glutamine.
  • the invention comprises a bovine PrP gene which codes for any basic amino acid i.e. codes for any of R- arginine, H-histidine or K-lysine at position 179.
  • the wild-type codon coding for Q at position 179 is replaced with a codon coding to R at position 179.
  • a mutated bovine PrP gene could be used to produce a transgenic cow which would be resistant to prion disease.
  • the wild-type gene could be mutated so that the codon coding for amino acid Q183 could be replaced with a codon coding for any basic amino, i.,e. any of R, H or K.
  • the Q230 can be replaced with any basic amino acid.
  • the I at 218 can be replaced with any basic amino acid.
  • a bovine (5) gene has a codon coding for Q in the 171, 175 and 222 positions and a codon coding for I in the 218 position. Any of these codons can be substituted with a codon coding for any of R, H or K — preferably R. Further, a transgenic cow can be produced with all or any of these substitutions in one or both alleles. Equivalent substitutions can be made in the PrP gene of any mammal and in particular in PrP genes of a pig, sheep, goat, horse, chicken, turkey, mule, deer, etc. PHARMACOPHORE DESIGN AND THE PrP c -PPMF INTERFACE
  • PrP c forms a complex with PPMF and PrP Sc resulting in a ternary complex of PPMF/ PrP c /PrP Sc .
  • the complex dissociates due to the lack of affinity of PrP So for PPMF.
  • pharmacophores fitting this geometric and chemical description are used to interfere with either the PrP s 7 PrP c or PPMF/ PrP c interface.
  • the inhibitors can be used to prevent the initial conversion of PrP c into prions, or later prevent the progression of prion formation.
  • the PrP Sc binding site on the surface of the PrP(90-231) NMR structure appears to form a rather large and discontinuous epitope (Scott, et al, 1997). Accordingly, we have focused on pharmacophores that preferably mimic the PPMF binding site on the surface of PrP c . Identifying pharmacophores of the invention requires the identification of small molecules, peptides, and the like that mimics the positive image of the residues that comprise the PPMF binding site on the surface of the PrP(90-231) NMR structure. A successful compound binds to PPMF, modifying its action, and thereby inhibiting prion replication.
  • deposition diseases such as the prion diseases appear to follow the form:
  • A is the normally synthesized gene product that carries out an intended physiologic role in a monomeric or oligomeric state
  • A* is an conformationally activated form of A that is competent to undergo a dramatic conformational change
  • B is the conformationally altered state that prefers multimeric assemblies
  • B n is the multimeric material that is pathogenic and relatively difficult to recycle.
  • PrP c and PrP Sc correspond to states A and B n where A is largely helical and monomeric and B n is ⁇ -rich and multimeric. Two types of kinetic barriers can be imagined that restrict the formation of B n .
  • the rate limiting step may be the formation of a stable multimeric nucleus (B n ).
  • PrP So formation is believed to require an escape from the kinetically trapped monomeric PrP c structure. See Figure 4.
  • PrP So formation is a first order process where the time from inoculation to disease doubles when the gene dose is changed in animals from the homozygous to the hemizygous state for the prion gene.
  • the time to disease is halved when the founder animals are crossed to yield a progeny carrying twice the transgene dose (Cohen & Prusiner, Anna Rev Biochem. 67:793-819 (1998)).
  • the conformational transformation of a monomeric chain follows first order kinetics, while the rate equation for nucleation events follows higher order kinetics dictated by the multimeric state of the nucleus.
  • PrP Sc providing a template to assist the conversion of nascent PrP c molecules implies that some PrP So templates should be more efficient at stabilizing the nascent PrP So molecule than others in the initial phase of disease propagation. This can be seen in the species barrier to prion transmission where SHa PrP Sc is less efficient than MoPrP Sc in causing disease in mice, and HuPrP Sc is even less efficient than SHa PrP Sc (Scott, etal, Cell 73:979-88 (1993); Telling, etal, Proc Natl Acad Sci US. 91:9936-40 (1994); Scott, et al , Proc Natl Acad Sci USA, 94:14279-84 (1997).
