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Title:
METHOD OF IDENTIFYING CRITICAL POINTS WITHIN PROTEIN-PROTEIN INTERACTIONS
Document Type and Number:
WIPO Patent Application WO/2001/087911
Kind Code:
A1
Abstract:
A method is disclosed for identifying compound defined by pharmacophores which compound affect protein-protein interactions. The method involves determining functional residues of at least one protein of a protein-protein interaction of interest. Three-dimensional structures are then developed based on the positions of the functional residues. The three-dimensional structures are then compared against compounds which have calculatable tertiary structures in order to identify compounds having a spatial orientation consistent with functionally relevant portions of a protein of the protein-protein interaction of interest.

Inventors:
PRUSINER STANLEY B (US)
COHEN FRED E (US)
JAMES THOMAS L (US)
KANEKO KIYOTOSHI (JP)
Application Number:
PCT/US2001/015832
Publication Date:
November 22, 2001
Filing Date:
May 15, 2001
Export Citation:
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Assignee:
UNIV CALIFORNIA (US)
PRUSINER STANLEY B (US)
COHEN FRED E (US)
JAMES THOMAS L (US)
KANEKO KIYOTOSHI (JP)
International Classes:
C07H17/00; C07K14/47; C12N15/10; G01N33/68; (IPC1-7): C07H17/00; C12Q1/68; G01N33/53
Other References:
GABRIEL J.-M. ET AL.: "Molecular cloning of a candidate chicken prion protein", PROC. NATL. ACAD. SCI. USA, vol. 89, October 1992 (1992-10-01), pages 9097 - 9101, XP002948406
DEMAIMAY R. ET AL.: "Late treatment with polyene antibiotics can prolong the survival time of scrapie-infected animals", J. VIROLOGY, vol. 71, no. 12, December 1997 (1997-12-01), pages 9685 - 9689, XP002948407
TELLING G.C. ET AL.: "Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein", CELL, vol. 83, 6 October 1995 (1995-10-06), pages 79 - 90, XP002948408
KANEKO K. ET AL.: "Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation", PROC. NATL. ACAD. SCI. USA, vol. 94, September 1997 (1997-09-01), pages 10069 - 10074, XP002948409
Attorney, Agent or Firm:
Bozicevic, Karl (Field & Francis LLP Suite 200 200 Middlefield Road Menlo Park, CA, US)
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Claims:
What is claimed is:
1. A mutated PrP gene, which gene comprises a condon mutation from a normal wild type codon to a different codon at least one position which position encodes a mutant amino acid located at a position where a PrP protein encoded by the mutated PrP gene interfaces with Prion Protein Modulator Factor (PPMF) and wherein the mutant amino acid prevents an interaction with PPMF which results in a conformational change of the PrP protein to a different conformation.
2. The mutated PrP gene of claim 1, wherein the gene is mutated from a normal wild type gene on an animal selected from the group consisting of a cow, a sheep, a pig and a human.
3. The mutated PrP gene of claim 2, wherein the mutation is from a wildtype codon to a codon coding for a naturally occurring basic amino acid selected from the group consisting of R, H and K.
4. The mutated PrP gene of claim 3, wherein the mutation is to a codon coding for R.
5. The mutated gene of claim 2, wherein the animal PrP gene and position of mutation are selected from the group consisting of : PrP gene of a Position I Position II Position III Position IV Bovine (5) Q171 Q175 1218 Q222 Bovine (6) Q179 Q183 1226 Q230 Sheep Q175 I218 Q222 Pig Q172 Q176 1219 Q223 Human E168 Q172 T215 Q219 wherein the mutated gene comprises only a single mutation at one position and wherein the mutation is to a codon coding for a basic amino acid.
6. The mutated PrP gene of claim 5, wherein the mutation is from a wildtype codon to a codon coding for a naturally occurring basic amino acid selected from the group consisting of R, H and K.
7. The mutated PrP gene of claim 6, wherein the mutation is to a codon coding for R.
8. The mutated PrP gene of claim 5, wherein the mutated gene is a PrP bovine (5) gene and the single mutation is from a wildtype codon at a position selected from Q171, Q175, I218 and Q222 to R at one of said position.
9. The mutated PrP gene of claim 5, wherein the mutated gene is a PrP bovine (6) gene and the single mutation is from a wildtype codon at a position selected from Q179, Q183, I226 and Q230 to R at one of said position.
10. A mutated bovine (5) PrP gene which gene comprises at least one mutated codon different from a wildtype codon at a position coding for an amino acid at a position selected from the group consisting of Q171 Q175 1218 Q222 wherein the mutated codon codes for an amino acid selected from the group consisting of R, H and K.
11. A mutated bovine (6) PrP gene which gene comprises at least one mutated codon different from a wildtype codon at a position coding for an amino acid at a position selected from the group consisting of Q179 Q183 1226 Q240 wherein the mutated codon codes for an amino acid selected from the group consisting of R, H and K.
12. A method of identifying compounds which affect proteinprotein interactions, comprising the steps of : providing a DNA sequence encoding at least a portion of at least one of two proteins involved in a proteinprotein interaction of interest; cloning the DNA sequence and producing a plurality of sequence clones; subjecting the clones to mutagenesis and producing a plurality of DNA sequence variants; operably inserting the variants into expression vectors and expressing the DNA sequence variants to produce a plurality of amino acid sequence variants; determining the effect of individual amino acid sequence variants on the proteinprotein interaction of interest.
13. The method of claim 12, further comprising: isolating an amino acid sequence variant determined to affect the proteinprotein interaction of interest.
14. The method of claim 13, further comprising : determining the sequence of the amino acid sequence variant; and determining differences between the sequence variant and at least a portion of a protein of the proteinprotein interaction of interest.
15. The method of claim 13, further comprising: providing DNA sequences encoding the amino acid sequence variant determined to affect the proteinprotein interaction of interest and operably inserting the DNA sequences into the genome of a transgenic animal; and observing the transgenic animal and determining differences between the transgenic animal and an animal without the DNA sequences inserted.
16. The method of claim 13, further comprising: determining the threedimensional shape of an amino acid sequence variant and analyzing that shape relative to at least a portion of a protein of the proteinprotein interaction of interest.
17. The method of claim 16, further comprising: using a computer program to analyze molecules which would be expected to affect the proteinprotein interaction of interest wherein the program selects molecules based on their three dimensional shape and the determined threedimensional shape of the amino acid sequence variant and creating a pharmacophore.
18. The method of claim 17, further comprising: assaying molecules which the computer indicates would be expected to affect the protein protein interaction of interest.
19. The method of claim 16, wherein the threedimensional shape is determined using an analytical methodology selected from the group consisting of NMR spectroscopy and Xray crystallography.
20. The method of claim 12, wherein the DNA sequence encodes the entire amino acid sequence of one protein of the proteinprotein interaction.
21. The method of claim 12, wherein the DNA sequence encodes a PrP protein.
22. A compound defined by a pharmacophore characterized by an ability to affect a proteinprotein interaction of interest said pharmacophore identified by the method of claim 12.
23. The compound of claim 22, wherein the compound is characterized by an ability to modulate conversion of PrPC to PrPSC in vivo.
24. The compound of claim 23, wherein the compound comprises a small molecule characterized by binding PPMF.
25. The compound of claim 23, wherein the compound comprises a small molecule characterized by binding PrP.
26. The compound of claim 23, wherein the compound comprises an epitope on PrPC which binds PPMF.
27. The compound of claim 26, wherein the compound comprises amino acids corresponding to residues 90231 of the human PrP protein.
28. The compound of claim 26, wherein the compound comprises an amino acid sequence corresponding to a sequence overlapping a residue selected from the group consisting of residues 168,172,215 and 219 of the human PrP protein.
29. The compound of claim 23, wherein the compound comprises an epitope on PPMF which binds PrP.
30. The compound of claim 23, wherein the compound is defined by a mimetic pharmacophore that facilitates conversion of PrP° to PrP".
31. The compound of claim 29, wherein the epitope of PrPC comprises an amino acid sequence corresponding to residues 89143 of the MoPrP protein.
32. The compound of claim 31, wherein the eptiope further comprises amino acids corresponding to residues 2331 of the MoPrP protein.
33. The compound of claim 31, wherein the eptiope further comprises amino acids corresponding to residues 225231 of the MoPrP protein.
34. The compound of claim 27, wherin the compound further comprises a mutation corresponding to the human GSS mutation P101L.
35. The compound of claim 23, wherein the wherein the compound is characterized by repressing conversion of PrP° to PrPSc.
36. The compound of claim 29, wherein the compound is a negative image of a human PrP structure encompassing residues 168,172,215 and 219 of human PrP°.
37. The compound of claim 23, wherein the compound is characterized by inhibiting sheet formation.
38. A method of facilitating conversion of PrPC to PrPSc, comprising administering a compound defined by the mimetic pharmacophore of claim 25.
39. A method of repressing conversion of PrPC to PrPSc, comprising administering an inhibitory compound of claim 35.
40. An assay to identify a compound defined by a PrP pharmacophore, said assay comprising the steps of : determining functional residues of the PrP protein involved in prion complex interactions; developing a plurality of three dimensional structures based on these functional residues; comparing the plurality of three dimensional structures with a series of compounds having calculatable tertiary structures; and identifying compounds having a spatial orientation consistent with binding PrP at the determined functional residues.
Description:
METHOD OF IDENTIFYING CRITICAL POINTS WITHIN PROTEIN-PROTEIN INTERACTIONS FIELD OF THE INVENTION 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.

