U.S. patent application number 10/256538 was filed with the patent office on 2004-04-15 for prpsc -interacting molecules and uses thereof.
Invention is credited to Cashman, Neil, Estey, Lisa, Francoeur, Greg, Francoeur, Susan, La Boissiere, Sylvie, Lawton, Robert, Paramithiotis, Eustache, Pinard, Marc.
Application Number | 20040072236 10/256538 |
Document ID | / |
Family ID | 32041771 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040072236 |
Kind Code |
A1 |
Cashman, Neil ; et
al. |
April 15, 2004 |
PrPSc -interacting molecules and uses thereof
Abstract
Peptides are disclosed which specifically bind to PrP.sup.Sc.
Methods of identifying these peptides by sequence-independent
and/or sequence-dependent screening assays are also disclosed.
Various applications of the method and of the peptides identified
are described, including their use for identifying drugs for
treating prion diseases.
Inventors: |
Cashman, Neil; (Toronto,
CA) ; Paramithiotis, Eustache; (Boucherville, CA)
; La Boissiere, Sylvie; (Montreal, CA) ; Lawton,
Robert; (Gorham, ME) ; Francoeur, Greg; (North
Yormouth, ME) ; Francoeur, Susan; (Portland, ME)
; Estey, Lisa; (Westbrook, ME) ; Pinard, Marc;
(Montreal, CA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
32041771 |
Appl. No.: |
10/256538 |
Filed: |
September 27, 2002 |
Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
G01N 2800/2828 20130101;
G01N 2469/20 20130101; A61P 37/02 20180101; G01N 33/6896 20130101;
A61P 25/28 20180101; G01N 2500/00 20130101 |
Class at
Publication: |
435/007.1 |
International
Class: |
G01N 033/53 |
Claims
We claim:
1. A method for identifying a peptide that binds to PrP.sup.Sc or a
fragment thereof, said method comprising the steps of: (a)
contacting a peptide of about 200 or fewer amino acids with a
PrP.sup.Sc polypeptide or fragment thereof under conditions that
allow for complex formation between said peptide and PrP.sup.Sc or
fragment thereof; and (b) detecting said complex, wherein the
presence of the complex identifies said peptide as one which
selectively binds to PrP.sup.Sc or a fragment thereof.
2. The method of claim 1, wherein said peptide is a fragment of
PrP.
3. The method of claim 1, wherein said peptide is about 100 to 150
amino acids in length.
4. The method of claim 1, wherein said peptide is about 50 to 100
amino acids in length.
5. The method of claim 1, wherein said peptide is about 25 to 50
amino acids in length.
6. The method of claim 1, wherein said peptide is about 9 to 25
amino acids in length.
7. The method of claim 1, wherein said peptide is a tripeptide.
8. The method of claim 1, wherein said peptide is a
tetrapeptide.
9. The method of claim 1, wherein said peptide is a
pentapeptide.
10. The method of claim 1, wherein said peptide is a
hexapeptide.
11. The method of claim 1, wherein said peptide is a
heptapeptide.
12. The method of claim 1, wherein said peptide is an
octapeptide.
13. The method of claim 1, wherein said peptide is a
nonapeptide.
14. The method of claim 1, wherein said peptide is a
decapeptide.
15. The method of claim 1, wherein said peptide includes a YYX or
YSA motif.
16. The method of claim 15, wherein said YYX or YSA motif is
repeated in said peptide.
17. The method of claim 16, wherein said YYX or YSA motif is
tandemly repeated in said peptide.
18. The method of claim 15, wherein said YYX motif is selected from
the group consisting of YYR, YYD, YYA, and YYQ.
19. The method of claim 15, wherein said motif includes the amino
acids YSA.
20. The method of claim 1, wherein said peptide includes an
YYXXYYXYY (SEQ ID NO: 1; where X is any amino acid) motif.
21. The method of claim 1, wherein said peptide is coupled to a
scaffolding agent.
22. The method of claim 21, wherein said scaffolding agent is 4-map
or 8-map.
23. The method of claim 1, wherein said peptide is covalently
coupled to a detectable-agent, solid support, or carrier.
24. The method of claim 1, wherein said complex is detected using
ELISA, RIA, western blotting, immunoprecipitation, fluorescence
polarization or flow cytometry.
25. A method for detecting a PrP.sup.Sc in a biological sample,
said method comprising the steps of: (a) contacting said biological
sample with a peptide of about 200 or fewer amino acids which
selectively binds to PrP.sup.Sc or a fragment thereof under
conditions that allow for complex formation between said peptide
and a PrP.sup.Sc polypeptide or fragment thereof, and (b) detecting
said complex as an indication that said PrP.sup.Sc is present in
said biological sample.
26. The method of claim 25, wherein said peptide does not
substantially bind PrP.sup.C.
27. The method of claim 25, wherein said peptide is 9 to 20 amino
acids in length.
28. The method of claim 25, wherein said peptide comprises a YYX or
YSA motif.
29. The method of claim 28, wherein said YYX motif is selected from
the group consisting of YYR, YYD, YYA, and YYQ.
30. The method of claim 25, wherein said peptide includes a
YSA.
31. The method of claim 26, wherein said peptide includes a
YYXXYYXYY (where X is any amino acid) motif.
32. The method of claim 25, wherein said biological sample
comprises a tissue or cell, a tissue or cell extract, a bodily
fluid, or a biopsy.
33. The method of claim 25, wherein said PrP.sup.Sc is from a
human, a livestock species, or a pet species.
34. The method of claim 28, wherein said peptide comprising said
YYX motif is covalently coupled to a detectable-label.
35. The method of claim 28, wherein said peptide comprising said
YYX or YSA motif is covalently coupled to a solid support or
carrier.
36. The method of claim 25, wherein said complex is detected using
an ELISA, RIA, western blotting, immunoprecipitation, fluorescence
polarization, or flow cytometry.
37. The method of claim 25, wherein said PrP.sup.Sc in said
biological sample is amplified PrP.sup.Sc.
38. A method for diagnosing a prion disease in a mammal, said
method comprising the steps of: (a) contacting a biological sample
of said mammal with a peptide of about 200 or fewer amino acids
which selectively binds to PrP.sup.Sc or a fragment thereof under
conditions that allow for complex formation between said peptide
and a PrP.sup.Sc polypeptide or fragment thereof; and (b) detecting
said complex, which if present indicates a prion disease in said
mammal.
39. The method of claim 38, wherein said prion disease is selected
from the group consisting of variant Creutzfeldt-Jakob Disease,
bovine spongiform encephalopathy, scrapie, transmissible spongiform
encephalopathy, and chronic wasting disease.
40. The method of claim 38, wherein said peptide does not
substantially bind
41. The method of claim 38, wherein said peptide comprises a YYX or
YSA motif.
42. The method of claim 41, wherein said YYX motif is selected from
the group consisting of YYR, YYD, YYA, and YYQ.
43. The method of claim 38, wherein said peptide includes a YSA
motif or a YAR motif.
44. The method of claim 38, wherein said biological sample
comprises a tissue or cell, a tissue or cell extract, a bodily
fluid, or a biopsy.
45. The method of claim 38, wherein said PrP.sup.Sc is from a
human, a livestock species, or a pet species.
46. The method of claim 41, wherein said peptide comprising said
YYX motif is covalently coupled to a detectable-agent.
47. The method of claim 41, wherein said peptide comprising said
YYX motif is covalently coupled to a solid substrate.
48. The method of claim 38, wherein said complex is detected using
an ELISA, RIA, western blotting, immunoprecipitation, fluorescence
polarization or flow cytometry.
49. A method for treating or preventing a prion disease in a
mammal, comprising administering to said mammal an effective amount
of a peptide of about 200 or fewer amino acids which selectively
binds to PrP.sup.Sc or a fragment thereof in a
pharmaceutically-acceptable carrier and under conditions that allow
for complex formation between said peptide and a PrP.sup.Sc
polypeptide or fragment thereof.
50. The method of claim 49, wherein said prion disease is selected
from the group consisting of variant Creutzfeldt-Jakob Disease,
bovine spongiform encephalopathy, and scrapie.
51. The method of claim 49, wherein said peptide does not
substantially bind PrP.sup.C.
52. The method of claim 49, wherein said peptide comprises a YYX or
a YSA motif.
53. The method of claim 52, wherein said YYX motif is selected from
the group consisting of YYR, YYD, YYA, and YYQ.
54. The method of claim 52, wherein said peptide includes a YSA
motif.
55. A method of inhibiting PrP.sup.Sc in a biological sample, said
method comprising: treating the biological sample with a peptide of
about 200 or fewer amino acids which selectively binds to
PrP.sup.Sc or a fragment thereof under conditions that allow for
complex formation between said peptide and a PrP.sup.Sc polypeptide
or fragment thereof and for a period of time sufficient to permit
the formation of a complex comprising said peptide and a
PrP.sup.Sc.
56. The method of claim 55, wherein said biological sample is a
bodily fluid, a tissue or organ.
57. The method of claim 55, wherein said biological sample is
perfused with said peptide.
58. The method of claim 56, wherein said peptide does not
substantially bind PrP.sup.C.
59. A method for decontaminating PrP.sup.Sc from a biological
sample, said method comprising the steps of: (a) treating the
biological sample with a peptide of about 200 or fewer amino acids
which selectively binds to PrP.sup.Sc or a fragment thereof under
conditions that allow for complex formation between said peptide
and a PrP.sup.Sc polypeptide or fragment thereof and for a period
of time sufficient to permit the formation of complex comprising
said peptide and PrP.sup.Sc; and (b ) recovering said complex from
said biological sample.
60. The method of claim 59, wherein said biological sample is a
tissue, bodily fluid, or organ.
61. The method of claim 59, wherein said biological sample is
perfused with said peptide.
62. The method of claim 59, wherein said peptide does not
substantially bind PrP.sup.C.
63. The method of claim 59, wherein said peptide comprises a YYX or
YSA motif.
64. The method of claim 63, wherein said YYX motif is selected from
the group consisting of YYR, YYD, YYA, and YYQ.
65. The method of claim 59, wherein said peptide includes a YSA
motif.