  • HuPrP Sc provides the most effective inoculum, but it has become clear the BoPrP Sc can cause disease in humans albeit at a much lower frequency (Hill, et al. , 1993). There is no evidence that SHaPrP Sc has ever caused disease in humans. It follows that one portion of the molecule is involved in the PrP Sc species specific features of the inoculum while a distinct surface of the molecule is available for interaction with a distinct species specific PPMF molecule.
  • the target size of the infectious particle is 55kDa, a feature corresponding to a dimer (Bellinger-Kawahara, et al, Virology 164:537-41 (1988)).
  • Identification has been made of four residues that are important to the human PrP c -PPMF interaction: 168, 172, 215 and 219.
  • residues 172, 215 & 219 form a continuous patch on the molecular surface.
  • Residues 170 & 171 are a part of this surface, but mutagenesis experiments demonstrate that they do not participate in this interaction while 168 is clearly not part of this surface in the known structure.
  • Pharmacophores of the present invention take advantage of this conversion process, and preferably have structural aspects that prevent the conversion of PrP c to its conversion competent state (e.g. inhibitory pharmacophores). I-n one example, by binding to PrP c or to a protein that interacts with PrP (e.g. PPMF at its PrP c binding domain) a pharmacophore of the present invention may block PrP c :protein interactions and prevent the conversion process.
  • a pharmacophore of the present invention may block PrP c :protein interactions and prevent the conversion process.
  • PrP POLYMORPHISMS AND MUTATIONS The PrP pharmacophores of the present invention optionally contain one or more polymorphisms or mutations known to facilitate prion formation. There are a number of mutations and polymorphisms existing with respect to the PrP gene of different species. A number of the mutations and polymorphisms are listed in the "Pathogenic Mutation Table" provided below. It is believed that additional mutations and polymorphisms exist in all species within the PrP gene. Substitutions in the replication inhibitor pharmacophore may be made with an amino acid which is biochemically quite different from the amino acid at that position which is known to render the animal susceptible to prion infection.
  • Acidic amino acids should be substituted with basic amino acids and vice versa.
  • Polar amino acids should be substituted with nonpolar amino acids and vice versa. Such mutations may increase efficacy of the pharmacophores for the uses described herein.
  • PrP genes For example, a chicken, bovine, sheep, rat and mouse PrP gene are disclosed and published within Gabriel et al., Proc. atl. Acad. Sci. USA 59:9097-9101 (1992). The sequence for the Syrian hamster is published in Basler et al., Cell 46:411-428 (1986). The PrP gene of sheep is published by Goldmann et al., Proc. Natl. Acad. Sci. USA 57:2476-2480 (1990). The PrP gene sequence for bovine is published in Goldmann et al., J. Gen. Virol. 72:201-204 (1991).
  • PrP gene sequence for chicken PrP gene is published in Harris et al., Proc. Natl. Acad. Sci. USA 55:7664-7668 (1991).
  • PrP gene sequence for mink is published in Kretzschmar et al., J. Gen. Virol. 73:2757-2761 (1992).
  • the human PrP gene sequence is published in Kretzschmar et al., DNA 5:315-324 (1986).
  • PrP gene sequence for mouse is published in Locht et al., Proc. Natl. Acad. Sci. USA 53:6372-6376 (1986).
  • the PrP gene sequence for sheep is published in Westaway et al., Genes Dev. 5:959-969 (1994).
  • PrP Sc formation is likely to take place in the caveolar space.
  • the inhibitors that follow the pharmacophores of the present invention may be lipidated to increase their efficacy.
  • Pharmacophore inhibitors can be membrane associated by attachment of a covalent linkage to a fatty acid. Prenylation, farnesylation, geranylgeranylation, palmitoylation and myristilation are exemplary modifications that would increase localization of the inhibitor of the invention to the membrane. Linkage to molecules such as cholesterol can also be used to affect localization of the protein.
  • Candidate molecules as inhibitory pharmacophores can encompass numerous chemical classes, including, but not limited to, peptides and small molecules.
  • Candidate pharmacophores can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate pharmacophores often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate inhibitor pharmacophores are also found among biomolecules including, but not limited to: polynucleotides, peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Candidate inhibitor pharmacophores can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized ohgonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacologically relevant scaffolds may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
  • Inhibitors that modulate molecules involved in prion complex formation and/or PrP Sc conversion can be identified using binding sites on molecules involved in the prion complex, e.g. PrP and PPMF.