BACKGROUND OF THE INVENTION Sequencing of the human genome indicates that it is comprised of approximately 30,000 genes which encode proteins (Venter (Celera) Science (2001)). Although the proteins will have some biological significance, it is estimated that a minority of the proteins expressed will, at least in the near term, be found to have significant pharmacological relevance. Thus, there are estimated to be about 6,000 to 12,000 proteins which have significant biological importance and as such the potential for providing commercially valuable drugs.

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. For example PrP proteins can exist in a normal PrP° configuration or in a disease related PrPSc 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).

From a clinical perspective, 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. 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. While the infectious prion diseases represents less than 1% of all cases, their link to mad cow disease in the U. K. (new variant CJD), growth hormone inoculations in the U. S. and France (iatrogenic CJD), and ritualistic cannibalism in the Fore tribespeople (Kuru) have raised the public awareness of this facet of the disease (Devillemeur, et al. (1996) Neurology, 47: 690-695; Hill, et al. (1997) Nature, 389: 448-450; Goodfield (1997) Nature, 387: 841-841).

A critical advance in our understanding of prion diseases came with the partial purification of a proteinaceous material that retained the ability to reinfect laboratory rodents (McKinley, et al.

(1983) Cell, 35: 57-62). Micro-sequencing and molecular biologic tools led to the cloning of the prion gene, a normal component of mammalian and avian genomes (Prusiner, et al. (1984) Cell, 38 : 127-134). 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, etal. J : Biol. Chem., 269: 16810-16820 (1994); Hegde, et al., Science 279: 827-34 (1998)). Biophysical characterization of the deglycosylated recombinant PrP refolded into a monomeric form resembling the normal cellular isoform (PrP°) reveals a two domain molecule with an N-terminal region (57-89) that binds 4 Cud atoms per chain (Viles, et al. (1999) Proc. Natl.

Acad. Sci. USA, 96: 2042-2047) and a C-terminal region (124-231) that contains 3 substantial helices and 2-3 residue p-strands joined by 2-3 hydrogen bonds (see Figure 1) (Riek, et al. (1996) Nature, 382: 180-182 ; James, et al. (1997) Proc. Natl. Acad. Sci. USA, 94: 10086-10091; Donne, et al. (1997) Proc. Natl. Acad. Sci. USA, 94: 13452-13457). By contrast, the disease causing form of the prion protein (PrPSc) is a multimeric assembly substantially enriched in p-sheet structure (40% p-sheet, 30% a-helices as judged by FTIR spectroscopy) (Pan, et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 10962-10966). Immunologic studies of PrPs suggest that the conformational change is largely in the region from residues 90-145 or perhaps 175 (Peretz, et al. (1997) J MoL Biol. 273: 614-622) These features have been codified in a model of PrPSc (see Figure 2) that emphasize the dramatic conformational distinction between PrPC and PrPsc.

A large number of genetic and transgenetic studies have helped to cement the role of the prion protein in the pathogenesis of this group of neurodegenerative diseases. First, a variety of genetic linkage studies of kindreds with familial prion diseases mapped the defect to the Prn-p locus.

Subsequent studies identified specific point mutations that caused inherited disease (Hsiao, et al.

(1989) Nature, 338: 342-345; Dlouhy, et al. (1992) Nat. Genet., 1: 64-67; Petersen, et al. (1992) Neurology, 42: 1859-1863 ; Poulter, et al. Brain 115: 675-85 (1992); Gabizon, et al. (1993) Am. J Hum. Genet., 53: 828-835). These loci are shown in Figure 3. Subsequently, the Prn-p gene was knocked out in mice with no obvious phenotypic sequelae (Biieler, et al. (1992) Nature, 356: 577- 582). While wild type mice will develop a prion disease-180d after intracerebral inoculation, the hemizygous animals require 400d to succumb to an infectious inoculum and the homozygous knockouts are resistant to prion infection (Prusiner, et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 10608-10612 ; Biieler, et al. (1994) Molecular Medicine, 1: 19-30). Transgenic mice carrying a sufficiently high number of copies of mutant gene (the human GSS mutation PI OIL) on the knockout background develop a spontaneous neurodegenerative disease that is faithful to the neuropathologic expectations developed from a study of the human kindreds. Knockout mice carrying a redacted form of the PrP transgene (90-141 ; 175-231) also develop a prion disease upon inoculation with full length RML prions (Supattapone, et al. (1999) Cell, 96: 869-878). The infection process is more efficient with the"mini"RML prion demonstrating that an artificial prion can be created and that replication efficiency demands fidelity at the amino acid sequence level.

While the predominantly a-helical PrPC and-sheet rich PrPSC have exceptionally different secondary and tertiary structures as judged by CD, FTIR, and NMR spectroscopy (Caughey, et al.

(1991) Biochemistry, 30: 7672-7680; Pan, et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 10962-10966; Riek, et al. (1996) Nature, 382 : 180-182; James, et al. (1997) Proc. Natl. Acad. Sci. USA, 94: 10086- 10091 ; Donne, et al. (1997) Proc. Natl. Acad. Sci. USA, 94: 13452-13457), they appear to share a common amino acid sequence and disulfide bridge (Cohen & Prusiner (1998) Annual Review of Biochemistry, 67: 793-819). Recent work has shown that a conformational change that is aided by an auxillary molecule is an obligatory step in PrPSc forrnation (Telling, et al. (1995) Cell, 83: 79-90; Kaneko, et al. (1997) R Mol. Biol. 270: 574-586). The exceptional stability of PrPsc and the marginal stability of PrPC together with a variety of transgenetic and cellular transfection studies have led to the conclusion that PrPC is a kinetically trapped intermediate in the folding of PrPsc (Cohen & Prusiner (1998) Annual Review of Biochemistry, 67: 793-819). This kinetic barrier can be reduced by exogenous administration of the PrPSc 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.

(1997) 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, et al. (1993) Nature, 362: 543-546; Come, et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 5959-5963; Zhang, et al. (1995) J MoL Biol., 250: 514-526; Nguyen,

et al. (1995) Biochemistry, 34: 4186-4192; Kaneko, et al. (1997) J MoL BioL 270: 574-586. In particular, peptides chosen from the region 90-145 are compatible with a-helical, irregularly coiled, and (3-sheet rich conformations when characterized under different conditions (Zhang, et al. (1995) J.

Mol. Biol., 250 : 514-526). Furthermore, catalytic amounts of (3-sheet rich peptides can facilitate the conformational conversion of peptides with distinct structures into 0-sheet rich isoforms (Gasset, et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 10940-10944;; Nguyen, et al. (1995) Biochemistry, 34: 4186-4192).