66. A method for identifying an agent for the treatment of a prion
disease, said method comprising the steps of: (a) combining a
peptide of about 200 or fewer amino acids which selectively binds
to PrP.sup.Sc or a fragment thereof, PrP.sup.Sc, and an agent under
conditions allowing for complex formation of said peptide and said
PrP.sup.Sc; and (b) determining whether complex formation is
increased or decreased in comparison to complex formation in the
absence of said agent, thereby identifying an agent for treating
said prion diseases.
67. The method of claim 66, wherein said prion disease is selected
from the group consisting of variant Creutzfeldt-Jakob Disease,
bovine spongiform encephalopathy, and scrapie.
68. The method of claim 66, wherein said peptide does not
substantially bind
69. The method of claim 66, wherein said peptide comprises a YYX or
YSA motif.
70. The method of claim 69, wherein said YYX motif is selected from
the group consisting of YYR, YYD, YYA, and YYQ.
71. The method of claim 66, wherein said peptide includes a YSA
motif.
72. The method of claim 66, wherein said prion disease affects a
human, a livestock species, or a pet species.
73. The method of claim 66, wherein said prion disease affects a
human, bovine, sheep, or goat.
74. The method of claim 66, wherein said agent increases complex
formation.
75. The method of claim 66, wherein said agent decreases complex
formation.
76. An apparatus for detecting PrP.sup.Sc in a biological sample,
the apparatus comprising: a peptide-fixed portion where a peptide
for trapping an amount of an analyte in a sample is present and in
a predetermined amount.
77. The apparatus of claim 76, wherein said peptide comprises a
YYX, YYR, YYD, or YYQ amino acid sequence, said peptide having
antigenicity as a PrP.sup.Sc.
78. The apparatus of claim 76, wherein said peptide is composed of
18 or fewer amino acids.
79. The apparatus of claim 76, wherein said peptide is composed of
12 or fewer amino acids.
80. The apparatus of claim 76, wherein said peptide is the
nonapeptide having the amino acid sequence YYRRYYRYY (SEQ ID NO:
2).
81. The apparatus of claim 77, wherein said peptide is composed of
8 or fewer amino acids.
82. The apparatus of claim 77, wherein said peptide is composed of
5 or fewer amino acids.
83. The apparatus of claim 77, wherein said peptide is the
tripeptide having the amino acid sequence YYR.
84. The apparatus of claim 77, wherein said analyte is a bodily
fluid.
85. A method for detecting an anti-PrP.sup.Sc antibody in a
biological sample, said method comprising the steps of: (a)
contacting said biological sample with a peptide of about 200 or
fewer amino acids which selectively binds to PrP.sup.Sc or a
fragment thereof under conditions that allow for complex formation
between said peptide and anti-PrP.sup.Sc antibody; and (b)
detecting said complexes as an indication that anti-PrP.sup.Sc
antibody is present in said biological sample.
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to molecules (for example, peptides)
that bind selectively to the disease-specific abnormal isoform of
the prion protein, herein generically designated PrP.sup.Sc, and
not to the normal isoform of this protein, designated PrP.sup.C.
These molecules are useful for detecting PrP.sup.Sc in a sample,
and for purifying PrP.sup.Sc. Additionally, the invention relates
to diagnostic aids for the detection of PrP.sup.Sc, pharmaceuticals
that inhibit the recruitment of normal PrP.sup.C to the
disease-specific PrP.sup.Sc, and methods for prion
decontamination.
[0002] The prion diseases are a group of rapidly progressive,
fatal, and untreatable neurodegenerative syndromes. Human prion
diseases include classical Creutzfeldt-Jakob disease (CJD), which
has sporadic, iatrogenic, and familial forms. More recently, a
variant CJD (vCJD) has been recognized in the United Kingdom,
France, the Republic of Ireland, Hong Kong, Italy and the United
States (Will et al., Lancet 347:921-25, 1996; Collinge, Lancet
354:317-23, 1999), likely derived from the consumption of cattle
tissues contaminated with the agent of bovine spongiform
encephalopathy ("BSE"; reviewed in Cashman, Can. Med. Assoc. J.
157:1381-5, 1997; Coulthart and Cashman, Can. Med. Assoc. J.
165:51-8, 2001). More than 173,000 cattle, primarily from Britain,
have developed symptomatic BSE, and many thousands more have
probably entered the food supply undetected. The primary epidemic
of vCJD has been predicted to range from dozens to hundreds of
thousands of afflicted individuals, based on various poorly
understood assumptions, such as the incubation period of the
disease. Moreover, classical CJD has been accidentally transmitted
between humans by contaminated cadaveric pituitary hormones, dura
mater transplantation, neurosurgical instrumentation, and corneal
transplantation (Brown et al., Neurology 55:1075-8, 2000). A
"secondary epidemic" of vCJD through blood and blood products,
general surgery, dentistry, vaccines, and cosmetics cannot be ruled
out at present. The detection of blood prion infectivity in
experimental BSE/vCJD infections of mice and sheep (Taylor et al.,
J. Hosp. Infect. 46:78-9, 2000; Houston et al., Lancet
356:999-1000, 2000), but not in classical CJD (Rickets et al.,
Emerg. Infect. Dis. 3:155-63,1997) suggests a special risk of
transmitting vCJD through blood and blood products. The United
States and Canada have now implemented a blood donor deferral for
individuals who resided in the UK during the BSE epidemic, and
Canada has also deferred donors from France. Donor deferrals
extended to Western Europe are now being implemented in the United
States and Canada.
[0003] Prions are the infectious agents that are associated with
the transmissible spongiform encephalopathies noted above. The
prion diseases are neurodegenerative syndromes characterized by
spongiform change (e.g., microcavitation of the brain, usually
predominant in gray matter), neuronal cell loss, astrocytic
proliferation disproportionate to neuronal loss, and accumulation
of an abnormal amyloidogenic protein, sometimes in discrete plaques
in the brain. The agents that transmit these diseases differ
markedly from viruses and viroids in that no chemical or physical
evidence for a nucleic acid component has been reproducibly
detected in infectious materials (Prusiner, Science 216: 136-144,
1982). A major step in the study of model scrapie prions was the
discovery and purification of a protein designated the
scrapie-associated prion protein (PrP.sup.Sc) (Bolton et al.,
Science 218:1309-11, 1982; Prusiner et al., Biochemistry
21:6942-50, 1982; McKinley et al., Cell 35:57-62, 1983). When
purified using proteinase K digestion, a 27-30 kD
protease-resistant protein was discovered in scrapie-affected
hamster brain, and was termed PrP 27-30, later found to be a
fragment of PrP.sup.Sc (Bolton et al., Science 218:1309-1311,
1982).
[0004] According to the prion hypothesis, prion infectivity resides
in PrP.sup.Sc, or a related conformational intermediate. PrP.sup.Sc
is the most prominent (or perhaps sole) macromolecule in
preparations of prion infectivity, and appears to be at least a
reliable surrogate for most prion infection. PrP.sup.Sc is a
conformational variant of a host-encoded cellular protein
designated PrP.sup.C (Oesch et al., Cell 40:735-746, 1985), which
is a glycosylphosphatidylinositol (GPI)-linked cell surface protein
with a molecular mass of 33-35 kD. PrP.sup.C has been isolated from
normal brain, and has been found to be protease-sensitive and not
associated with scrapie disease-producing activity (Bolton and
Bendheim Ciba Found. Symp. 135:164-177, 1988). According to the
prion theory, PrP.sup.C converts into PrP.sup.Sc in a
template-directed process initiated by contact with PrP.sup.Sc
(Prusiner, Proc. Natl. Acad. Sci, USA 95:13363-83, 1998).
[0005] PrP.sup.C is an evolutionarily conserved membrane protein
for which the actual biological or physiological function is
unclear. Mice devoid of PrP.sup.C are viable and show no obvious
signs of neurological and physical impairment (Bueler et al.,
Nature 356:577-582, 1992), except for an ataxic syndrome in certain
PrP knockout mouse strains due to upregulation in brain of the
prion homolog protein dopple (Moore et al., J Mol. Biol.
292:797-817, 1999). Prnp knockout mice are not susceptible to prion
infection, underscoring the central importance of PrP.sup.C in the
replication of infectivity (Bueler et al., Cell 73:1339-1347, 1993;
Prusiner et al., Proc. Natl. Acad. Sci., USA 90:10608-10612, 1993).
Targeted investigations of Prnp knockout mice revealed impaired
synaptic function (Collinge et al., Nature 370:295-297, 1994) and
altered sleep regulation (Tobler et al., J. Neurosci. 17:1869-79,
1997). Moreover, antibody-mediated ligation of PrP.sup.C at the
cell surface has been shown to depress T cell activation (Cashman
et al., Cell 61:185-192, 1990; Li et al., Cell. Immunol. 207:49-58
2001), suggesting a role for the protein in immune function.
[0006] PrP.sup.C is present in large excess to PrP.sup.Sc in
accessible peripheral tissues and organs of animals and humans
afflicted with prion diseases. The availability of reagents that
distinguish PrP.sup.Sc from PrP.sup.C would therefore be of great
value in development of a test for prion infection in blood or
other tissues accessible to sampling. Furthermore, in view of the
epidemic nature of BSE and vCJD, a pressing need exists for
therapeutic agents that prevent and/or treat prion diseases.
Accordingly, a need exists in the art for testing samples for the
presence of prions, methods for treating prion diseases, as well as
for developing anti-prion pharmaceuticals and decontaminants.
SUMMARY OF THE INVENTION
[0007] As is described in greater detail below, we have developed a
method to exploit the distinctive physiochemical properties of
PrP.sup.Sc (and its protease-resistant fragment PrP 27-30) to
identify molecules (e.g., peptides) which selectively bind to these
conformationally abnormal isoforms, without binding to the normal
isoform which is usually present in great excess in biological
fluids and tissues.