  • molecules involved in the prion complex e.g. PrP and PPMF.
  • residues 168, 172, 215 and 219 on the surface of the human PrP c molecule are known to contribute to the integrity of the PrP c -PPMF interface, and thus these molecules define functional residues on a binding site of PrP.
  • Identification of structural aspects of proteins involved in prion complex formation, such as the side chains involved in the PPMF/PrP c interaction, can define a tertiary structure to be used in an assay to design pharmacophores that modulate molecules and/or protein:protein interactions in the prion complex.
  • a dataset of compounds (small molecules, peptides, etc) having a particular tertiary structure can be identified using techniques known in the art, such as medicinal chemistry, combinatorial chemistry and molecular modeling, to determine molecules that are likely to bind to the atoms or groups of atoms of a protein involved in prion complex formation and/or conversion of PrP c to PrP Sc .
  • factors such as hydrophobicity and hydrophilicity, placement of the functional residues in a structural motif, and mutations involved in prion mediated disorders may also be taken into account.
  • the assay involves (1) matching compounds in a library with the binding site regarding spatial orientation; (2) screening candidate compounds visually using computer generated molecular display software; and (3) experimentally screening actual compounds against PrP c in the presence of PrP Sc to determine compounds which inhibit or enhance conversion of PrP c to PrP Sc .
  • This methods is shown schematically in Figure 7.
  • this portion of the molecule can serves as a template for comparison with known molecules, e.g., in a database such as Available Chemicals Database (ACD, Molecular Design Labs, 1997), or it may be used to design molecules de novo.
  • ACD Available Chemicals Database
  • the initial group of identified molecules may contain tens or hundreds of thousands or more of different non-peptide organic compounds.
  • a different or supplemental group may contain millions of different peptides which could be produced synthetically in chemical reactions or via bacteria or phage. Large peptide libraries and methods of making such are disclosed in U.S. Patents 5,266,684, issued November 30, 1993, and 5,420,246, issued May 30, 1995, which are incorporated herein by reference.
  • Non-peptide organic molecules are disclosed in PCT publication WO 96/40202, published December 19, 1996, incorporated herein by reference.
  • the initial library of molecules is screened via computer generated modeling, e.g. , computer models of the compounds are matched against a computer model of the PPMF binding site on PrP c to find molecules which mimic the spatial orientation and basic polymorphism of PPMF. This screening should substantially reduce the number of candidate molecules relative to the initial group.
  • the screened group is then subjected to further screening visually using a suitable computer program which makes viewable images of the molecules.
  • the resulting candidate molecules are then actually tested for their ability to inhibit PrP Sc formation.
  • a collection of small molecules or peptides can be screened for their ability to affect prion conversion or to mitigate an undesirable phenotype (e.g., a symptom) associated with prion-mediated disease, e.g. neuropathy.
  • the candidate pharmacophores can be screened in either non-transgenic animals or in animals that are transgenic for an alteration in PrP c , and preferably in a transgenic animal with an ablated, endogenous PrP gene, and even more preferably a transgenic animal with an ablated, endogenous PrP gene and an expressed, exogenous PrP gene from a genetically diverse animal that is the target of the pharmacophore, e.g. a PrP 00 Tg(MHuM).
  • the candidate pharmacophore is initially tested in an ex vivo cellular array of prion replication optimized using the tools of medicinal chemistry and then administered to a non-human, transgenic animal, and the effects of the candidate determined.
  • the candidate pharmacophore can be administered in any manner desired and/or appropriate for delivery of the small molecules or peptides in order to effect a desired result.
  • the candidate pharmacophore can be administered by injection (e.g. , by injection intravenously, intramuscularly, subcutaneously, or directly into the tissue in which the desired affect is to be achieved), orally, or by any other desirable means, and preferably is administered intercerebrally.
  • the in vivo screen will involve a number of animals receiving varying amounts and concentrations of the candidate therapeutic (from no therapeutic candidate to an amount of the candidate that approaches an upper limit of the amount that can be delivered successfully to the animal), and may include delivery of the pharmacophore in a different formulation.