More than a million cattle infected with bovine spongiform encephalopathy (BSE) have entered the food chain in the U. K., and fears that BSE has been transmitted to man were raised when new variant (CJD) appeared in the U. K. Since it is hard to predict the number of cases of this disease that may arise in the future, initiation of the search for an effective therapy is essential. No systematic drug discovery efforts have been attempted owing to difficulties in developing a robust screening assay. Many isolated observations with potential therapeutic implications have been made.

For example, several compounds are known to inhibit PrPSc formation in scrapie-infected neuroblastoma cells such as sulfated glycans and the amyloid stain Congo Red (Caughey & Raymond (1991) J : Biol. Chem., 266: 18217-18223). However, these compounds are unable to cross the blood-brain barrier, and therefore have no therapeutic benefit after the infection has reached the central nervous system (Caughey, et al. (1993) J Yirol., 67: 6270-6272; Ehlers & Diringer (1984) R Gen. Virol., 651325-1330; Farquhar & Dickinson (1986) J : Gen. Virol., 67 : 463-473). Other candidates such as polyene antibiotics (Demaimay, et al. (1997) R Virol., 71: 9685-9589) and anthracyclines (Tagliavini, et al. (1997) Science, 276: 1119-1122) have very low therapeutic indices.

Tetrapyrroles inhibit PrPsc formation and there is some evidence that they can cross the blood-brain barrier (Caughey, et al. (199) Proc. Natl. Acad. Sci. USA, 95: 12117-12122), but at this time, the mechanism of action and in vivo efficacy of these compounds is unknown.

There is a need in the art for molecules with the ability to prevent and/or halt the progression of prion-mediated disorders.

SUMMARY OF THE INVENTION Dominant negative mutations in a PrP gene are described which result in inhibition of prion formation. Understanding information regarding these mutations results in (1) improving rational drug design technology thereby providing drugs which inhibit prion formation; and (2) improving gene therapy technologies making it possible to produce genes which can produce prion resistant livestock. The drugs and the proteins of the mutated genes have similarity in their 3-dimensional shapes which result in similar physiological characteristics in terms of inhibiting prion formation.

A method of identifying compounds which affect protein-protein interactions is disclosed.

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.

In a preferred embodiment 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. Using the structure 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.

Thus, 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. Further 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 PrPC to PrPsc. The molecules disclosed can be small molecules, peptides or protein analogs, e. g. analogs of PrPC. 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 (3-sheet formation. In another embodiment, the molecules of the invention promote PrPC conversion to PrPSc, e. g. by binding to PrPC and facilitating a conformational change from PrPC to PrPSC. The molecules may be designed to be species specific, meaning that the molecule will only bind to PrPC or Prion Protein Modulator Factor (PPMF) of the same or a genetically similar species.

The presence of PPMF is needed to convert PrPC to PrPSc. Alternatively, the molecules of the invention may be designed to bind to PrPC 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 prPc or PPMF can present the interaction needed to bring about the conversion of PrP° to PrPsc.

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 PrPsc 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. 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.

In a preferred embodiment, 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. Optionally or alternatively, 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.

An advantage of the present invention is that infectivity of prions in a sample can be determined rapidly.

In another aspect of the invention, 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 PrPC to PrPSc and/or progression of neurodegeneration by any number of mechanisms, including but not limited to binding a molecule involved in prion complexing, e. g. PrPC or PPMF or inhibiting p-sheet formation or elongation Yet another aspect of the invention features a method of repressing conversion of PrPC to PrPSC, 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 PrP3' 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 PrPSc replication complex (PrP°, PrPSc, PPMF) at these functional residues.

These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the molecules as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a ribbon drawing of the NMR of rSHa PrP (90-231). Residues 90-115 are not shown. a-helical regions are shown in mauve and 0-strands are shown in cyan.

Figure 2 is a model of the putative structure of PrPSc highlighting the dramatic increase in the (3-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 a-helices A, B and C and the two 0-strands S 1 and S2. Above the line of the human sequence are mutations that cause prion disease. Below the lines are polymorphisms, some but not all of which are known to influence the onset as well as the phenotpye of disease.

Figure 4 is an illustration of the distinction between thermodynamic and kinetic models for the energetics of the conversion of PrP° carrying the wild-type (WT) and mutant (MUT) sequences into PrP". AG is the free energy difference between the PrPC and PrP"states and AG is the activation energy barrier separation these two states. A5GWTMUT is the difference between AGWT and AGMUT. The free energy diagrams for the wild-type sequences are shown (solid lines), as are the mutant sequences (broken lines).

Figure 5 is an approach to small molecules in computer screening for inhibitors of PrPSc replication based on blocking the PrPC : PPMF interaction.

Figure 6 shows dependence of Cp-60 inhibition on (MHM2) PrP" concentration using the Time Resolved Fluorescence (TRF) technique of Safar et al (1998). 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 immunoassay 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, 1OB and 10C are images of gels run to demonstrate the presence or lack of protease resistant PrPSc.

Figures 11A and 11B are images of gels run to show the presence of or a lack of a protease resistant PrP" Figures 12A-12L are images of brains showing the presence of a plaque due to the presence of a PrPsc.

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" in different transgenic mice brains.

Figure 15A and 15B are each images of gels run showing the presence or lack thereof of PrPSc in different transgenic mice brains.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Before the present peptides, small molecules, and assay methodology are described, it is to be understood that this invention is not limited to particular peptides, small molecules, assay methods, described and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates

of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DEFINITIONS The term"PPMF"is used for Prion Protein Modulator Factor, which is a protein which can be glycosylated and is characterized by binding to PrPC and facilitating a conformational change from PrPC to PrPSc. 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.

The term"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. Thus, as used here 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 PrPsc activity by interaction with (1) the discontinuous epitope on PrPC to which PPMF binds or (2) the surface of PPMF which binds to PrPC. 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 PrPsc, PPMF, PrP°, or other proteins involved in the prion complex that facilitate the conversion of PrPC to PrP". The term pharmacophore defines properties of peptides, peptide analogs and small molecules.

The term"small molecule"as used herein 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.

The terms"treatment","treating"and"treat"and the like are used herein to generally mean 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. The"treatment"as used herein covers any treatment of a disease in a mammal, particularly a cow, pig, sheep, mouse or human, and includes: (a) preventing prion disease or symptoms from occurring in a subject which may be predisposed to the disease or symptom or infected with prion particles but has not yet been diagnosed as having a prion disease which can include the use of gene therapy; (b) inhibiting prion disease symptoms, i. e., arresting the development of prion disease ; or

(c) relieving a prion disease symptom, i. e., causing regression of prion disease or prion disease symptoms.

The term"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.

The term"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. For example, the 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° (for five different proteins) are shown in Table 1.

The term"FVB"refers to a mouse strain commonly used in the production of transgenic mice. For purposes of this invention it should be noted that the mouse prion protein (PrP) gene is intact and mouse PrP is therefore expressed at normal levels.

The term"Prnp°/°"or"Prnp-Abl"refers to a transgenic animal which has its PrP gene ablated with the"°'°"indicating that both alleles are ablated whereas"°'+"indicates only one is ablated.

Specifically, 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.

The term"sporadic CJD"abbreviated as"sCJD"refers to the most common manifestation of Creutzfeldt-Jakob Disease (CJD). This disease occurs spontaneously in individuals with a mean age of approximately 60 at a rate of 1 per million individuals across the earth.

The term"Iatrogenic CJD"abbreviated as"iCJD"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.

The term"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.

The term"Gerstmann-Strassler-Scheinker Disease"abbreviated as"GSS"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 GSS.

The term"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 PrPSc molecules encoded by a PrP gene which expresses PrPC which changes conformation to become PrPSc. 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. Four 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). As used herein 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.

The terms"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. The term"PrP gene"refers generally to any gene of any species which encodes any form of a PrP amino acid sequences including any prion protein.

Some commonly known PrP sequences are described in Gabriel et al., Proc. Natl. Acad. Sci. USA 89 : 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. Thus, 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.

The terms"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. For example, 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.

The terms"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. For example, 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.

The terms"genetically diverse animal"and"genetically diverse mammal"are used to describe an animal which includes a native PrP codon sequence of the host animal which differs from the genetically diverse test animal by 17 or more codons, preferably 20 or more codons, and most preferably 28-40 codons. Thus, 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. In general, prions of a given animal will not infect a genetically diverse animal and PPMF of a given animal will not bind to PrPC of a genetically diverse animal.