[0008] The invention therefore relates to methods for identifying
molecules (e.g., peptides) that selectively bind to PrP.sup.Sc,
exploiting surface hydrophobicity, charge interactions, and
beta-sheet converting potential of peptides in their affinity
reactions with PrP.sup.Sc. For example, a subset of these
molecules, such as peptides, are defined by amino acid sequences
within the prion protein sequence itself and/or by evolutionarily
conserved amino acid substitutions to these sequences. Other
peptides are those that bind selectively to regions of PrP.sup.Sc
(such as those regions of the protein that include YYX amino acid
residues) based on physicochemical interactions such as hydrophobic
interactions, pi-stacking, or beta sheet interactions. Peptides
selectively binding PrP.sup.Sc may be constrained by cyclization,
and by formation of hairpins or loops by introduction of cysteines
to form disulfide bonds upon oxidation. For use in a diagnostic
test of prion infection, peptides can be covalently coupled to a
solid substrate, such as agarose beads, magnetic beads, or ELISA
plates, reacted with a sample containing PrP.sup.Sc, cleared of
PrP.sup.C by washing (with or without proteinase K), so that
detection of bound PrP.sup.Sc can be efficiently and specifically
accomplished with a PrP.sup.Sc-specific antibody, or with a
non-distinguishing antibody or molecule binding both PrP.sup.Sc and
PrP.sup.C. Such peptides are also useful in concentrating
PrP.sup.Sc from biological samples.
[0009] Peptides of the invention are suitable for therapeutic,
diagnostic, or decontamination purposes. In a preferred embodiment,
the molecular weight of the peptide is from about 40,000 Daltons to
about 300 Daltons. Other peptides may fall in a narrower range, for
example, 30,000 to about 1,000 Daltons, or from about 20,000 to
about 2,000 Daltons. Peptides of the invention may be synthesized
according to standard methods known in the art.
[0010] In a related embodiment, the invention is directed to a
detectably-labeled peptide as described herein, the peptide
preferably having a covalently attached label capable of
detection.
[0011] In still other embodiments, the peptides are linked to a
spacer having multiple side chain amines, such as poly(lysine), can
be used to "amplify" the available surface functionalities.
Multiple antigen peptide system ("MAPS") peptides typically consist
of a branched lysine core matrix (Tam, Proc Natl Acad Sci USA 85:
5409-13, 1988; Tam and Zevala, J. Immunol Methods 124: 53-61, 1989;
Tam, J. Immunol Methods 196: 17-32, 1996). The branched lysine core
provides a scaffolding to support multiple copies of any of the
peptides described herein.
[0012] Accordingly, in a first aspect, the invention features a
method for identifying a peptide that binds to PrP.sup.Sc or a
fragment thereof. This method includes the steps of (a) contacting
a peptide of about 200 or fewer amino acids with a PrP.sup.Sc
polypeptide or fragment thereof under conditions that allow for
complex formation between the peptide and PrP.sup.Sc or fragment
thereof, and (b) detecting the complex, wherein the presence of the
complex identifies the peptide as one which selectively binds to
PrP.sup.Sc or a fragment thereof. In preferred embodiments, the
complex is detected using ELISA, RIA, western blotting,
immunoprecipitation, fluorescence polarization or flow
cytometry.
[0013] In preferred embodiments, the peptide is a fragment of PrP.
In additional preferred embodiments, the peptide can range in chain
length from 100 to 150 amino acids, more preferably 50 to 100 amino
acids, or 25 to 50 amino acids, and most preferable 9 to 25 amino
acids. The peptide can also be a tripeptide, a tetrapeptide, a
pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a
nonapeptide, or a decapeptide.
[0014] In preferred embodiments of the first aspect, the peptide
can include a YYX (where X is any amino acid) or YSA motif, which
can be repeated in the peptide (e.g., in tandem). The YYX motif can
include the amino acids YYR, YYD, YYA, and YYQ. The peptide can
also include a YYXXYYXYY (SEQ ID NO: 1) where X is any amino acid)
motif. An example of a peptide with a YYXXYYXYY motif is the
peptide YYRRYYRYY (SEQ ID NO: 2).
[0015] In additional preferred embodiments, the peptide is coupled
to a scaffolding agent, for example, a 4-map or an 8-map. The
peptide can also be covalently coupled to a detectable-agent, solid
support, or carrier.
[0016] The invention also features a method for detecting a
PrP.sup.Sc in a biological sample. This method includes the steps
of (a) contacting the biological sample with a peptide of about 200
or fewer amino acids which selectively binds to PrP.sup.Sc or a
fragment thereof under conditions that allow for complex formation
between the peptide and a PrP.sup.Sc polypeptide or fragment
thereof, and (b) detecting the complex as an indication that
PrP.sup.Sc is present in the biological sample.
[0017] In preferred embodiments, the peptide does not substantially
bind PrP.sup.C. The peptide can be 9 to 20 amino acids in length
and can include a YYX, YYXXYYXYY, or YSA motif. Preferred sequences
for the YYX motif are YYR, YYD, YYA, and YYQ. In preferred
embodiments, the peptide comprising the YYX sequence is covalently
coupled to a detectable-label. The peptide comprising the YYX or
YSA motif can also be covalently coupled to a solid support or
carrier.
[0018] The biological sample includes any tissue or cell, tissue or
cell extract, bodily fluid or biopsy. In preferred embodiments, the
PrP.sup.Sc is from a human, a livestock species, or a pet species.
In additional preferred embodiments, the PrP.sup.Sc from the
biological sample is amplified PrP.sup.Sc. Preferred methods of
detection include ELISA, RIA, western blotting,
immunoprecipitation, fluorescence polarization, and flow
cytometry.
[0019] The present invention also includes methods for diagnosing a
prion disease such as variant Creutzfeldt-Jakob Disease, bovine
spongiform encephalopathy, scrapie, transmissible spongiform
encephalopathy, and chronic wasting disease. This method includes
the steps of (a) contacting a biological sample from a mammal with
a peptide of about 200 or fewer amino acids which selectively binds
to PrP.sup.Sc or a fragment thereof under conditions that allow for
complex formation between the peptide and a PrP.sup.Sc polypeptide
or fragment thereof and (b) detecting the complex which, if
present, indicates a prion disease in the mammal.
[0020] In preferred embodiments, the peptide does not substantially
bind PrP.sup.C. In additional preferred embodiments, the peptide
comprises a YYX, YSA, or YAR motif, wherein the YYX motif is
selected from the group consisting of YYR, YYD, YYA, and YYQ. The
peptide comprising a YYX motif can optionally be covalently coupled
to a detectable-agent or a solid substrate.
[0021] For the diagnostic methods, it is preferred that the
biological sample comprise a tissue or cell, a tissue or cell
extract, a bodily fluid, or a biopsy and the PrP.sup.Sc is from a
human, a livestock species, or a pet species. Detection of the
complex is preferably achieved through the use of ELISA, RIA,
western blotting, immunoprecipitation, fluorescence polarization,
or flow cytometry.
[0022] The present invention also features methods for treating or
preventing a prion disease in a mammal. This method includes the
steps of administering to the mammal an effective amount of a
peptide of about 200 or fewer amino acids which selectively binds
to PrP.sup.Sc or a fragment thereof in a
pharmaceutically-acceptable carrier and under conditions that allow
for complex formation between the peptide and a PrP.sup.Sc
poplypeptide or fragment thereof. A prion disease can be selected
from the group consisting of variant Creutzfeldt-Jakob Disease,
bovine spongiform encephalopathy, and scrapie.
[0023] In preferred embodiments, the peptide does not substantially
bind to PrP.sup.C. The peptide preferably comprises a YYX or YSA
motif, wherein the YYX motif is selected from the group consisting
of YYR, YYD, YYA, and YYQ.
[0024] Additional features of the invention include methods of
inhibiting PrP.sup.Sc in a biological sample. This method comprises
treating the biological sample with a peptide of about 200 or fewer
amino acids which selectively binds to PrP.sup.Sc or a fragment
thereof under conditions that allow for complex formation between
the peptide and a PrP.sup.Sc polypeptide or fragment thereof and
for a period of time sufficient to permit the formation of a
complex comprising the peptide and a PrP.sup.Sc.
[0025] In preferred embodiments, the biological sample is a bodily
fluid, a tissue, or an organ. The biological sample is also
preferably perfused with the peptide, which, in preferred
embodiments, does not substantially bind to PrP.sup.Sc.
[0026] The present invention also features a method for
decontaminating PrP.sup.Sc from a biological sample. This method
includes the steps of (a) treating the biological sample with a
peptide of about 200 or fewer amino acids which selectively binds
to PrP.sup.Sc or a fragment thereof under conditions that allow for
complex formation between the peptide and a PrP.sup.Sc polypeptide
or fragment thereof and for a period of time sufficient to permit
the formation of a complex comprising the peptide and PrP.sup.Sc,
and (b) recovering the complex from the biological sample. The
biological sample is preferably a tissue, bodily fluid or an organ
and is perfused with the peptide.
[0027] In preferred embodiments, the peptide does not substantially
bind PrP.sup.C. In additional preferred embodiments, the peptide
comprises a YYX or YSA motif, wherein the YYX motif is selected
from the group consisting of YYR, YYD, YYA, and YYQ.
[0028] The present invention also features a method for identifying
an agent for the treatment of a prion disease selected from the
group consisting of variant Cruetzfeldt-Jakob Disease, bovine
spongiform encephalopathy, and scrapie. The method includes the
steps of (a) combining a peptide of about 200 or fewer amino acids
which selectively binds to PrP.sup.Sc or a fragment thereof,
PrP.sup.Sc, and an agent under conditions allowing for complex
formation of the peptide and the PrP.sup.Sc, and (b) determining
whether complex formation is increased or decreased in comparison
to complex formation in the absence of the agent, thereby
identifying an agent for treating the prion disease. The agent used
can decrease or increase complex formation.
[0029] In preferred embodiments, the prion disease affects a human,
a livestock species, or a pet species. Non-limiting examples of
animals that can be affected by the prion disease include a human,
a bovine, a sheep, or a goat.
[0030] In preferred embodiments, the peptide does not substantially
bind PrP.sup.C. In additional preferred embodiments, the peptide
comprises a YYX or YSA motif, wherein the YYX motif is selected
from the group consisting of YYR, YYD, YYA, and YYQ.