  • the pharmacophore can be administered singly or can be combined in combinations of two or more, especially where administration of a combination of pharmacophores may result in a synergistic effect.
  • the present invention also encompasses pharmaceutical compositions comprising small molecules or peptides fitting the chemical and geometric constraints of the pharmacophores for reducing, inhibiting, or otherwise mitigating plaque formation or prion replication in a subject susceptible to neuronal degenerative disorders associated with protein deposit formation.
  • compositions of the invention preferably contain small molecules or peptides of the present invention. They may also be used in appropriate association with other pharmaceutically active compounds.
  • the following methods and excipients are merely exemplary and are in no way limiting.
  • the small molecules or peptides fitting the chemical and geometric constraints of the pharmacophores can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • the compounds can be formulated into preparations for injections by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol.
  • aqueous or nonaqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol.
  • the formulations may also contain conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • the compounds can be utilized in aerosol formulation to be administered via inhalation.
  • the compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
  • the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
  • bases such as emulsifying bases or water-soluble bases.
  • the compounds of the present invention can be a ⁇ t ⁇ iinistered rectally via a suppository.
  • the suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
  • the compounds may be formulated with other pharmaceutically active agents, particularly other agents that can modulate onset or symptoms of the condition to be treated.
  • the polycation compound can be co-administered with one or more biologically active agents that reduce protein deposit formation and/or prevent protein deposit formation.
  • biologically active agents include nonsteroid anti-inflammatory drugs (NSAIDs) or aspirin-like drugs (J.R. Vane, Semin Arthritis Rheum 26:2-10 (1997)), selective inhibitors of COX-2 (J.R. Vane Int J Tissue React, 20:3-15
  • parenteral actaiinistration of a solution of the formulations of the invention is preferably nontoxic at a dosage of 0.1 mg/mouse, which is an LD 50 of less than one at 40 mg/Kg.
  • Administration of a compound of the invention may be accomplished by any convenient means, including parenteral injection, and direct intracerebral injection or continuous (e.g., long-term or chronic) infusion.
  • the compounds of this invention can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, etc., administration.
  • the active agent may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation.
  • Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds of the present invention.
  • unit dosage forms for injection or intravenous administration may comprise the compound of the present invention in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • Implants for sustained release formulations are well-known in the art. Implants are formulated as microspheres, slabs, etc. with biodegradable or non-biodegradable polymers. For example, polymers of lactic acid and/or glycolic acid form an erodible polymer that is well-tolerated by the host.
  • the implant containing sensitizer is placed in proximity to the site of protein deposits (e.g., the site of formation of amyloid deposits associated with neurodegenerative disorders), so that the local concentration of active agent is increased at that site relative to the rest of the body.
  • the formulations can also be administered by infusion into the brain, and may be administered in either a continuous (e.g., sustained) or non-continuous fashion.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the compound for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
  • the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, are readily available to the public.
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
  • the compounds of the invention will generally be administered in dosages of 0.001 mg to 5 mg/kg body weight per day.
  • the range is broad, since in general the efficacy of a therapeutic effect for different mammals varies widely with doses typically being 20, 30 or even 40 times smaller (per unit body weight) in man than in animal models (e.g. , in the transgenic mice described herein).
  • the mode of administration can have a large effect on dosage.
  • oral dosages in the mouse may be ten times the injection dose.
  • Still higher doses may be used for localized routes of delivery.
  • a typical dosage may be: a solution suitable for intravenous administration; a tablet taken from two to six times daily; or a one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient, etc.
  • the time-release effect may be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.
  • dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Some of the specific compounds are more potent than others. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means. A preferred means is to measure the physiological potency of a given compound. EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used ( e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
  • Recombinant PrP Sc was distinguished from endogenous wild-type (wt) Mo PrP Sc by using the SHa/Mo chimeric PrP designated MHM2 that contains a binding site for the anti-SHa PrP 3F4 monoclonal antibody (mAb).