The terms"ablated prion protein gene,""disrupted PrP gene,""ablated PrP gene,""PrP°/°" and the like are used interchangeably herein to mean 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.

Examples of nonfunctional PrP genes and methods of making such are disclosed in Bueler, H., et al "Normal development of mice lacking the neuronal cell-surface PrP protein"Nature 356,577-582 (1992) which is incorporated herein by reference. Both alleles of the genes are disrupted.

The terms"susceptible to infection"and"susceptible to infection by prions"and the like are used interchangeably herein to describe a transgenic or hybrid test animal which develops a prion disease if inoculated with prions which would normally only infect a genetically diverse test animal.

The terms are used to describe a transgenic or hybrid animal 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).

The terms"resistant to infection","resistant to infection with prions"and the like mean 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 PrPC/PPMF binding site.

The term"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.

ABBREVIATIONS USED HEREIN INCLUDE : BSE for bovine spongiform encephalopathy; CJD for Creutzfeldt-Jakob Disease; CNS for central nervous system;

FFI for fatal familial insomnia; GSS for Gerstmann-Strassler-Scheinker Disease; Hu for human; 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; MHu2MPrPSc for the scrapie isoform of the chimeric human/mouse PrP gene; Mo for mouse; MoPPMF for mouse Prion Protein Modulator Factor; Mo PrP for a mouse PrP protein; MoPrPSc 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°/° for ablation of both alleles of an endogenous PrP protein gene, e. g., the Mo PrP gene; PrPcJD for the CJD isoform of a PrP gene; PrPsc for the scrapie isoform of the PrP protein; ScN2a for persistently infected scrapie mouse neuroblastoma cells also expressing (MHM2) PrPC ; SHa for a Syrian hamster; SHa PrP for a Syrian hamster PrP protein; Tg for transgenic ; Tg (BovPrP) for transgenic mice containing the complete cow PrP gene; Tg (HuPrP) for transgenic mice containing the complete human PrP gene; Tg (HuPrP)/Prnp°/° for a hybrid mouse obtained by crossing a mouse with a human PrP protein gene (HuPrP) with a mouse with both alleles of the endogenous PrP protein gene disrupted; Tg (MHu2M) mice are transgenic mice of the invention which include the chimeric MHu2M gene ; Tg (MHu2M)/Prnp°/° 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+/°) 81/Prn-p+/° for a particular line (81) of transgenic mice expressing SHa PrP, +/0 indicates heterozygous; and 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.

However, 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.

Preferably, 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.

The method can be carried out in a variety of different ways. For example, 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.

Further, it is possible to determine 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.

Using the information regarding the three-dimensional shape it is possible to use computer programs in order to analyze molecules which would be expected to effect the protein-protein interaction of interest wherein the program selects molecules based on their three-dimensional shape and

determines three-dimensional amino acid variants and creates pharmacophores. Using 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. Where the three- dimensional shape is determined it is possible to use 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. For example, the invention includes compounds which are characterized by the ability to modulate the conversion of PrP° to PrPSc in vivo. Such molecules include the small molecules characterized by binding PPMF and molecules which bind to PrP".

In a specific version of the invention 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 PrPC at the determined functional residues.

In one aspect of the invention 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. By making certain mutations in the PrP gene the resulting PrP protein can prevent the formation of the pathogenic form of the PrP protein i. e. prevent prion formation.

Accordingly, 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 PrPC to PrPS.

It is understood, that with respect to humans that transgenic humans would not, in general be produced. However, 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.

MUTATED PrP GENES An important aspect of the invention described here is a 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.

In a hamster PrP gene the four positions of interests are Q168, Q172, Q215 and Q219.

However, there are differences between PrP genes and as such the position of interest will be different positions for different species of animals. For example, the polymorphisms at positions equivalent to 168,172,215 and 219 in a hamster as follows: TABLE OF RESIDUE POSITIONS AT PrPC/PPMF INTERFACE ANIMAL POSITION I POSITIONII POSITIONIII POSITIONIV mouse Q167 Q171 V214 Q218 hamster Q168 Q172 T215 Q219 human E168 Q172 1215 E219 bovine (6) Q179 Q183 I226 Q230 bovine (5) Q171 Q175 1218 Q222 sheep Q171 Q175 1218 Q222 pig Q172 Q176 1219 Q223 Using these position coordinates those skilled in the art will be able to determine equivalent positions in the PrP protein of other animals and specifically in other livestock animals such as goats, chickens, horses, etc. 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. glutamin. 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.

Preferably the wild-type codon coding for Q at position 179 is replaced with a codon coding to R at position 179. Such a mutated bovine PrP gene could be used to produce a transgenic cow which would be resistant to prion disease. Alternatively, 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. Similarly, the Q230 can be replaced with any basic amino acid. Still further, the I at 218 can be replaced with any basic amino acid.

Based on the above it will be understood that for each wild-type PrP gene there are four different mutants of the invention which could be used to produce an animal resistant to prion infection. One gene would have a substitution at the hamster equivalent of 168, one gene would have a substitution at the hamster equivalent of 219, one gene would have a substitution at the hamster equivalent of T215 and final one would have a substitute at the hamster equivalent 219. The substitution is preferably Q to R or I to R. However, the substitution (s) can be Q to any of R, H or K or I to any of R, H. or K. This results in multiple possibilities for mutant PrP genes useful in producing prion resistant mammals.

For example 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 PrPC-PPMF INTERFACE PrPC forms a complex with PPMF and PrPsc resulting in a ternary complex of PPMF/PrP° /PrPsc. After the conversion of PrPC to PrPsc, the complex dissociates due to the lack of affinity of PrPSc for PPMF. To inhibit this PrPSc replication cycle, pharmacophores fitting this geometric and chemical description are used to interfere with either the PrPSc/PrPC or PPMF/PrPC interface. The inhibitors can be used to prevent the initial conversion of PrP° into prions, or later prevent the progression of prion formation.

The PrPsc 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 PrPC. 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.

CONVERSION THEORY In general, deposition diseases such as the prion diseases appear to follow the form: Ae A* e B < Bn where 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 and Bn is the multimeric material that is pathogenic and relatively difficult to recycle. For the prion diseases, PrPC and PrP"correspond to states A and Bn where A is largely helical and monomeric and Bn is p-rich and multimeric.

Two types of kinetic barriers can be imagined that restrict the formation of Bn If the protein in question is relatively large and the conformations of A and B are quite different, then a largely enthalpic kinetic barrier could exist where B can act as a template to reduce the barrier to the conformational change. Alternatively, if a smaller peptide is undergoing the dramatic conformational reorganization (as with Ap in Alzheimer's disease (Jarrett & Lansbury, Ann N YAcad Sci. 695: 144-8 (1993) or calcitonin in medullary carcinoma of the thyroid or a smaller conformation change is sufficient (as in sickle cell hemoglobin fibril formation (Mirchev & Ferrone, JMol Biol. 265: 475-9 (1997)) the rate limiting step may be the formation of a stable multimeric nucleus (Bn).

Without being bound to any particular theory, PrPSc formation is believed to require an escape from the kinetically trapped monomeric PrPC structure. See Figure 4. For example, PrPsc 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. In transgenic animals carrying a mutant gene that causes a spontaneous prion disease, the time to disease is halved when the founder animals are crossed to yield a progeny carrying twice the transgene dose (Cohen & Prusiner, Annu 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.