[0031] The present invention also features an apparatus for
detecting PrP.sup.Sc in a biological sample. The apparatus
comprises a peptide-fixed portion where a peptide for trapping an
amount of an analyte in a sample is present and in a predetermined
amount. In preferred embodiments of this aspect, the analyte is a
bodily fluid.
[0032] In preferred embodiments, the peptide comprises a YYX, YYR,
YYD, or YYQ amino acid sequence and has antigenicity as a
PrP.sup.Sc. The peptide can also have the amino acid sequence
YYRRYYRYY or YYR. The peptide is composed of 18 or fewer amino
acids, preferably 12 or fewer amino acids, more preferably 8 or
fewer amino acids, and most preferably 5 or fewer amino acids.
[0033] The invention also features a method for detecting an
anti-PrP.sup.Sc antibody in a biological sample. This method
comprises the steps of (a) contacting the biological sample with a
peptide of about 200 or fewer amino acids which selectively binds
to PrP.sup.Sc or a fragment thereof under conditions that allow for
complex formation between the peptide and anti-PrP.sup.Sc antibody,
and (b) detecting the complexes as an indication that
anti-PrP.sup.Sc antibody is present in the biological sample.
[0034] By "peptide" is meant a molecule comprised of a chain of
amino acid residues joined by peptide (i.e., amide) bonds and
includes proteins and polypeptides. Peptides can be expected to
possess conformational preferences and to exhibit a
three-dimensional structure. Both the conformational preferences
and the three-dimensional structure are typically defined by the
polypeptide's primary (i.e., amino acid) sequence and/or the
presence (or absence) of disulfide bonds or other covalent or
non-covalent intrachain or interchain interactions. Exemplary
peptides of the invention are those having a chain of 200 or fewer
amino acids. Other peptides range from between 3 to 100 amino
acids, or 9 to 25 amino acids. Exemplary peptides are obtained from
PrP and typically include consensus sequences such as "YYX," "YYR,
"YYQ," "YYD," or "YSA." The peptide can, if desired, be linked
(e.g., covalently) to a detectable label for use as an affinity
probe to immobilized PrP.sup.Sc (or a PrP.sup.Sc fragment). Other
peptides can be random amino acid sequences that specifically bind
to PrP.sup.Sc based on physicochemical interaction, for example, to
regions of PrP.sup.Sc that include amino acid residues YYX. As is
disclosed in greater detail below, the invention provides peptides
that specifically bind to a PrP.sup.Sc protein and preferably bind
to a native non-denatured PrP.sup.Sc protein at high affinity.
Standard procedures for carrying out such affinity measurements are
known in the art and can be directly applied to measure the
affinity of a peptide of the invention for its binding to
PrP.sup.Sc. Preferred peptides of the invention are those that
specifically bind to PrP.sup.Sc, but do not substantially bind to
PrP.
[0035] The selective binding affinity between a peptide and
PrP.sup.Sc (or a fragment thereof) generally falls in the range of
about 1 nM to about 1 mM. In preferred embodiments of the present
invention, the selective binding is on the order of about 10 nM to
about 100 .mu.M, more preferably on the order of about 100 nM to
about 10 .mu.M, and most preferably on the order of about 100 nM to
about 1 .mu.M.
[0036] Systematic substitution of one or more amino acids of a
consensus sequence with a D-amino acid of the same type (e.g.,
D-lysine in place of L-lysine) may also be used to generate more
stable peptides. In addition, so-called constrained peptides
comprising a consensus sequence, for example those described
herein, or a substantially identical consensus sequence variation
may be generated by methods known in the art. For example, by
adding internal cysteine residues capable of forming intramolecular
disulfide bridges which cyclize the peptide as is described by Tam
et al. (Eur. J. Biochem. 267:3289-3300, 2000). The common modes of
cyclization also include side chain to side chain cyclization or
side chain to end-group cyclization as is described by Houston et
al. (J. Peptide Res. 52:81-88, 1998). For this purpose, amino acid
side chains are connected together or to the peptide backbone.
Another common cyclization as is the end-to-end cyclization as is
described in Kondejewski et al. (J. Biol. Chem. 274:13181-13192,
1999).
[0037] By "detectable label" is meant a material, which when
covalently attached to the peptides of this invention, permits
detection of the peptide. Suitable detectable labels are well known
in the art and include, by way of example, radioisotopes,
fluorescent labels (e.g., fluorescein), enzymes, epitope tags (e.g.
FLAG and Myc) and the like. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, beta-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.111In or .sup.99Tc. The
particular detectable label employed is not critical. Selection of
the label relative to such factors is well within the skill of the
art. Covalent attachment of the detectable label to the peptide is
accomplished by conventional methods well known in the art. For
example, when the 125I radioisotope is employed as the detectable
label, covalent attachment of 125I to the peptide can be achieved
by incorporating the amino acid tyrosine into the peptide and then
iodating the peptide. If tyrosine is not present in the peptide,
incorporation of tyrosine to the N or C terminus of the peptide can
be achieved by well-known chemistry. Similarly, .sup.32P can be
incorporated onto the peptide as a phosphate moiety through, for
example, a hydroxyl group on the peptide using conventional
chemistry. Other methods for detectably-labeling a peptide of the
invention are well known in the art.
[0038] By "prion diseases" is meant a group of prion-mediated,
rapidly progressive, fatal, and untreatable brain degenerative
disorders including, without limitation, Creutzfeldt-Jakob disease
(CJD), variant CJD, iatrogenic CJD, familial CJD, Kuru,
Gerstmann-Straussler syndrome, and fatal familial insomnia in
humans (Prusiner, Science 252:1515-1522, 1991), scrapie in sheep
and goats, and spongiform encephalopathy in cattle, as well as
recently described prion diseases in other ruminants and cats (see,
for example, Pattison, Emerg. Infect. Dis. 4:390-394, 1998).
[0039] By "treatment of prion diseases" is meant the ability to
reduce, prevent, stabilize, or retard the onset of any symptom
associated with prion diseases, particularly those resulting in
spongiform change, neuronal cell loss, astrocytic proliferation,
accumulation of PrP.sup.Sc protein, dementia, or death.
[0040] By "YYX" is meant a peptide having the sequence
Tyrosine-Tyrosine-X, where X is any amino acid. By"YYR" is meant a
peptide having the sequence Tyrosine-Tyrosine-Arginine. By "YYQ" is
meant a peptide having the sequence "Tyrosine-Tyrosine-Glutamine."
By "YYD" is meant a peptide having the sequence
"Tyrosine-Tyrosine-Aspartic acid." By "YSA" is meant a peptide
having the sequence Tyrosine-Serine-Alanine. By "YYA" is meant a
peptide having the sequence Tyrosine-Tyrosine-Aspartic acid." By
"YYRRYYRYY" is meant a peptide having the sequence
Tyrosine-Tyrosine-Arginine-Arginine-Tyrosine-Tyrosine-Arginine-Tyrosine-T-
yrosine."
[0041] Amino acid residues in peptides are abbreviated as follows:
Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile
or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or
S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A;
Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q;
Asparagine is Asn or N; Lysine is Lys or K; Asparcic Acid is Asp or
D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is
Trp or W; Arginine is Arg or R; and Glycine is Gly or G.
[0042] By a "therapeutic composition" is meant a composition
appropriate for administration to an animal, for example, a mammal,
such as a human, a livestock species (for example, a bovine, goat,
pig, or sheep), or a pet species.
[0043] By a "small molecule" is meant a compound with a molecular
weight of less than or equal to 10,000 Daltons, preferably, less
than or equal to 1000 Daltons, and, most preferably, less than or
equal to 500 Daltons.
[0044] Exemplary solid supports useful in the methods of the
invention include magnetic beads or other beads (e.g., agarose),
membranes (e.g. nitrocellulose, nylon), or plate wells (e.g.,
ELISA, or derivatized surfaces), carriers would generally be
soluble substrates, such as proteins (such as albumin or
thyroglobulin) or other soluble molecules.
[0045] The compounds (e.g., peptides) described herein are useful
for the prevention and treatment of prion diseases, for example,
those mediated by PrP.sup.Sc.
[0046] The invention also provides for pharmaceutical compositions
comprising one or more of the compounds described herein and a
physiologically acceptable carrier. These pharmaceutical
compositions can be in a variety of forms including oral dosage
forms, as well as inhalable powders and solutions and injectable
and infusible solutions.
[0047] Synthetic peptides can also be used directly to treat prion
diseases, and medications useful in the prevention and treatment of
prion diseases can be screened by inhibition of interactions
between PrP.sup.Sc and PrP.sup.Sc-binding peptides. PrP.sup.Sc can
be adsorbed from biological fluids and tissues to neutralize prion
infectivity prior to human use or as a prion disease prophylactic
in animal feed. For example, the present invention further provides
methods for identifying potential new drug candidates (and
potential lead compounds) and methods for determining the
specificities of these compounds. For example, knowing that a
peptide exhibits a selective affinity to PrP.sup.Sc enables the
identification of a compound that effects the binding between these
molecules, e.g., either as an agonist or as an antagonist
(inhibitor) of the interaction. With this assay, then, one can
screen one or more libraries of candidate compounds to identify a
compound exhibiting the most desired characteristic, e.g., the most
efficacious in disrupting the interaction or in competing with the
peptide for binding to PrP.sup.Sc.
[0048] Such synthetic peptides also have utility as diagnostic
reagents, and in therapy of prion diseases.
[0049] The invention further includes a solid support which
includes a peptide that selectively binds PrP.sup.Sc in the form of
a detection apparatus or device which comprises a chamber, one or
more inlet ports, one or more outlet ports, and a matrix within the
chamber to which the peptide is adsorbed or chemically crosslinked.
In an illustrative embodiment, a peptide of the invention is
adsorbed to a solid support, a solution containing PrP.sup.Sc is
passed over the support, and non-binding components from the
solution are removed, e.g., by washing. Methods for washing such a
column, device, or apparatus to remove contaminating materials, and
then subsequently eluting the bound PrP.sup.Sc from the peptide
matrix using eluants such as chaotropic reagents are well known in
the art. The apparatus of the invention provides detection systems
for detecting, monitoring, quantitating or analyzing PrP.sup.Sc
specifically retained on the surface of the platform, in a
detection chamber comprising a specific binding reagent (e.g., a
peptide that specifically binds PrP.sup.Sc). Detection systems
useful in the manufacture and use of the platforms of the invention
include, but are not limited to, fluorescent, chemiluminescent, or
radioactive measurements.