  • MHMHuA Mo residues 214, 218 and 219 were replaced with the corresponding human residue
  • MHMHuB Mo residues 226 through 230 replaced with Hu
  • MHMHu(A+B) combined replacements
  • EXAMPLE 2 IDENTIFICATION OF INHIBITORY PHARMACOPHORES Alanine scanning mutagenesis (Wells, 1991, Methods Enzymol 202:390-411) has been used to estimate experimentally the binding contribution of single residues to a protein-protein interaction.
  • residues 168, 172, 215 and 219 on the surface of the human PrP c molecule contribute the most to the integrity of the PrP-PPMF interface. Indeed, substitution for basic residues at this site increases the affinity of PrP for PPMF sufficiently to block the replication cycle.
  • those side chain sites define a plausible 3D pharmacophore target for mimetic design.
  • the sidechain coordinates of residues Q168, Q172, T215 and Q219 from the PrP(90-231) NMR structure as well as the coordinates of residue Q168 when helix B is extended to residue 166.
  • the graph theory algorithm of Ullman was employed. This approach has been utilized to aid comparison of protein structures and to search for ligands and sidechain patterns in the Protein Data bank (PDB).
  • PDB Protein Data bank
  • Our algorithm has two main stages. Firstly, the covalent connectivity of the pharmacophore sidechains are compared to the compounds. If at least one match for each of these sidechains is located within the compound, then the distances and angles between these substructures are then compared to the pharmacophore. This approach confers considerable flexibility to the program and facilitates the search for substructures within a 3D pharmacophore that are connected both covalently and non- covalently.
  • Transiently transfected ScN2a cells were incubated with lOuM KM-00561for 3 days. Protein-immunoblotting analysis of the lysates were performed before (PK-) and after (PK+) proteinase K digestion. For immunoblotting, the monoclonal antibody mAb3F4 was used. Compounds were purchased from the appropriate supplier, dissolved in 5% DMSO and incubated with scrapie infected neuroblastoma cells at a variety of concentrations. Administration of 5% DMSO with no compound was used as a negative control, and mevastatin in 5% DMSO was used as a positive control.
  • FIG. 9 shows compound 60 (the original lead compound) and the preliminary screening data at a single concentration for six of the related compounds that are commercially available.
  • Substituted pyridines are a relatively easy target for analog development.
  • the quantitative assay results can be used to determine IC 50 's for each of the analogs in Figure 10 and then to develop a synthetic plan for building a structure activity relationship.
  • mice were identified by PCR screening for transgene integration by using a Beckman robotic workstation. Tg mice from the F2 generation were sacrified and the level of the MoPrP 0 expression in the brain was determined by immunoblot by using 2 fold serial dilutions of the homogenate that were compared with FVB and Tg(MoPrP-A)4053 brain homogenates.
  • Tg(MoPrP,Q167R)Prnp + + and Tg(MoPrP,Q218K)Prnp +/+ mice were produced by repeated back-crossed of Tg(MoPrP,Q 167R)Prnp 00 and Tg(MoPrP,Q218K)Prnp 0/0 mice with FVB mice, until we obtained the F3 generation.
  • both the transgene and the wt Prnp genes were identified by PCR screening.
  • mice and Tg mice were inoculated intra-cerebrally with 30 ⁇ l of a 1% RML preparation or 10% brain homogenate prepared in PBS by using a 27-gauge disposable hypodermic needle inserted into the right parietal lobe. Beginning 50 days after inoculation, the well being of the mice was monitored daily, while the neurologic status was assessed semi-weekly as previously described (Carlson G.A., Mol. Cell Biol. , 8:5528-5540 (1988)).
  • mice were scored positive for prion disease when two or three signs of neurologic dysfunction were present and progressive deterioration of the animal was apparent by using 16 diagnostic criteria previously described (Carlson G.A., Cell, 46:503-511(1986); Scott, Cell, 59:847-857 (1989)). Once clinical signs were detected, animals were sacrified when death was clearly j-mminent. Whole brain were removed for histological and immunoblot analysis to confirm the diagnosis of scrapie.
  • mice brain homogenates Preparation of mice brain homogenates and their analysis by immunoblot.