The concept of PrPSc providing a template to assist the conversion of nascent PrPC molecules implies that some PrPSc templates should be more efficient at stabilizing the nascent PrPScmolecule than others in the initial phase of disease propagation. This can be seen in the species barrier to prion transmission where SHa PrPSc is less efficient than MoPrPsc in causing disease in mice, and HuPrPsc is even less efficient than SHa PrPsc (Scott, et al., Cell 73 : 979-88 (1993); Telling, et al., Proc Natl

Acad Sci US. 91: 9936-40 (1994); Scott, etal, Proc NatlAcadSci USA, 94: 14279-84 (1997). For humans, HuPrPsCprovides the most effective inoculum, but it has become clear the BoPrPSc can cause disease in humans albeit at a much lower frequency (Hill, et al., 1993). There is no evidence that SHaPrPSchas ever caused disease in humans. It follows that one portion of the molecule is involved in the PrPSc species specific features of the inoculum while a distinct surface of the molecule is available for interaction with a distinct species specific PPMF molecule. Dominant negative mutations to the PrP gene have been identified that prevent wild type PrPSc replication apparently by sequestering PPMF (Telling, et al., Cell 83 : 79-90 (1995); Kaneko, et al., Proc Natl Acad Sci USA 94: 10069-74 (1997)). Epidemiologic studies have suggested the existence of dominant negative mutations in humans and sheep that act via this mechanism (Prusiner, et al., Proc Natl Acad Sci U S A 95 : 13363-83 (1998)).

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 PrPC-PPMF interaction: 168,172,215 and 219. In the NMR structures of various recombinant forms of PrPC, 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. However, if the second a-helix is extended by one turn toward the N-terminus to include residue 168, a continuous surface is formed (James, et al., Proc Natl Acad Sci USA 9410086-91 (1997). Wallace, et al., PNAS (1999) have suggested that this reorganization of the molecule creates an A* state that is conversion competent.

Pharmacophores of the present invention take advantage of this conversion process, and preferably have structural aspects that prevent the conversion of PrP° to its conversion competent state (e. g. inhibitory pharmacophores). In one example, by binding to PrPC or to a protein that interacts with PrP (e. g. PPMF at its PrPC binding domain) a pharmacophore of the present invention may block PrPC : 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. Thus, if a basic and/or polar amino acid is present at the

critical site that site could be replaced with an acidic and/or nonpolar amino acid. With these criteria in mind some trial and error would be required. 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.

There are a number of known pathogenic mutations in the human PrP gene. Further, there are known polymorphisms in the human, sheep and bovine PrP genes. The following is a list of such mutations and polymorphisms: PATHOGENIC MUTATION TABLE Pathogenic Human Sheep Bovine human mutations Polymorphisms Polymorphisms Polymorphisms 2 octarepeat insert Codon 129 Met/Val Codon 171 Arg/Gln 4 octarepeat insert Codon 219 Glu/Lys Codon 136 Ala/Val 5 octarepeat insert Codon 154 Arg/His 5 octarepeat insert 6 octarepeat insert 6 octarepeat insert 7 octarepeat insert 7 octarepeat insert 8 octarepeat insert 9 octarepeat insert Codon 102 Pro-Leu Codon 105 Pro-Leu Codon 117 Ala-Val Codon 145 Stop Codon 178 Asp-Asn Codon 180 Val-Ile Codon 198 Phe-Ser Codon 200 Glu-Lys Codon 210 Val-Ile Codon 217 Asn-Arg Codon 232 Met-Ala In order to provide further meaning to the above chart demonstrating the mutations and polymorphisms, one can refer to the published sequences of PrP genes. For example, a chicken, bovine, sheep, rat and mouse PrP gene are disclosed and published within Gabriel et al., Proc. Natl.

Acad. Sci. USA 89 : 9097-9101 (1992). The sequence for the Syrian hamster is published in Basler et al., Cell 46 : 417-428 (1986). The PrP gene of sheep is published by Goldmann et al., Proc. Natl.

Acad. Sci. USA 87 : 2476-2480 (1990). The PrP gene sequence for bovine is published in Goldmann et al., J. Gen. Virol. 72: 201-204 (1991). The sequence for chicken PrP gene is published in Harris et al., Proc. Natl. Acad. Sci. USA 88 : 7664-7668 (1991). The 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). The PrP gene sequence for mouse is published in Locht et al., Proc. Natl. Acad. Sci. USA 83 : 6372-6376 (1986). The PrP gene sequence for sheep is published in Westaway et al., Genes Dev. 8 : 959-969 (1994). These publications are all incorporated herein by reference to disclose and describe the PrP gene and PrP amino acid sequences.

LOCALIZATION OF PHARMACOPHORES WHICH INHIBIT PrPSCREPLICATION PrPSc formation is likely to take place in the caveolar space. Thus, 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.

ASSAYS TO IDENTIFY INHIBITOR PHARMACOPHORES 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 oligonucleotides 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 PrPSc conversion can be identified using binding sites on molecules involved in the prion complex, e. g.

PrP and PPMF. For example, residues 168,172,215 and 219 on the surface of the human PrPC molecule are known to contribute to the integrity of the PrPC-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/PrPC 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. Specifically, 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 PrPC to PrPsc. Optionally, 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.

In a preferred embodiment of the assay of the invention, 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 PrPC in the presence of PrPsc to determine compounds which inhibit or enhance conversion of PrP° to PrPSC. This methods is shown schematically in Figure 7.

Once the functional residues of the target protein (e. g. PPMF) are identified, 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. In one example, 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. Libraries of 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 PrPC 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 PrPSc formation.

Screening of Candidate Pharmacophores In Vivo 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°, 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°Tg (MHuM).

In general, 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. For example, 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. Normally, 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.

To speed development the pharmacophore is tested initially in a cellular system such as ScN2a cells or the cellular system disclosed in USSN 09/318,888, which is incorporated herein by reference.

PHARMACEUTICAL COMPOSITIONS 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.

Formulations Pharmaceutical formulations 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.

For oral preparations, 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.

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. 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.

Furthermore, the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered 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.

For use in the subject methods, 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. For example, to treat Alzheimer's disease or CAA, 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. Examples of such compounds 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 JTissue React, 20: 3-15 (1998)), protein phosphatases that act on microtubule-associated protein tau protein phosphatases (K.

Iqbal, Ann N YAcad Sci 777 : 132-8 (1996)), modulators of APP proteolytic enzymes and apoE activity (P. T. Lansbury Jr, Arzneimittelforschung 45: 432-4 (1995)), inhibitors of polysaccharides, such as glycosaminoglycan and proteoglycans, (B. Leveugle et al., Neuroreport 5: 1389-92 (1994)) and the like. The additional active ingredients may be conjugated to the pharmacophore or may be contained separately within a formulation.

The formulations of the invention have the advantage that they are non-toxic in tested forms of administration. For example, parenteral administration of a solution of the formulations of the invention is preferably nontoxic at a dosage of 0.1 mg/mouse, which is an LD50 of less than one at 40 mg/Kg.

Administration 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. As such, 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. Similarly, 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. Methods, formulations, and devices suitable for delivery to the brain in a continuous (e. g., chronic) or non- continuous (e. g., single, discrete dose per administration) fashion are described in, for example, U. S.

Patents 5,711,316; 5,832,932; 5,814,014; 5,782,798; 5,752,515; 5,735,814; 5,713,923; 5,686,416; 5,624,898; 5,624,894; 5,124,146 ; and 4,866,042 (delivery of genetic material).

The term"unit dosage form,"as used herein, 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. Moreover, 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.

Dosage Depending on the patient and condition being treated and on the administration route, 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). Similarly the mode of administration can have a large effect on dosage. Thus for example 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.

Those of skill will readily appreciate that 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.

EXAMPLE 1 BINDING SITE IN PrPC for PPMF An examination of the amino acids which distinguish Hu PrP from Mo PrP shows only seven residues at the C-terminus (168-231) that are different. Four of these residues are close to the glycophosphatidylinosytol (GPI) anchor attached to Ser231 while the remaining three residues were within or near the C-terminus of a postulated a-helix which has been confirmed by NMR structural studies. To identify the critical binding site within PrPC for PPMF the seven residues were divided into two groups: those at the C-terminal end of the last a-helix (HuA) and those at the extreme C- terminus (Hub). The Mo residues were replaced with Hu counterparts in positions that were critical for binding of PrPC to Mo PPMF to determine the effect of such on inhibiting the formation of recombinant PrPS. Recombinant PrPSc was distinguished from endogenous wild-type (wt) Mo PrP" by using the SHa/Mo chimeric PrP designated MHM2 that contains a binding site for the anti-SHa PrP 3F4 monoclonal antibody (mAb).