[0050] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1 shows that the YYRRYYRYY peptide captures PrP.sup.Sc
in hamster brain lysates.
[0052] FIG. 2 shows that the YYRRYYRYY peptide captures PrP.sup.Sc
in bovine brain lysates.
[0053] FIG. 3 shows that peptides related to the YYRRYYRYY peptide
capture PrP.sup.Sc in mouse brain lysates.
[0054] FIG. 4 shows that that the YYAAYYAYY (SEQ ID NO: 3) and
YSAASYASY (SEQ ID NO: 4) peptides compete with YYRRYYRYY-linked
beads for capture of mouse PrP.sup.Sc.
DETAILED DESCRIPTION OF INVENTION
[0055] We describe below a method by which PrP.sup.Sc-interacting
molecules (e.g., peptides) can be recognized. Preferably,
PrP.sup.Sc-interacting molecules will be identified that exploit
general physicochemical properties of PrP.sup.Sc which distinguish
it from PrP.sup.C, or that exploit the sequence- and
structure-specific molecular mechanism of prion isoform interaction
and/or conversion. Accordingly, the present invention provides
methods for identifying compounds that bind to and inactivate PrP
or otherwise behave as a prion antagonist. These compounds include
"lead" peptide compounds and "derivative" compounds constructed so
as to have the same or similar molecular structure or shape as the
lead compounds, but that differ from the lead compounds either with
respect to susceptibility to hydrolysis or proteolysis and/or with
respect to other biological properties, such as increased affinity
for PrP.sup.Sc. The present invention also provides compositions
comprising an effective amount of a prion antagonist, and more
particularly a compound, that is useful for treating prion
diseases.
[0056] Discovery of Peptides That Selectively Bind to
PrP.sup.Sc
[0057] The present invention provides methods for the
identification of one or more peptides that binds to PrP.sup.Sc or
a fragment. The invention provides for the rapid identification of
peptides having the ability to interact with PrP.sup.Sc. By
screening individual peptides or other sources of peptides (e.g.,
peptide libraries) for binding to PrP.sup.Sc, the invention allows
for the identification of highly disparate protein sequences
possessing equivalent functional activities, for example, the
ability to identify peptides that bind PrP.sup.Sc using (1)
peptides whose sequence or composition are not determined by the
sequence or composition of PrP or (2) peptides whose sequence or
composition are determined by the sequence or composition of PrP.
The ability to identify and isolate peptides that bind to
PrP.sup.Sc or a fragment thereof will prove invaluable in bringing
new compounds into prion disease drug discovery programs.
[0058] PrP.sup.Sc
[0059] PrP.sup.Sc is defined as a misfolded, prion
disease-associated conformational isoform of the prion protein.
Natural and experimental prion infections are recognized in (but
not limited to) humans, sheep, goats, elk, deer, cattle, mice, and
hamsters. The set of abnormal conformations (designated as, for
example, PrP.sup.Sc, PrP.sup.BSE, PrP.sup.CWD, PrP.sup.CJD
PrP.sup.vCJD, depending upon the species of origin and prion
strain; PrP.sup.Sc herein used generically) may possess partial
protease resistance and high .beta. sheet content. PrP.sup.Sc
fragments are typically defined as portions of PrP.sup.Sc that are
sufficiently large as to retain prion infectivity. The "protein
only" theory of prion infectivity posits that molecules of
PrP.sup.C, a normal cell surface membrane protein, are converted to
PrP.sup.Sc by a template-directed process catalyzed by the abnormal
isoform.
[0060] Peptides
[0061] Peptides of the invention are, in general, molecules having
an isoform-selective affinity for PrP.sup.Sc or a fragment thereof,
with little or no binding to PrP.sup.C. As described further below,
the peptides of the invention are typically a fragment of PrP that
preferably contains between 3 to 75 amino acid residues of PrP, or
multimers thereof. Exemplary PrP amino acid sequences deposited at
SwissProt include P04156 (human), P04925 (mouse), P97895 (golden
hamster), P23907 (sheep), P52113 (goat), O02841 (white-tailed
deer), P79142 (American elk), P10279 (bovine), Q01880 (bovine) and
O18754 (cat). In other embodiments, the peptides of this invention
include multimers of PrP sequences, composed of either sequences
derived directly from reported PrP sequences, or containing amino
acid substitutions homologous to the native sequence, as indicated
by physicochemical similarity (e.g., Bacon and Anderson, J. Mol.
Biol. 191: 153-61, 1986) or likelihood of substitution in evolution
(e.g., Dayhoff et al., Atlas Protein Seq. Struc. 5: 345-352, 1978).
These sequences include, but are not limited to, sequences
containing PrP repeat motifs such as "YYX," "YYR," "YYQ," or "YYD."
In particular embodiments, the peptides are in the form of MAPS,
which are prepared according to standard methods (Tam, Proc Natl
Acad Sci USA 85: 5409-13, 1988; Tam and Zevala, J. Immunol Methods
124: 53-61, 1989; Tam, J. Immunol Methods 196: 17-32, 1996),
including 4-map and 8-map formats. One or more N-terminal cysteines
for use as a coupling moiety may be added to the peptide sequence.
Peptides can be produced not only by recombinant methods, but also
by using chemical methods well known in the art. Solid phase
peptide synthesis may be carried out in a batchwise or continuous
flow process which sequentially adds .alpha.-amino- and side
chain-protected amino acid residues to an insoluble polymeric
support via a linker group. A linker group such as
methylamine-derivatized polyethylene glycol is attached to
poly(styrene-co-divinylbenzene) to form the support resin. The
amino acid residues are N-a-protected by acid labile Boc
(t-butyloxycarbonyl) or base-labile Fmoc
(9-fluorenylmethoxycarbonyl). The carboxyl group of the protected
amino acid is coupled to the amine of the linker group to anchor
the residue to the solid phase support resin. Trifluoroacetic acid
or piperidine are used to remove the protecting group in the case
of Boc or Fmoc, respectively. Each additional amino acid is added
to the anchored residue using a coupling agent or pre-activated
amino acid derivative, and the resin is washed. The full length
peptide is synthesized by sequential deprotection, coupling of
derivitized amino acids, and washing with dichloromethane and/or N,
N-dimethylformamide. The peptide is cleaved between the peptide
carboxy terminus and the linker group to yield a peptide acid or
amide. These processes are described, for example, in Merrifield,
J. Am. Chem. Soc. 85:2149-2154, 1963; Atherton, Solid Phase Peptide
Synthesis, IRL Press, Oxford (1989); and Erickson and Merrifield,
In: The Proteins, (Neurath, H. and Hill, R. L., eds.) Academic
Press, New York, vol. 2, pp. 255-527, 1976. Automated synthesis may
also be carried out on machines such as the ABI 431A peptide
synthesizer (ABI). A protein or portion thereof may be purified by
preparative high performance liquid chromatography and its
composition confirmed by amino acid analysis or by sequencing
(Creighton, Proteins, Structures and Molecular Properties, W H
Freeman, New York N.Y., 1984).
[0062] According to the methods of the invention, a peptide (which
optionally is conjugated to a detectable label) or a plurality of
peptides are contacted with PrP.sup.Sc to identify one or more
peptides that selectively binds to PrP.sup.Sc. If desired, random,
combinatorial or conformationally-constrained peptide libraries can
be used as a source of peptides, which can be screened to identify
peptides that bind to, for example PrP.sup.Sc. Many libraries are
known in the art that can be used, e.g., chemically synthesized
libraries or in vitro translation based libraries. Conformationally
constrained libraries that can be used include but are not limited
to those containing invariant cysteine residues which, in an
oxidizing environment, cross-link by disulfide bonds to form
cystines, modified peptides (e.g., incorporating fluorine, metals,
isotopic labels, are phosphorylated, etc.), peptides containing one
or more non-naturally occurring amino acids, non-peptide
structures, and peptides containing a significant fraction of
.gamma.-carboxyglutamic acid. Screening of peptide libraries or
individual peptides for a peptide that selectively binds to
PrP.sup.Sc or a fragment thereof, but not to PrP, is accomplished
using any of a variety of commonly known methods.
[0063] The step of contacting PrP.sup.Sc or a fragment thereof with
a peptide or with a plurality of polypeptides may be effected in a
number of ways. For example, PrP.sup.Sc can be immobilized on a
solid support and a solution of the plurality of polypeptides is
contacted with the immobilized PrP.sup.Sc. This procedure is
similar to an affinity chromatographic process, with the affinity
matrix being comprised of the immobilized PrP.sup.Sc. The peptides
having a selective affinity for PrP.sup.Sc are then purified by
affinity selection. The nature of the solid support, process for
attachment of PrP.sup.Sc to the solid support, solvent, and
conditions of the affinity isolation or selection procedure are
carried out according to conventional methods and are well known to
those of ordinary skill in the art.
[0064] If the amino acid sequence is to be determined from the
polypeptide itself, one may use microsequencing techniques. The
sequencing technique may, if desired, include mass
spectroscopy.
[0065] In certain situations, it may be desirable to wash away any
unbound peptide from a mixture of the PrP.sup.Sc and the plurality
of polypeptides prior to attempting to determine or to detect the
presence of a selective affinity interaction (i.e., the presence of
a recognition unit that remains bound after the washing step).
[0066] The degree of selective binding between PrP.sup.Sc (or a
fragment thereof) and its interacting peptide may vary, generally
falling in the range of about 1 nM to about 1 mM. In preferred
embodiments of the present invention, the selective binding is on
the order of about 10 nM to about 100 .mu.M, more preferably on the
order of about 100 nM to about 10 .mu.M, and most preferably on the
order of about 100 nM to about 1 .mu.M. Binding is said to be
selective when under similar experimental conditions a peptide
exhibits higher affinity for PrP.sup.Sc than for PrP.sup.C. The
binding affinity may be quantitatively or qualitatively assessed by
any method known in the art.