  • 10% brain homogenates in PBS were prepared by using 5 ml volume syringe coupled to gauge needles of decreasing diameters. Repeated suctions and extrusions of the solution were done in order to crush the brain tissue. The solution is spin down for 5 min at 2000 rpm in a Beckman model centrifuge and the supernatant is then collected. Protein concentration of the brain homogenates was measured with the BCA reagent (Pierce) and corresponds to 10 mg/ml. Volumes of 500 ⁇ l of 1% brain homogenates were prepared in PBS, 2% sarcosyl, and digested with 20 ⁇ g/ml of proteinase K at a ratio of 1:50 protease to protein for 1 h at 37°C.
  • Samples non digested by proteinase K were prepared by mixing an aliquot of 10 % brain homogenate with an equal volume of SDS loading buffer. Samples were boiled for 5 min before loadinglO ⁇ l on a 12% SDS/PAGE Precast BioRad gels. Immunoblot analysis was performed according to a protocol described previously (Scott, Protein Sc., 1:986-997 (1992)). Rl and D13 Hu antibodies at l ⁇ g/ml were used to detect modified and wt MoPrP on the membrane.
  • the immunoassay was performed according to the technique described previously by Safar et al., on the SHaMoPrP (Safar, Nature Med, 4:1157-1165 (1998)).
  • the reaction is stopped by a mixture of protease inhibitors (5 rnM PMSF, aprotinin and leupeptin at 4 ⁇ g/ml).
  • the samples are precipitated with sodium phosphotungtate (NaPTA) at a final concentration of 0.32% in MgCl 2 , during 1 hour at 37°C on a shaker. After 30 min of centrifugation at 20, 000 g, the pellet is resuspended in 50 ⁇ l of PBS, 0.2% sarcosyl and divided into 2 aliquots of equal volume of 25 ⁇ l. One of the aliquot corresponds to the native measures.
  • NaPTA sodium phosphotungtate
  • the other aliquot is mixed with 25 ⁇ l of 8M guanidine-HCl and boiled for 5 min at 80°C, and corresponds to the denatured measures.
  • the samples are then quantified by immunoassay using the TRF, described elsewhere (Safar, Nature Med., 4:1157-1165 (1998)).
  • the europium-labelled Hu-D 13 antibody was used to detect modified and wt MoPrP proteins.
  • Brain tissue was immersion-fixed in 10% buffered formalin solution immediately after the mice were sacrified. The brains were embedded in paraffin and histological sections were prepared and stained with hematoxylin and eosin (H & E) for evaluation of spongiform degeneration. Brain tissue sections were also incubated with antibodies directed against glial fibrillary acidic protein (GFAP) to evaluate the degree of reactive astrocytic gliosis. Development of the sections was obtained with a secondary antibody coupled with peroxidase. The hydrolytic autoclaving technique was performed as described previously (Muramoto, Am. J. Pathol. , 140: 1411- 1420 (1992)).
  • GFAP glial fibrillary acidic protein
  • Histoblots Histoblots were performed according to the protocol described previously (Taraboulos, Proc. Natl. Acad. Sci. USA, 89:7620-7624 (1992)).
  • Residue 171 in sheep PrP corresponds to codon 167 in mice PrP (MoPrP) and codon 168 in human PrP (HuPrP).
  • Residue 219 in HuPrP corresponds to codon 218 in MoPrP.
  • mice After the microinjection step, few founders were born and 2 lines Tg(MoPrP,Q167R)1437Prap 0/0 and Tg(MoPrP,Q167R)12320Prnp 00 , were selected for further breeding.
  • the PrP expression level of those mice was established by comparing serial dilutions of their brain homogenates with that of normal FVB mice (IX PrP level) and that of normal Tg(MoPrP- A)4053 (8X PrP level) by immunoblot.
  • 16X PrP the Tg(MoPrP,Q167R)1437Prnp 0/0 .
  • Tg(MoPrP,Q167R)1437/Pr « /+ lx lx RML >409 0/10 Tg(MoPrP,Q167R)1437/P / + lx lx None >256 0/6
  • mice inoculated with RML prions were healthy more than 550 days post-inoculation and some of the mice were sacrificed in order to analyze their brain by immunoblot. The object was to determine if those mice were in a subclinical state of the disease. Indeed, mice seemed healthy but we could detect in their brain some PrP So form, the marker for prion replication and infectivity.