Three chimeric constructs, denoted as MHMHuA (Mo residues 214,218 and 219 were replaced with the corresponding human residue), MHMHuB (Mo residues 226 through 230 replaced with Hu), and MHMHu (A+B) (combined replacements), were transiently transfected into scrapie infected Mo neuroblastoma (ScN2a) cells. Neither MHMHu (A+B) nor MHMHuA was converted into PrPSc as judged by the acquisition of protease resistance. By contrast, MHMHuB was converted into PrPsc as efficiently as the control MHM2. These results indicate that Mo PPMF did not bind to MHMHu (A+B) or to MHMuA but did bind to MHMHuB and MHM2, both of which were converted into PrPsc.

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.

For prion replication, residues 168,172,215 and 219 on the surface of the human PrPC 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. Thus, those side chain sites define a plausible 3D pharmacophore target for mimetic design. In particular, the sidechain coordinates of residues Q 168, Q 172, 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.

Since basic residues at Q168, Q172 and Q219 and acidic or hydrophobic residues at T215 increase the affinity of PPMF for PrP°, Arg, Lys, His, Asp, Glu and Trp were modeled onto the relevant residue positions of the PrP (90-231) NMR structure using the program SCWRL. Bower, Cohen & Bunbrack, R Molec. Biol. (1997). Using these coordinates, a dataset of 3D pharmacophores were created in a combinatorial fashion using all the atoms comprising a sidechain as well as only the functional atoms. The fact that residue Q168 may be part of helix B rather than occupy the position suggested by the PrP (90-231) NMR structure was also taken into account.

Mutational data suggests that substitution of more than one basic residue at the PPMF site led to PrP molecules that could not inhibit scrapie formation in a dominant negative fashion. With this mind, the relevant 3D pharmacophores that had more than one basic residue were filtered from the 3D pharmacophore dataset as necessary. This resulted in-1000 templates that were compared with the 210, 000 compounds present in the Available Chemicals Database (ACD, Molecular Design Labs, 1997) for compounds that mimic both the spatial orientation and basic polymorphism present in the dataset of 3D pharmacophores.

To speed up the search through the compound database, 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). 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. 45 minutes of CPU time were required to perform a single search with a 3D pharmacophore so-15 days were required using a Silicon Graphics Indigo 2 workstation to search with-1000 3D pharmacophores templates against the ACD. Compounds were scored according to the number of 3D pharmacophore elements that they matched as well as the number of functional groups that they present (where 4 is the optimal solution which is equal to the number of residues present on the PPMF binding site on PrPC).

Following visual screening of the-1000 compounds for a variety of structural medicinal and toxicologic concerns, a total of 63 compounds were selected for screening. The effect of the 63 compounds tested at a 20, uM concentration on transiently transfected ScN2a cells is summarized below in Table 1.

Nucleic Acid derivatives Number Compound Name Results 3 a--Adenosine Negative 7 1, N6-Ethenoadenosine-5'-Positive Monophosphate 10 Cytidylyl (3'5') Guanosine Negative 17 2'3'-Di-0-Acetylguanoside Negative 18 Purine Riboside Positive 26 Hydroxyguanidino-purine Negative Riboside 27 Adenylyl (3'5') Cytidine Negative 28 Guanylyl (2'5') Guanosine Negative 32 2'3'-Di-O-Acetyladenosine Positive 29 Adenosine 2'3'cyclic Negative monophosphase sodium salt 33 Guanosine 2'3'Negative 35 Adenosine 5'-Carboxylic Acid Negative 36 Inosine 3'-Monophosphate Negative 40 2'-O-Anthramiloy Indenosine Negative 3'5'-Cyclic Monophosphate 51 2,5 Dimethoxy-Negative phenylguanidine Carbonate Amino acid andpeptide derivatives Number Compound Name Results 2 Naphtha (Terbutoxycarbonyl)-Negative L-Arginine 6 Chloroacetyl-DL-Nortencine Negative 34 MTH-DL-Arginine HCI Negative<BR> 45 N Phrhaloyl-DL-Histidine Negative<BR> 48 n-alphabenzoyl-Histidinol Negative 5 H-Ala-Arg-Oh Acetate Negative 20 PGlu-Gly-Arg-Phe Amide Negative 30 [Mei (0) 4, D-Lys, Pho9]-Negative Fragment 4-9 49 BOC-Val-Gly-Arg-ßNa-AcOH Negative<BR> 54 Z-ARG-OBZL (P-NO2) HBR Negative<BR> 55[Glu]-TRH Negative 57 Leu-Asp-Val-Pro-Ser Negative Antibiotics Number Compound Name Results 9 Neamine Negative 15 Butirosin Disulfate Salt Negative 16 Puromycin Aminonucleoside Negative<BR> 19 Geneticin Negative 21 Ribostamycin Negative 22 Dibekacin Negative 23 Sisomycin Negative 39 Amikacin Negative<BR> 41 Trimellitic Acid Amide Negative 42 Streptomycin Negative Diverse compounds Number Compound Name Results 1 Urocanic Acid Negative 13 Caffeine Negative 14 4', 6-Diamidino-2 Phenylindole Negative 25 Amidinophenyl (6 Amidino-2-Negative indolyl) Phenyl Ether 37 Amino Imidazole Negative Carobxamidoxime 38 Allentoic Acid Negative 44 1, 1, Thiobis 5,5'Dimethyl Negative hydratoin 46 7-acotoxy mthyl6 benzamido Negative hexah 7 methyl 5 oxoimidazo 47 3 mitro 4,4' Negative methylamediamiline 53 Z-Phenyl-Arginyl-7-Amido-4-Negative Methylcoumarin Hcl 64 Isoxazol diazaspito Negative 65 4 Nitro Phenyl Methoxy Negative Benzoyl 66 6-Morpholino-5-Negative Nitroimidazole 56 KM-04966* Negative 59 RJF-00556 Negative 60 KM-00561 Positive 61 NRB-04485 Negative 62 KM-06274 Positive 69 Amiloride-HCI Negative 70 KM-06272 Negative 71 KM-06273 Negative 72 KM-06278 Negative 73 KM-06280 Negative 74 KM-06281 Negative 74 KM-06281 Negative 75 CD-05250 Negative 76 SEW-105 Negative * Mawbridge catalog numbers

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. Experiments showing compounds that present an inhibitory effect on PrPSc formation in ScN2a cells have been repeated at least 3 times.

The production of proteinase K resistant PrPSc can be followed as a function of concentration of the various compounds. Compounds 18,32,62 and 60 were effective at inhibiting PrPSc formation at 20-40RM concentrations. Figure 8 illustrates a dose response curve looking at the effect of compound 60 on PrPSc production using the quantitative assay of Safar, et al., (1998) which shows an IC50=20FM. While nanomolar potency is normally sought in enzyme and receptor based screening assays, our results are close to the single digit micromolar concentrations frequently accepted as "hits"in complex cell based screening assays (e. g., antimicrobial agents).

A limited structure/activity relationship was obtained by screening a series of nine similar compounds. Figure 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 IC50's for each of the analogs in Figure 10 and then to develop a synthetic plan for building a structure activity relationship.

EXAMPLE 3 Materials and Methods Laboratory animals and inoculum.

RML inoculum used was described previously (Chandler, Lancet, 1: 1378-1379 (1961)).

Normal FVB mice were obtained from Charles River Laboratories. Tg (MoPrP-A) 4053 mice have been described (Telling, Genes Dev, 10: 1736-1750 (1996)). MoPrP, Q167R and MoPrP, Q218K mutant genes previously made by Kaneko et al., were subcloned into the cosTet vector for microinjection (Kaneko, Proc. Natl. Acad. Sci. USA, 94: 10069-10074 (1997)). Founders Tg (MoPrP, Q167R) Prnp°/° and Tg (MoPrP, Q218K) Prnp°/° 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° 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, Q167R) Prnp°/° and Tg (MoPrP, Q218K) Prnp°/° mice with FVB mice, until we obtained the F3 generation. In the FVB back-crossed animals, both the transgene and the wt Prnp genes were identified by PCR screening.

Determination of the incubation period in transgenic animals.

Control mice and Tg mice were inoculated intra-cerebrally with 30, ul 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 imminent. Whole brain were removed for histological and immunoblot analysis to confirm the diagnosis of scrapie.