[0067] Screening for Interactions of PrP.sup.Sc With Peptides Whose
Sequence or Composition are Not Determined By the Sequence or
Composition of PrP
[0068] The interaction of PrP.sup.Sc with a peptide may be due to
physicochemical forces between the molecules unrelated to a
specific sequence contained within PrP. Examples of such
interactions include the following.
[0069] Hydrophobic Interactions
[0070] PrP.sup.Sc displays increased hydrophobicity in comparison
to PrP.sup.C, probably contributing to the poor solubility and
increased aggregation tendency of the abnormal isoform. Moreover,
increased molecular surface hydrophobicity is observed in
recombinant PrP.sup.C induced by low pH and denaturants to develop
increased beta sheet content reminiscent of PrP.sup.Sc (Swietnicki
et al, J. Biol. Chem. 272:27517-27520, 1997). Hydrophobic moieties
will bind to other hydrophobic moieties in aqueous environments,
due to Van der Waals interactions induced by molecular proximity
and an increase in entropy of water associated with the reduction
of hydrophobic molecular surface (Lodish et al., eds., Molecular
Cell Biology, Palgrave Publishers, 2000). Thus, peptides containing
hydrophobic amino acids will bind selectively to PrP.sup.Sc.
Hydrophobic amino acids, graded from most to least hydrophobic
(Kyte and Doolittle, J. Mol. Biol. 157:105-32, 1982), are
phenylalanine, methionine, isoleucine, leucine, valine, cysteine;
other hydrophobic amino acids (Engleman et al., Annu. Rev. Biophys.
Biophys. Chem. 15:321-53, 1986) are tryptophan, alanine, threonine,
glycine, and serine. It should be noted that excessive
hydrophobicity of selected peptides may render them unusable in
PrP.sup.Sc-selective binding, for reasons of generalized protein
adherence (including PrP.sup.C) and neutralization of PrP.sup.Sc
binding due to self-adherence prior to interaction with a sample.
Thus, peptides containing amino acids of intermediate
hydrophobicity may prove superior for this application, or
hydrophobic amino acids interspersed with hydrophilic amino acids
to enhance solubility in an aqueous sample.
[0071] Pi-Stacking Interactions
[0072] We have demonstrated that recombinant PrP subjected to
denaturants and low pH displays more tyrosyl groups at the
molecular surface than PrP.sup.C, and that mono- and polyclonal
antibodies directed against the PrP repeat motif
tyrosine-tyrosine-arginine bind to PrP.sup.Sc but not PrP.sup.C
from a number of species (see, for example, WO/0078344). PrP
refolded in a disease-specific form is therefore believed to
possess more solvent accessible tyrosine side chains than does
PrP.sup.C. Increased accessibility of surface tyrosyls can be
exploited for isoform-specific affinity interactions by pi-stacking
with aromatic amino acids contained in peptide reagents of this
invention. Pi-stacking occurs through interaction of an
electron-rich aromatic ring circumference with an electron poor
aromatic ring center, which may be mediated by a displaced parallel
interaction, or an edge-to-face or "T" interaction conformation
(McGaughey et al, J. Biol. Chem. 273:15458-63, 1998). Aromatic
residues classically thought to undergo pi stacking include
tyrosine, tryptophan, and phenylalanine. Peptides containing
aromatic amino acids will specifically interact with PrP.sup.Sc
through pi-stacking with tyrosine or other aromatic residues
exposed on the molecular surface of PrP.sup.Sc.
[0073] Charge Interactions
[0074] PrP.sup.Sc displays more surface hydrophobicity than
PrP.sup.C, but charged or polar moieties can participate in
specific interactions. Such charged and polar moieties potentially
exposed to solvent on PrP.sup.Sc at physiological solutions and pH
include glycans (particularly the negatively charged sialic acid
residues terminating many glycan antennae; Endo et al.,
Biochemistry 28:8380-8,1989) and charged amino acid side chains
such as arginine, lysine, aspartate, and glutamate.
[0075] Beta Sheet Interactions
[0076] PrP.sup.Sc contains much more beta sheet conformation than
does PrP.sup.C. It is possible that peptides could adopt a beta
strand conformation which would then be "incorporated" into the
beta sheet-rich structure of PrP.sup.Sc. Moreover, peptides
constrained in a beta sheet conformation may constitute an affinity
reagent selectively interacting with PrP.sup.Sc. Parallel and
antiparallel beta sheets are stabilized by hydrogen bonding between
C.dbd.O groups on one strand and NH groups on an adjacent strand.
Moreover, amino acids of beta strands in proteins often alternate
between hydrophobic side chains buried in the protein interior, and
hydrophilic side chains at the molecular surface.
Sequence-constrained properties may therefore be mimicked by
synthetic peptides or by structurally constrained peptides, which,
in turn, may be exploited for specific affinity interactions with
PrP.sup.Sc. In this manner, the synthetic peptide may be regarded
as an exogenous beta strand for domain swapping with the relevant
endogenous PrP strand. 3D domain swapping has been recently
recognized as a mechanism by which monomeric proteins may form
multimeric assemblies (reviewed in Bennett et al, Protein Science,
4: 2455-2468, 1995).
[0077] Screening for Interactions of PrP.sup.Sc With Peptides Whose
Sequence or Composition are Determined By the Sequence or
Composition of PrP
[0078] In another series of examples, the interaction of PrP.sup.Sc
with a peptide may be due to physicochemical forces resulting from
prion protein interaction with itself, dependent upon a specific
sequence contained within PrP. It is possible that such self-self
interactions drive the PrP.sup.C to PrP.sup.Sc conversion central
to the propagation of prion infectivity.
[0079] In one example of the invention, peptides are constructed to
mimic a domain of PrP that is involved in the molecular process of
recognition and recruitment of PrP.sup.C by PrP.sup.Sc. These
interactions can utilize a number of intermolecular forces, such as
ionic interactions, hydrogen bonding, hydrophobic interactions, and
pi-stacking of aromatic residues. The invention particularly
relates to diagnostic aids that contain a PrP motif, either as a
peptide with a single motif, or tandem repeats of the motif. One
such motif repeated three times in the PrP sequence comprises two
sequential tyrosines in association with a C-terminal arginine at
two sites (YYR), and a C-terminal glutamine or aspartate at the
third site (YYQ/D). YYX motif sequences are conserved across a
number of species including, but not limited to, bovine, man,
sheep, mouse, and hamster. Poly- and monoclonal antibodies directed
against YYR have been shown to immunoprecipitate PrP.sup.Sc
specifically, establishing that motif side chains become
differentially solvent accessible in the conformational conversion
of the prion protein. See, for example, WO/0078344. The specific
interaction of solvent exposed YYR motifs in PrP with a synthetic
polyamino acid chain can therefore be exploited for selective
recognition of PrP.sup.Sc in a biological sample.
[0080] In another aspect, the invention relates to short synthetic
prion peptides (e.g., three to ten amino acids or four to twelve
amino acids, inclusive) including amino acid side chains which are
differentially exposed to solvent in PrP.sup.Sc but not PrP.sup.C.
Critical amino acid residues participating in this interaction can
be identified and a specific artificial sequence (peptide) can be
constructed to selectively bind PrP.sup.Sc and not PrP.sup.C.
Examples of motifs participating in the conversion of PrP.sup.C to
PrP.sup.Sc (and thus potentially differentially solvent accessible
in PrP.sup.Sc) are to be found in the medical and biological
literature (see, for example, Horiuchi et al., J. Biol. Chem.
276:15489-97, 2001).
[0081] Notably, amino acids from several groups often possess the
potential to interact with proteins by several intermolecular
forces. For example, tryptophan may interact by means of
hydrophobic and pi-stacking interactions, and tyrosine may interact
by aromatic pi-stacking and hydrogen bonding involving its hydroxyl
group. Thus, another example of the invention is interaction with
PrP.sup.Sc via a peptide modeled on the prion protein sequence, but
with evolutionarily conserved amino acid substitutions.
[0082] To demonstrate the interaction of a PrP peptide with
PrP.sup.Sc, fifty .mu.g of normal (N) or scrapie (Sc) hamster brain
lysate was incubated with 100 .mu.l of 17D10 antibody-coated
tosyl-activated magnetic beads (17D10; FIG. 1, lanes 2 and 3), 100
.mu.l of YYRRYYRYY peptide-coupled sulfolink resin (10-mer; FIG. 1,
lanes 4 and 5), or 100 .mu.l of a FIP control peptide-coupled
sulfolink resin (control; FIG. 1, lanes 6 and 7) in binding buffer
(PBS, 3% Igepal CA630 and 3% Tween-20) for 2.5 hours at room
temperature. The beads (or resin) were then washed with wash buffer
(PBS, 2% Igepal CA630 and 2% Tween-20) three times, and resuspended
in gel loading buffer. The samples were run on 16% Tris Glycine
gels, and blots developed with monoclonal 6H4 and goat anti-mouse
IgG-HRP conjugate. With this repeat motif derived from the prion
protein amino acid sequence, precipitation of the prion protein,
PrP.sup.Sc derived from scrapie infected mouse brain, is observed,
but not the normal isoform, PrP.sup.C derived from normal mouse
brain (FIG. 1). The FIP control peptide precipitated neither
isoform.
[0083] In addition, the interaction of the YYRRYYRYY peptide with
bovine PrP.sup.Sc was examined (FIG. 2). Varying concentrations of
BSE-positive (Sc) or negative (N) bovine brain lysate were
incubated with 100 .mu.l of tosyl-activated magnetic beads coated
with cys-YYRRYYRYY ("YYR" 10-mer) or cys-YARRAYRAY ("YAR"; SEQ ID
NO: 5; 10-mer) in IP binding buffer for 2.5 hours at room
temperature. The beads were then washed with IP wash buffer three
times, and resuspended in gel loading buffer. The samples were run
on 4-12% NuPage MES gels, and blots developed with monoclonal 6H4
and goat anti-mouse IgG-HRP conjugate. The lane assignments are as
follows: Lane 1, YYR 10-mer and 10 .mu.g normal lysate; lane 2, YYR
10-mer and 10 .mu.g BSE lysate; lane 3, YAR 10-mer and 10 .mu.g
normal lysate; lane 4, YAR 10-mer and 10 .mu.g BSE lysate; lane 5,
YYR 10-mer and 20 .mu.g BSE lysate; lane 6, YYR 10-mer and 10 .mu.g
BSE lysate; lane 7, YYR 10-mer and 5 .mu.g BSE lysate; lane 6, YYR
10-mer and 1 .mu.g BSE lysate. These data indicate that the
YYR-related affinity precipitation of PrP.sup.Sc is not
species-restricted. It should be noted that although the YAR 1
0-mer coupled to tosyl beads failed to capture PrP.sup.Sc from BSE
positive samples, biotinylated versions of this peptide coupled to
strepavidin coated magnetic beads do function as effective capture
reagents.