  • PK treated brain homogenate from normal FVB mouse inoculated with RML prions gave a strong PrP So band (Figure 10B, lane 11).
  • PK treated samples from transgenic animals inoculated with RML prions presented no MoPrP Sc Q167R band detectable by immunoblot, and showed no difference with the non inoculated transgenic mice (lanes 4-6) or the non inoculated FVB mice (lane 10), as well as the knock-out animals (lanes 7-9). Prolonged exposure of the film, up to 15 min did not allow for the detection of a MoPrP So Q167R signal.
  • mice None of the 10 Tg(MoPrP,Q167R)1437Prnp +/+ mice inoculated with RML prions showed sign of sickness at 409 days post-inoculation as shown in the data provided above. Those mice carried both wt MoPrP and mutant MoPrP,Q167R at IX level.
  • the brain tissue showed some light astrocytic gliosis which is consistent with aging as the aged-match Tg(MoPrP,Q167R)1437Prnp 0/0 mice non- inoculated present the same pattern ( Figure 12F and H).
  • hippocampus sections from Tg(MoPrP,Q167R)1437Prnp +/+ mice that remain healthy more than 409 days post-inoculation revealed the presence of numerous vacuolations associated to a severe astrocytic gliosis already at 300 days ( Figure 121 and J).
  • the neurodegeneration looks as severe as the normal FVB mice inoculated with RML ( Figure 12A and B).
  • a calibration curve was created using RML-infected FVB mice brain homogenates (Figure 14A).
  • the data showed a linear relation between the MoPrP Sc level and the dilution of the brain homogenates up to 0.00032 % (32 x 10 "5 ).
  • No PrP So is detected after dilution 10 "6 which corresponds to a ratio den/natPrP of 1.78 ⁇ 0.04.
  • Control with brain homogenates from FVB normal mice give a ratio den/natPrP of 1.09 ⁇ 0.04 which is under the cut-off value of 1.8 for infectivity ( Figure 14B).
  • a brain homogenate at a concentration of 5% was determined at 82 ⁇ 2.38. The value we found for those transgenic mice is quite high considering the fact that animals were still healthy when they were sacrificed (300 days).
  • mice non inoculated like Tg(MoPrP,Q167R)1437Prnp + + , Tg(MoPrP,Q167R)1437Prnp 0 0 and NonTg/PrnpO/0 were all equivalent, with ratio den/natPrP between 0.58 ⁇ 0.04 and 1.2 ⁇ 0.01.
  • Tg(MoPrP,Q218K)22500Prnp 0 0 and Tg(MoPrP,Q218K)21603Prnp 0/0 which expressed 32X and 16X PrP level respectively.
  • Those transgenic lines were successively back-crossed with normal FVB mice, the F3 generation of Tg(MoPrP,Q218K)22500Prnp +/+ and Tg(MoPrP,Q218K)21603Prnp + + mice were obtained.
  • Independent inoculation experiments with RML prions were started and 1/16 mice in the
  • Tg(MoPrP,Q218K)22500Prnp 00 line showed signs of scrapie around 300 days. It is likely that this animal developed a spontaneous disease because at the same time, one non-inoculated mouse also died as shown in the data provided below: Table 2. Susceptibility of Tg(MoPrP,Q218K) to RML prions
  • Tg(MoPrP,Q218K)22500Prnp 0/0 line is protected against RML inoculation but might be susceptible to a spontaneous disease due to overexpression.

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Abstract

L'invention porte sur un procédé d'identification d'un composé défini par des pharmacophores, ce composé affectant des interactions protéines-protéines. Le procédé consiste à déterminer des résidus fonctionnels d'au moins une protéine d'une interaction de protéines. Des structures tridimensionnelles sont développées à partir des positions des résidus fonctionnels et sont ensuite comparées aux composés qui comportent des structures tertiaires calculables afin d'identifier des composés ayant une orientation spatiale correspondant aux parties appropriées fonctionnelles d'une protéine induite dans l'interaction protéine-protéine.
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