Antibodies.

Humanized antibodies, D13 that recognized (97-106) PrP and RI that recognized (225- 231) PrP were used for the immunoblot. The polyclonal R073 antiserum was raised in a rabbit against SDS/PAGE purified SHaPrP27-30 that reacts with SHa as well as MoPrP (Serban, Neurology, 40: 110-117 (1990)).

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 gl 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. The digestion is stopped with 5 mM of freshly made PMSF. Then, Aliquots were taken out and mixed with an equal volume of SDS loading buffer

and boiled for 5 min. 30 u1 of PK-treated sample is loaded on a 12% SDS/PAGE Precast gels (Criterion system from BioRad).

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 loadingl0 ul 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)). RI and D13 Hu antibodies at 1 Rg/rnl were used to detect modified and wt MoPrP on the membrane.

Quantification of PrPsc by immunoassay using Time-Resolved Fluorescence (TRF) spectroscopy.

The immunoassay was performed according to the technique described previously by Safar et al., on the SHaMoPrP (Safar, NatureMed., 4: 1157-1165 (1998)).

Preparation of the calibration curve : Brains from normal or RML-inoculated FVB mice were resuspended to 5% solution, in PBS, 2% sarcosyl according to the protocol described in the paragraph above. Then, 5-fold serial dilutions were performed by diluting the 5% RML-FVB brain homogenate with 5% normal FVB brain homogenate. 10 points dilutions were done starting at 1%, 0.2%, 0.04%, 0.008% etc. Then, aliquots of 1 ml were digested with 25 gg/nd of proteinase K at a ratio of 1: 200 protease to protein, for 1 h at 37°C, on a shaker. The reaction is stopped by a mixture of protease inhibitors (5 mM PMSF, aprotinin and leupeptin at 4 yg/ml). The samples are precipitated with sodium phosphotungtate (NaPTA) at a final concentration of 0.32% in MgClz, during 1 hour at 37°C on a shaker. After 30 min of centrifugation at 20,000 g, the pellet is resuspended in 50 N, 1 of PBS, 0.2% sarcosyl and divided into 2 aliquots of equal volume of 25 ul.

One of the aliquot corresponds to the native measures. The other aliquot, is mixed with 25 ul 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-D13 antibody was used to detect modified and wt MoPrP proteins.

Preparation of modifiedMoPrP proteins : ThelO% brain homogenates in PBS, previously analyzed by immunoblots, were also tested by immunoassay using the TRF spectroscopy. The brain homogenates were diluted to 5% solution in PBS, 2% sarcosyl and rehomogenized with a syringe to break aggregates. Aliquots of 1 ml were digested with 25 Rg/ml of proteinase K for 1 h at 37°C, on a shaker. Then the reaction is stopped with a mixture of protease inhibitors and the sample is precipitated with NaPTA, as described in the subsection above. Then, samples are processed exactly the same way as the calibration curve samples.

Neuropathology. 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. R Pathol., 140: 1411- 1420 (1992)).

Histoblots Histoblots were performed according to the protocol described previously (Taraboulos, Proc. Natl. Acad. Sci. USA, 89: 7620-7624 (1992)).

Nomenclature. 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.

Results Transmission studies of RML prions in Tg (MoPrP, Q167R).

After the microinjection step, few founders were born and 2 lines Tg (MoPrP, Q167R) 1437Prnp°/° and Tg (MoPrP, Q167R) 12320Prnp°/°, 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 (1X PrP level) and that of normal Tg (MoPrP- A) 4053 (8X PrP level) by immunoblot. It was estimated that Tg (MoPrP, Q167R) 1437Prnp°/° and Tg (MoPrP, Q167R) 12320Prnp°/° expressed 1X and 16X PrP level, respectively. There were difficulties in breeding the higher expressor animals e. g. 16X PrP and as such inoculation experiments were started only with the Tg (MoPrP, Q167R) 1437Prnp°/°. With the aim to verify the dominant negative inhibition effect of MoPrP, Q167R towards wt MoPrP protein observed in the ScN2a cells, the Tg (MoPrP, Q167R) 1437Prnp°/° were successively back-crossed with normal FVB mice, until the results obtained the F3 generation of Tg (MoPrP, Q167R) 1437Pmp"' mice.

Those mice expressed both wt MoPrP and mutant MoPrP, Q167R at 1X level.

Two independent inoculation experiments with RML prions were started and none of the 12 Tg (MoPrP, Q167R) 1437Prnp°/° mice showed signs of scrapie disease after 550 days as shown in the table of susceptibility data below:

Table 1. Susceptibility of Tg (MoPrP, Q167R) to RML prions Host PrP Expression Levela Inoculum Incubation Period n/nob Mutant Wild-type (Days + SEM) FVB 0 lx RML 127i 1 50/50 Tg (MoPrP, Q167R) 1437/P° Ix 0 RML >550 0/8 Tg (MoPrP, Q167R) 1437/P° Ix 0 RML >557 0/4 Tg (MoPrP, Q167R) 1437/P° Ix 0 None >557 0/10 Non TglPrnp°'° 0 0 RML >557 0/7 Tg (MoPrP, Q167R) 1437/P Ix Ix RML >409 0/10 Tg (MoPrP, Q167R) 1437/Prnp+ lx lx None >256 0/6 °Expression levels were determined by comparing serial dilutions of transgenic mouse brain<BR> homogenates with that of normal FflB mice (Ix PrP level) by immuoblot. bn, number of sick mice ; no, number of inoculated mice.

However, FVB control mice expressing wt PrP at the same PrP level, died at 1271 days post-inoculation with RML prions. Moreover, none of the 10 Tg (MoPrP, Q167R) 1437Prnp+/+ mice inoculated with RML prion showed signs of sickness at 409 days post-inoculation. Control experiment with Tg (MoPrP, Q167R) 1437Prnp°/° mice non inoculated was started in order to determine the effect of PrP expression level on mice behaviour. Those animals showed no sign of spontaneous degeneration, as expected. Control experiment with Non Tg/Pmpo'o mice, inoculated with RML prions strain was also done and animals presented no sign of scrapie disease as shown in the data provided above.

Immunoblot analysis of Tg (MoPrP, Q167R) inoculated with RML prions.

All of the Tg (MoPrP, Q167R) 1437Prnp°/° 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 PrPSc form, the marker for prion replication and infectivity.

Immunoblot before proteinase K digestion was done as a control to check the PrP expression level (Figure 10A). Each samples loaded on the gel were equal in protein amounts as shown with the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody.

PK treated brain homogenate from normal FVB mouse inoculated with RML prions, gave a strong PrPSc band (Figure 10B, lane 11). However, PK treated samples from transgenic animals inoculated with RML prions (lanes 1-3) presented no MoPrPScQl67R 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 MoPrPScQl67R signal. Moreover, a second identical western blot was probed with the D13 antibody which recognized (97-106) PrP epitope compared to RI which recognized (225-231) PrP epitope. Here again, immunoblot probed with D 13 showed no MoPrPScQ 167R band for transgenic mice inoculated with RML, as in the case of RI antibody.

As the Q 167R mutation introduced into MoPrP might have created a higher susceptibility of the protein towards the PK, the samples were digested with lower concentrations of enzyme, ranging from 1 to 20 ug/ml (Figure 10C). Normal FVB mice inoculated with RML prions showed PrPSC signal that increase when PK concentration is lowered in the mixture. However, no PrPSc band was present in the brain extracts from Tg (MoPrP, Q167R) 1437Prnp°/° mice inoculated or not with RML, even at 1 Rg/ml of PK. According to the immunoblots, the MoPrP, Q 167R is unable to convert into a MoPrPScQl67R form, resistant to PK digestion. This mutation protects mice from becoming infected with prions in the RML innoculent.