[0084] To determine whether peptides related to the YYRRYYRYY
(10-mer) peptide facilitate the capture of PrP.sup.Sc, fifty .mu.g
of scrapie (Sc) mouse brain lysate was incubated with 100 .mu.l of
tosyl-activated magnetic beads coated with cys-YYRRYYRYY ("(YYR)3;
FIG. 3, lanes 1 and 6), cys-AYRRYARYA ("(AYR).sub.3"; SEQ ID NO: 6;
FIG. 3, lane 2), cys-YARRAYRAY ("(YAR).sub.3"; FIG. 3, lane 3),
cys-YYAAYYAYY ("(YYA).sub.3; FIG. 3, lane 4), or cys-YSAASYASY
("(YSA).sub.3"; FIG. 3, lane 5) in IP binding buffer for 2.5 hours
at room temperature. The beads were then washed with IP wash buffer
three times, and resuspended in gel loading buffer. The samples
were run on 16% Tris-glycine gels, and blots developed with
monoclonal 6H4 and goat anti-mouse IgG-HRP conjugate. As shown in
FIG. 3, precipitation of PrP.sup.Sc was observed with the YYR
repeat sequence, and with YYA and YSA substitutions, but not with
YAR or AYR repeats. These data suggest a critical role for tyrosine
pairs, and/or a hydroxyl moiety of one tyrosine, in the affinity
precipitation using peptides coupled to tosyl-activated beads.
Biotinylated versions of the (YAR).sub.3 peptide, however, are
effective capture reagents when coupled to streptavidin magnetic
beads. Other peptide sequences that have been evaluated and shown
to selectively bind PrP.sup.Sc in this format include, YARYARYAR,
YRAARYRAY, bovine PrP (158-183) and bovine PrP (130-147). Peptides
negative for PrP.sup.Sc selective capture include NHSTHNTGH (SEQ ID
NO: 7), DRYYWYFDV (SEQ ID NO: 8) and DEAYYKGWFAY (SEQ ID NO:
9).
[0085] The specificity of the peptide: PrP.sup.Sc was also studied
in the following competition experiments. To demonstrate that the
YYAAYYAYY and YSAASYASY (SEQ ID NO: 10) peptides compete with
YYRRYYRYY beads for the capture of mouse PrP.sup.Sc, 100 .mu.l of
tosyl-activated magnetic beads coated with cys-YYRRYYRYY were
incubated over night at 4.degree. C. with 500 .mu.g/ml free peptide
in IP binding buffer. Fifty .mu.g of normal (N) or scrapie (Sc)
mouse brain lysate was added to each sample, and the beads were
then incubated for 2.5 hours at room temperature. The samples were
then washed with IP wash three times, and resuspended in gel
loading buffer. The samples were run on 16% Tris Glycine gels, and
blots developed with monoclonal 6H4 and goat anti-mouse IgG-HRP
conjugate. The data are shown in FIG. 4: lane 1, normal mouse
lysate and no competing peptide; lane 2, Scrapie lysate and no
competing peptide; lane 3, scrapie lysate and cys-YARRAYRAY
("(YAR).sub.3"); lane 4, scrapie lysate and cys-YYAAYYAYY
("(YYA).sub.3"); lane 5, scrapie lysate and cys-YSAASYASY
("(YSA).sub.3"); lane 6, Scrapie lysate and cys-AYRRYARYA
("(AYR).sub.3"). These data show that free YYAAYYAYY and YSAASYASY
compete with tosyl-activated YYRRYYRYY beads for capture of
PrP.sup.Sc. Surprisingly, free YARRAYRAY peptide failed to compete
for binding even though biotinylated forms of the peptide are
effective ligands for PrP.sup.Sc. In any case, these data extend
those exemplified in FIG. 2 in implicating paired tyrosines,
particularly the hydroxyl moiety of the internal tyrosine, in the
specific affinity interaction of YYR-related peptides with
PrP.sup.Sc.
[0086] Use
[0087] The peptides described herein may be used, for example, for
the following diagnostic, therapeutic, vaccination, and
decontamination purposes, as well as for screening for novel
compounds that can be utilized to diagnose or combat prion diseases
or decontaminate prion samples.
[0088] Diagnostics
[0089] The peptides disclosed herein find diagnostic use generally
in the detection or monitoring of prion diseases. For example, the
YYRRYYRYY peptide may be used to monitor the presence or absence of
PrP.sup.Sc in a biological sample (e.g., a tissue biopsy, a cell,
or fluid) using standard and/or amplified detection assays. Such
assays and methods may involve direct detection of PrP.sup.Sc, and
are particularly suited for screening large amounts of samples for
the presence of PrP.sup.Sc. For example, any of the peptides
described herein may be detectably-labeled to measure peptide: PrP
complex formation. If desired, because of the specificity of the
peptides described herein for capturing PrP.sup.Sc, pretreatment of
a test sample with protease prior to analysis is optional. Any
appropriate label which may be directly or indirectly visualized
may be utilized in these detection assays including, without
limitation, any epitope tag, radioactive, fluorescent, chromogenic
(e.g., alkaline phosphatase or horseradish peroxidase), or
chemiluminescent label, or a hapten (for example, digoxigenin or
biotin) which may be visualized using a labeled, hapten-specific
antibody or other binding partner (e.g., avidin). For example,
using the peptides described herein, PrP.sup.Sc may be readily
detected at the cell surface (e.g., a leukocyte) using standard
flow cytometry methods such as those described herein. Samples
found to contain increased levels of labeled complex compared to
appropriate control samples are taken as indicating the presence of
PrP.sup.Sc, and are thus indicative of a prion-related disease.
[0090] In addition, novel compounds useful for diagnosing prion
disease may be identified using the peptides of the invention. For
example, combinatorial chemical libraries or small molecule
libraries are screened to identify compounds having the ability to
inhibit the binding interaction of one or more of the peptides
described herein according to standard methods.
[0091] Such libraries may be derived from natural products,
synthetic (or semi-synthetic) extracts, or chemical libraries
according to methods known in the art. Those skilled in the field
of drug discovery and development will understand that the precise
source of compounds is not critical to the screening procedure(s)
of the invention. Examples of natural compound sources include, but
are not limited to, plant, fungal, prokaryotic, or animal sources,
as well as modification of existing compounds. Numerous methods are
also available for generating random or directed synthesis (e.g.,
semi-synthesis or total synthesis) of any number of chemical
compounds, including, but not limited to, saccharide-, lipid-,
peptide-, and nucleic acid-based compounds, i.e. aptamers.
Synthetic compound libraries may be obtained commercially or may be
produced according to methods known in the art. Furthermore, if
desired, any library or compound is readily modified using standard
chemical, physical, or biochemical methods.
[0092] Compounds that inhibit binding of a peptide at lowest
concentration are referred to as "high affinity competitors" and
are useful in the diagnostic methods of the invention. Such high
affinity competitors that mimic the activity of the peptide are
subsequently tested for efficient recognition and binding of
PrP.sup.Sc. Once identified, high affinity competitors may be
coupled to solid substrates (for example, ELISA wells or beads) for
use in the capture phase of virtually any diagnostic test for prion
infection or, alternatively, in blocking format assays.
Vaccines
[0093] Peptides of the invention and mixtures and combinations
thereof are also useful as active components of vaccines capable of
inducing a prophylactic or therapeutic immune response against
prion diseases in a host susceptible to and/or harboring infection.
Routes of administration, antigen doses, number and frequency of
injections will vary from species to species and may parallel those
currently being used in the clinic and/or experimentally to provide
immunity or therapy against other infectious diseases or cancer.
For example, the vaccines are pharmaceutically acceptable
compositions containing the peptide of this invention, its
analogues or mixtures or combinations thereof, in an amount
effective in the mammal, including a human, treated with that
composition to raise immunity sufficient to protect the treated
mammal from prion infection for a period of time. It is also
possible that PrP.sup.Sc-specific immunity prompted by immunization
with peptides that include YYX amino acid residues (or YYR, YYD, or
YYQ amino acid residues) or related compounds are useful to favor
the degradation of PrP.sup.Sc or alleviate manifestations of the
disease without affecting the expression or function of PrP.sup.C
in the brain and other tissues, resulting in improvement of
clinical status in clinically symptomatic humans with prion
disease.
[0094] Different types of vaccines can be developed according to
standard procedures known in the art. For example, a vaccine may be
peptide-based, nucleic acid-based, bacterial- or viral-based
vaccines. More specifically, with regard to peptide vaccines,
peptides corresponding to the PrP.sup.Sc-specific epitope or a
functional derivatives thereof can be utilized as a prophylactic or
therapeutic vaccine in a number of ways, including: 1) as monomers
or multimers of the same sequence, 2) combined contiguously or
non-contiguously with additional sequences that may facilitate
aggregation, promote presentation or processing of the epitope
(e.g., class I/II targeting sequences) and/or additional antibody,
T helper or CTL epitopes to increase the immunogenicity of the
PrP.sup.Sc-specific epitope as a means to enhance efficacy of the
vaccine, 3) chemically modified or conjugated to agents that would
increase the immunogenicity or delivery of the vaccine (e.g., fatty
acid or acyl chains, KLH, tetanus toxoid, or cholera toxin), 4) any
combination of the above, 5) the above in combination with
adjuvants, including but not limited to aluminum salts, saponins or
triterpenes, MPL, and cholera toxin, and/or delivery vehicles,
including but not limited to liposomes, VPLs or virus-like
particles, microemulsions, attenuated or killed bacterial and viral
vectors, and degradable microspheres, 6) administered by any route
or as a means to load cells with antigen ex-vivo.