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, Q 167R at 1X level. Experiments were carried out to determine if the RML inoculated Tg (MoPrP, Q167R) 1437Prnp+/+mice were able to replicate or not the wt MoPrP protein, because it was observed that the mutant protein MoPrP, Q 167R could not be converted into MoPrP"Q167R form in the Tg (MoPrP, Q167R) 1437Prnp°/° animals. Two healthy Tg (MoPrP, Q167R) 1437Prnp+/+animals were sacrificed at 300 days post-inoculation and their brain homogenates were analyzed on immunoblot (Figure 11). The PK-treated brain homogenates from Tg (MoPrP, Q167R) 1437Prnp+/+mice revealed the presence of low amounts of PK resistant form, both with equivalent intensity (Figure 11B, lanes 1-2). The intensity of those bands corresponds to about 1/10 the signal obtained with normal FVB mice inoculated with RML prions (lane 10) as if the efficiency of conversion was decreased.

Pathology analysis of brain tissue from Tg (MoPrP, Q167R) mice inoculated with RML prions.

Pathology analysis was done on the other midbrain of the transgenic animals that were previously analyzed by immunobloting (Figures 10-11). The purpose was to determine if the transgenic animals were in the incubation period and would develop a prion disease soon or if they are protected from RML prions. The brain analysis from Tg (MoPrP, Q167R) 1437Prnp°/° mice that remain healthy 550 days post-inoculation, showed no vacuolation in the hippocampus as in the case of the non inoculated animals (Figure 12E and G). The brain tissue showed some light astrocytic

gliosis which is consistent with aging as the aged-match Tg (MoPrP, Q167R) 1437Prnp°° mice non- inoculated present the same pattern (Figure 12F and H). However, 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 12I and J). The neurodegeneration looks as severe as the normal FVB mice inoculated with RML (Figure 12A and B). However, in the Tg (MoPrP, Q167R) 1437Prnp++mice, the neurodegeneration is localized only in the ventral part of the hippocampus whereas in the FVB mice the neurodegeneration is present in different areas of the brain and both in ventral and dorsal hippocampus, althought more intense in the dorsal area. Brain analysis from non inoculated Tg (MoPrP, Q167R) 1437Prnp+/+mice looks as normal (Figure 12K and L) as the non inoculated FVB mice (Figure 12C and D). The pathology analysis was at least done for 2 independent animals of every experiment. According to these data, there is a correlation between the immunoblots results and the neuropathology studies.

Localization of PrPSc in the brain of Tg (MoPrP, Q167R) mice.

Histoblotting demonstrated some light PrPSc deposits only in the brain of Tg (MoPrP, Q167R) 1437Prnp+/+mice (Figure 13E). Moreover, the PrPSC deposits are localized mainly in the left part of the brain, especially the ventral hippocampus area. However, the histoblot of Tg (MoPrP, Q167R) 1437Prnp°° mouse brain inoculated with RML presents no PrPSG staining (Figure 13B) as in the case of non inoculated transgenic animal (Figure 13C) as well as knock-out mouse inoculated with RML prions (Figure 13D).

Detection of infectivity of the Tg (MoPrP, Q167R) mice brain by Conformational Dependent Immunoassay.

In order to detect traces of infectivity that we might not be able to detect by immunoblot, samples were also analyzed by using the Conformational Dependent Immunoassay (CDI). This technique allows the quantification of the different isoforms of PrP by immunofluorescence (Safar, Nature in Med., 4: 1157-1165 (1998)). Safar et al. showed in their calibration curve using scrapie- infected SHa brain homogenates, that a ratio of antibody binding to denatured/native PrP (den/natPrP) greater than 1. 8 indicated the presence of SHaPrPsc and infectivity.

A calibration curve was created using RML-infected FVB mice brain homogenates (Figure 14A). The data showed a linear relation between the MoPrPSc level and the dilution of the brain homogenates up to 0.00032 % (32 x 10-5). No PrPsc is detected after dilution 10-5which 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).

The only samples where we detect some infectivity was detected, were the RML-inoculated

Tg (MoPrP, Q167R) 1437Prnp++mice. The 2 brains extracts that were previously analyzed by immunoblot obtained by CDI (see U. S. Patents 5,891,641 and 6,214,565), a ratio den/natPrP of 46. 2 1.57 and 45.7 i 1.69 which corresponds to 0.05 ug/ml ofrPrP. Compared to the calibration curve, a brain homogenate at a concentration of 5% was determined at 82 i 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). Controls with mice non inoculated like Tg (MoPrP, Q167R) 1437Prnp++, Tg (MoPrP, Q 167R) 1437Prnp°/° and NonTg/PrnpO/0 were all equivalent, with ratio den/natPrP between 58i0. 04and 1. 2i0. 01.

Transmission studies of RML prions in Tg (MoPrP, Q218K).

A partial dominant negative effect was observed with the RML inoculated Tg (MoPrP, Q167R) Prop+/+ expressing low level of MoPrP, Q167R. The objective was to determine if a higher expression level of the transgene could lead to a complete dominant negative inhibition of the wt-MoPrP. Because there were difficulties establishing a transgenic line which expresses higher level of PrP, Q167R, micro-injection was carried out on another dominant negative mutation, the Q218K. Two lines were established Tg (MoPrP, Q218K) 22500Prnp°/° and Tg (MoPrP, Q218K) 21603Prnp°'°, 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) 2250OPmpo/o 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 Host PrP Expression level Inoculum Incubation Period nln b Mutant Wild-type (Days i SEM) Tg (MoPrP-A) 4053/Prnp°'° 0 8x RML 50 2 16/16 Non Tg/Pmpo/o 0 0 RML >228 0/11 Tg (MoPrP, Q218K) 22500/Prnp°/° 32x 0 RML >375 1/16 Tg (MoPrP, Q218K) 22500/Prnp°/° 32x 0 None >354 1/10 Tg (MoPrP, Q218K) 22500/Pmp-"" 32x lx RML >277 0/8 Tg (MoPrP, Q218K) 22500/Prnp+'+ 32x lx None >151 0/6 Tg (MoPrP, Q218K) 21603/Prnp°/° 16x 0 RML >298 0/8 Tg (MoPrP, Q218K) 21603/Prnp°/° 16x 0 RML >243 0/10 Tg (MoPrP, Q218K) 21603/Prnp°/° 16x 0 None >256 0/10 Tg (MoPrP, Q218K) 21603/Prnp°/° 16x lx RML >277 0/9 Tg (MoPrP, Q218K) 21603/Prnp°'° 16x lx None >207 0/10

'Expression levels were determined by comparing serial dilutions of transgenic mouse brain homogenates with that of hormal FfB mice (IXPrP level) by immunoblot. bn, number of sick mice ; no, number of inoculated mice.

The above results are probably due to the high level of PrP expressed by the transgene.

Similar results were obtained with a Tg (MoPrP, Q218K, #T189) 20250 Prnp°/° line which expressed the transgene at 32X level. All the mice inoculated with RML died after 315 33 days post- inoculation and the non inoculated mice died after 322 22- 30 days. Pathologic analysis of those mice revealed numerous vacuolations and prononced astrocytic gliosis. Pathologic profiles were undistinguishable between inoculated and non-inoculated animals. However PrPSc was not detected on immunoblot.

The Tg (MoPrP, Q218K) 22500Prnp°/° line is protected against RML inoculation but might be susceptible to a spontaneous disease due to overexpression.

None of the eight Tg (MoPrP, Q218K) 21603Prnp°'° mice inoculated with RML prions showed signs of scrapie disease after almost 300 days, which is remarkable due to the 16X expression level of the transgene. The wild-type Tg (MoPrP-A) 4053 that expressed 8-fold the level of the transgene usually developed prion disease after 50 days. Identically, all of the Tg (MoPrP, Q218K) 21603Pmp+/+ mice inoculated with RML prions looks healthy at 277 days.

Immunoblots analysis of Tg (MoPrP, Q218K) inoculated with RML prions.

Experiments were carried out to determine if the back-crossed animals harboring both copies of wt MoPrP and mutant MoPrP, Q218K were able to replicate wt PrPSc as in the case of the lower expressor Tg (MoPrP, Q167R) Prnp+/+. For each series of experiment listed in the above data two animals were sacrificed and an immunoblot was performed on their brain homogenates before and after proteinase K digestion (Figure 15). The assay did not detect any wt-MoPrPSc or MoQ21 XKPrPSc in the brain tested (Figure 15B). All of those samples were also processed by CDI, and all of the ratio den/natPrP measured, were under 1.8 the cut-off value for infectivity.

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.