[0095] Examples of uses of nucleic-acid based vaccines as a
prophylactic or a therapeutic include: 1) any nucleic acid encoding
the expression (transcription and/or translation) of the
PrP.sup.Sc-specific epitope, 2) additional nucleic acid sequences
that facilitate processing and presentation, aggregation,
secretion, targeting (to a particular cell type) of the
PrP.sup.Sc-specific epitope, either translational fusions or
independent transcriptional units, 3) additional nucleic acid
sequences that function as adjuvants/immunomodulators, either
translational fusions or independent transcriptional units, 4)
additional antibody, T helper or CTL epitopes that increase the
immunogenicity of the PrP.sup.Sc-specific epitope or efficacy of
the vaccine, either translational fusions or independent, 5) any
combination of the above, 6) the above administered in saline
(`naked` DNA) or in combination with an adjuvant(s), (e.g. aluminum
salts, QS-21, MPL), immunomodulatory agent(s) (e.g. rIL-2, rGM-CSF,
rIL-12), and/or nucleic acid delivery agents (e.g. polymer-,
lipid-, peptide-based, degradable particles, microemulsions, VPLs,
attenuated bacterial or viral vectors) using any route or ex vivo
loading.
[0096] Attenuated or killed bacterial or viral vectors can be used
to deliver either the antigen or DNA/RNA that codes for the
expression of the antigen. These can also be used as a means to
load cells with antigen ex vivo.
[0097] Vaccines are prepared according to standard methods known in
the art, and will be readily applicable to any new or improved
method for vaccine production.
[0098] Decontamination
[0099] PrP.sup.Sc can be adsorbed from biological fluids and
tissues to neutralize prion infectivity prior to human use or as a
prion disease prophylactic in animal feed using the peptides
disclosed herein.
[0100] In still another aspect, the invention features methods for
decontaminating PrP.sup.Sc from a biological sample. In a preferred
embodiment, the method involves the steps of: (a) treating the
biological sample with the peptide (or a fragment or analog
thereof), the treatment permitting PrP.sup.Sc complex formation
with the peptide; and (b) recovering the PrP complex from the
biological sample. Such a decontamination procedure may also
involve the use of perfusing a biological sample with peptide (or a
fragment or analog thereof) coupled to biotin, hapten or epitope
tag, such as FLAG or Myc, for the removal via streptavidin, or
antibody affinity column chromatography or direct inactivation of
PrP.sup.Sc by binding.
[0101] Accordingly, the methods and compositions described herein
are useful for the decontamination of biological samples that are
known or suspected of being contaminated with a prion, e.g.
intended for transplantation. In particular, biological samples may
be incubated with a peptide of the invention, and the complexes
removed using standard methods. Alternatively, a peptide of the
invention may be incubated with biological samples to complex with,
and thereby inhibit the infectivity of prion.
[0102] Therapeutics
[0103] The invention further features a method of treating or
preventing a prion disease in an animal (for example, a human, a
bovine, sheep, pig, goat, dog, or cat). In one preferred
embodiment, the method involves administering to the animal a
therapeutically effective amount of PrP peptide identified
according to the methods disclosed herein that blocks the
conversion of PrP.sup.C to PrP.sup.Sc, inhibits PrP.sup.Sc:PrP
aggregate formation, or blocks the recruitment of PrP.sup.C to
PrP.sup.Sc. In another aspect, the invention features a
pharmaceutical preparation for the therapy and prevention of prion
diseases comprising a PrP peptide of the invention or structurally
related compounds, for example, compounds which exploit the
PrP.sup.Sc-specific exposure of peptides including amino acid
residues YYX (or YYR, YYD, or YYQ amino acid residues) can be
rationally designed or obtained from combinatorial libraries which
mimic the interaction of a YYX containing peptide with anti-YYX
peptides. These compounds are useful in prion diagnostics or as
therapies for prion diseases. If desired, the peptides of the
invention can be provided in the form of pharmaceutically
acceptable salts. Examples of preferred salts are those with
therapeutically acceptable organic acids, e.g., acetic, lactic,
maleic, citric, malic, ascorbic, succinic, benzoic, salicylic,
methanesulfonic, toluenesulfonic, or pamoic acid, as well as
polymeric acids such as tannic acid or carboxymethyl cellulose, and
salts with inorganic acids such as the hydrohalic acids, e.g.,
hydrochloric acid, sulfuric acid, or phosphoric acid. In addition,
any of the peptides of the invention may be administered to a
mammal, particularly a human, in one of the traditional modes
(e.g., orally, parenterally, transdermally, or transmucosally), in
a sustained release formulation using a biodegradable biocompatible
polymer, or by using micelles, gels, and liposomes.
[0104] Moreover, small molecules derived from the structure of the
YYR epitope(s), including but not limited to tyrosine side-chain
derivatives, may block the conversion reaction. Finally, direct
chemical modification of critical residues, such as enzymatic lysis
of tyrosine rings, or covalent derivatization of tyrosine rings
with bulky substitutions, may also disrupt the PrP.sup.C to
PrP.sup.Sc conversion reaction.
[0105] For example, such compounds may be identified using the
antibodies of the invention. Accordingly, combinatorial libraries
or small molecule libraries or both (infra) are screened to
identify compounds having the ability to inhibit the binding
interaction of one or more of the peptides described herein
according to standard methods. Compounds that inhibit binding of
such molecules are useful in the therapeutic methods of the
invention. Once identified, such compounds are tested for their
ability to combat prion diseases in any appropriate model
system.
[0106] Evaluation of whether a test antagonist (e.g., a peptide
described herein) confers protection against the development of a
prion disease in vivo generally involves using an animal known to
develop such a disease (e.g., Chandler, Lancet 6:1378-1379, 1961;
Eklund et al., J. Infectious Disease 117:15-22, 1967; Field, Brit.
J. Exp. Path. 8:129-239, 1969). An appropriate animal (for example,
a mouse or hamster) is treated with the test compound according to
standard methods, and a reduced incidence or delayed onset or
progression of a prion-related illness, compared to untreated
control animals, is detected as an indication of protection. The
test compound may be administered to an animal which has previously
been injected with a prion agent or, alternatively, the test
compound may be tested for its ability to neutralize a prion agent
by pre-incubating the prion and the compound and injecting the
prion/compound mixture into the test animal. A molecule (e.g., an
antagonist as described above) that is used to treat or prevent a
prion disease is referred to as an "anti-prion therapeutic."
[0107] An anti-prion therapeutic according to the invention may be
administered with a pharmaceutically-acceptable diluent, carrier,
or excipient, in unit dosage form. For example, conventional
pharmaceutical practice may be employed to provide suitable
formulations or compositions to administer such anti-prion
therapeutics to animals suffering from or presymptomatic for a
prion disease, or at risk for developing a prion disease. Any
appropriate route of administration can be employed, for example,
parenteral, intravenous, subcutaneous, intramuscular, intracranial,
intraorbital, ophthalmic, intraventricular, intracapsular,
intraspinal, intracisternal, intraperitoneal, intranasal, aerosol,
or oral administration.
[0108] Methods well known in the art for making formulations are
found in, for example, "Remington's Pharmaceutical Sciences."
Formulations for parenteral administration can, for example,
contain excipients, sterile water, or saline, polyalkylene glycols
such as polyethylene glycol, oils of vegetable origin, or
hydrogenated napthalenes. Biocompatible, biodegradable lactide
polymer, lactide/glycolide, or polyoxyethylene-polyoxypropylene
copolymers can be used to control the release of the compounds.
Other potentially useful parenteral delivery systems for anti-prion
therapeutic compounds include ethylene-vinyl acetate copolymer
particles, osmotic pumps, implantable infusion systems, and
liposomes. Formulations for inhalation can contain excipients, for
example, lactose, or can be aqueous solutions containing, for
example, polyoxyethylene-9-lauryl ether, glycocholate and
deoxycholate, or can be oily solutions for administration in the
form of nasal drops, or as a gel.
[0109] The methods of the present invention may be used to reduce
or prevent the disorders described herein in any animal, for
example, humans, domestic pets, zoo animals (such as tigers, exotic
ruminants, and nonhuman primates), or livestock. Where a non-human
animal is treated, the anti-prion therapeutic employed is
preferably specific for that species.
[0110] In related aspects, the invention features therapeutic and
diagnostic compounds identified according to any of the
aforementioned methods.
[0111] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
Sequence CWU 1
1
11 1 9 PRT Artificial Sequence VARIANT 3, 4, 7 Xaa = Any Amino Acid
1 Tyr Tyr Xaa Xaa Tyr Tyr Xaa Tyr Tyr 1 5 2 9 PRT Artificial
Sequence Synthetic 2 Tyr Tyr Arg Arg Tyr Tyr Arg Tyr Tyr 1 5 3 9
PRT Artificial Sequence Synthetic 3 Tyr Tyr Ala Ala Tyr Tyr Ala Tyr
Tyr 1 5 4 9 PRT Artificial Sequence Synthetic 4 Tyr Ser Ala Ala Ser
Tyr Ala Ser Tyr 1 5 5 9 PRT Artificial Sequence Synthetic 5 Tyr Ala
Arg Arg Ala Tyr Arg Ala Tyr 1 5 6 9 PRT Artificial Sequence
Synthetic 6 Ala Tyr Arg Arg Tyr Ala Arg Tyr Ala 1 5 7 9 PRT
Artificial Sequence Synthetic 7 Tyr Ala Arg Tyr Ala Arg Tyr Ala Arg
1 5 8 9 PRT Artificial Sequence Synthetic 8 Tyr Arg Ala Ala Arg Tyr
Arg Ala Tyr 1 5 9 9 PRT Artificial Sequence Synthetic 9 Asn His Ser
Thr His Asn Thr Gly His 1 5 10 9 PRT Artificial Sequence Synthetic
10 Asp Arg Tyr Tyr Trp Tyr Phe Asp Val 1 5 11 11 PRT Artificial
Sequence Synthetic 11 Asp Glu Ala Tyr Tyr Lys Gly Trp Phe Ala Tyr 1
5 10
* * * * *