Patent 6,673,332

The present invention provides methods and compositions for the treatment of Alzheimer's disease. In particular, the present invention provides methods and compositions suitable to assess, characterize, and identify inhibitors of neuronal transport of Alzheimer's amyloid precursor protein.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for the treatment of Alzheimer's disease. In particular, the present invention provides methods and compositions suitable to assess inhibitors of neuronal transport of Alzheimer's amyloid precursor protein.

The present invention provides methods for identifying modulators of transport of amyloid precursor protein comprising the steps of: providing kinesin-I, amyloid precursor protein, and at least one test compound suspected of having modulating activity; combining kinesin-I, amyloid precursor protein, and the test compound(s) under conditions such that the kinesin-I and amyloid precursor protein will bind to produce a kinesin-I/amyloid precursor protein complex, in the absence of an inhibitor. In preferred embodiments, the binding of the kinesin-I and amyloid precursor protein is detected. In some embodiments, the TPR domain of the light chain of kinesin-I interacts with the amyloid precursor protein. In some preferred embodiments, the binding of the kinesin-I and amyloid precursor protein is inhibited, while in other preferred embodiments, the binding of the kinesin-I and amyloid precursor protein is enhanced. In some embodiments, the binding is detected using any method suitable for the detection of such binding. In some embodiments, the methods used include, but are not limited to co-immunoprecipitation methods, co-immunoprecipitation followed by Western blotting, sucrose gradient centrifugation, microtubule binding assays, column chromatography methods, gel overlays, ATPase assays, and surface plasmon resonance (e.g., BIACORE). In some particularly preferred embodiments, the method comprises a co-immunoprecipitation method. In alternative embodiments, the method further comprises Western blotting. In still further embodiments, the methods involve microtubule binding assays, while in other embodiments, the methods involve ATPase assays, and in additional embodiments, the methods involve sucrose gradient centrifugation. In some preferred embodiments, biochemical methods are used. In alternative preferred embodiments, embodiments, the method is conducted in vivo. In some preferred in vivo methods, the methods are conducted within cells, while in other embodiments, the methods are conducted within an animal (e.g., an animal model of disease). In some embodiments, the methods further comprise the step of exposing an animal to the complex of kinesin-I and amyloid precursor protein. In still further embodiments, the methods are conducted in vitro. The present invention further provides compounds identified using these methods. In some particularly preferred embodiments, the compound(s) identified using the methods of the present invention are provided to an animal to treat neurological illness. In some embodiments, the animal is suffering from a neurological illness. In particularly preferred embodiments, the neurological illness is Alzheimer's disease. In alternative particularly preferred embodiments, the animal is a human.

The present invention also provides methods for identifying compounds that facilitate transport of amyloid precursor protein comprising the steps of: providing an animal capable of producing amyloid precursor protein, wherein the animal has at least one mutation in at least one subunit of kinesin-I, and at least one test compound; administering the test compound(s) to the animal, and detecting the binding of the kinesin-I and amyloid precursor protein. In one embodiment, the test compound inhibits the binding of kinesin-I and amyloid precursor protein, while in an alternative embodiment, the test compound enhances the binding of kinesin-I and amyloid precursor protein. In some embodiments, the kinesin-I encoded by the mutant kinesin-I subunit is functionally normal, while in other embodiments, the kinesin-I encoded by the mutant kinesin-I subunit is mutated. In still further embodiments, the animal having the mutant kinesin-I subunit fails to produce functional kinesin-I. It is not intended that the mutation be limited to any particular mutation, nor is it intended that the mutation particularly affect any specific portion of the kinesin-I molecule. In alternative embodiments, the animal produces abnormal amyloid precursor protein. The present invention further provides compounds identified using these methods.

The present invention also provides methods for treating neurological illness by administering a compound identified using any of the methods described above to an animal suffering from a neurological illness. In some embodiments, the compound administered to the animal inhibits the binding of kinesin-I and amyloid precursor protein, while in alternative embodiments, the compound administered to the animal enhances the binding of kinesin-I and amyloid precursor protein. In particularly preferred embodiments, the compound administered to the animal inhibits or prevents the neuronal transport of amyloid precursor protein. In some preferred embodiments, the neurological illness is Alzheimer's disease. In particularly preferred embodiments, the animal is a human.

DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for the treatment of Alzheimer's disease. In particular, the present invention provides methods and compositions suitable to assess inhibitors of neuronal transport of Alzheimer's amyloid precursor protein.

As indicated above, Alzheimer's disease (AD) is a progressive, neurodegenerative disorder characterized by extracellular deposition of amyloid protein. The amyloid deposits are mainly composed of an insoluble peptide, the amyloid beta peptide (A.beta.), which is derived from proteolytic cleavage of the amyloid precursor protein (APP). Numerous studies have suggested that aberrant trafficking or processing of APP may play a causative role in AD (reviewed in Selkoe, Nature 39 (Suppl) A23-30 [1999]; Sinha and Lieberburg, Proc. Natl. Acad. Sci. USA 96:11049-11053 [1999]; and De Strooper and Annaert, J. Cell. Sci., 113:1857-1870 [2000]). Thus, methods and compositions to elucidate the normal mechanisms of axonal transport and trafficking of APP are essential to elucidating how APP participates in the development of AD and in the identification and characterization of treatment regimens to alleviate the signs and symptoms of Alzheimer's disease.

In neurons, APP is transported within axons by fast anterograde axonal transport from the neuronal cell bodies to the distal nerve terminals (Koo et al., Proc. Natl. Acad. Sci. USA 87:1561-1565 [1990]; and Sisodia et al., J. Neurosci., 13:3136-3142 [1993]). Anti-sense inhibition experiments using oligonucleotides complementary to kinesin heavy chain coding sequences in hippocampal neurons suggested that axonal transport of APP requires the microtubule-dependent motor protein kinesin-I (Amaratunga et al., J. Neurochem., 64:2374-2376 [1995]; Ferreira et al., J. Neurosci., 12:3112-3123 [1993]; Kaether et al., Mol. Biol. Cell 11:1213-1224 [2000]; and Yamazaki et al., J. Cell. Biol., 129:431-442 [1995]). However, a prominent gap is the lack of information about whether, or how APP interacts with components of the neuronal kinesin-I transport machinery.

Kinesin-I

Kinesin-I was the first member of the kinesin superfamily to be identified (Brady, Nature 317:73-75 [1985]; and Vale et al., Cell 42:39-50 [1985]), and is responsible for ATP-dependent movement of vesicular cargoes within cells (reviewed in Goldstein and Philip, Ann. Rev. Cell Dev. Biol., 15:141-183 [1999]; Goldstein and Yang, Ann. Rev. Neurosci., 23:39-72 [2000]; and Kamal and Goldstein, Curr. Opin. Cell Biol., 12:503-508 [2000]). Kinesin-I is composed of two kinesin heavy chain (KHC) and two kinesin light chain (KLC) subunits. In the mouse, there are three genes encoding KHC (KIF5A, KIF5B, and KIF5C) and three genes encoding KLC (KLC1, KLC2, and KLC3) (See e.g., Rahman et al., J. Cell. Biol., 146:1277-1288 [1998]; and Xia et al., Genomics 52:209-213 [1998]). These KHC and KLC subunits appear to associate in all possible combinations (Rahman et al., [1998], supra; Xia et al., [1998], supra). KIF5A and KIF5C are neuron specific isoforms, whereas KLC1 is neuronally enriched. KIF5B and KLC2 are ubiquitously expressed; the expression pattern of KLC3 is unknown.

Both KHC and KLC have distinct conserved domains. KHC has an N-terminal motor domain (Yang et al., Cell 56:879-889 [1989]), a central alpha-helical coiled-coil stalk domain (de Cuevas et al., J. Cell Biol., 116:957-965 [1992]; Gauger and Goldstein, J. Biol. Chem., 268:13657-13666 [1990]; and Hirokawa et al., Cell 56:867-878 [1989]), and a globular C-terminal tail domain, perhaps involved in cargo-binding (Bi et al., J. Cell. Biol., 138:999-1008 [1997]; Seiler et al., Nat. Cell Biol., 2:333-338 [2000]; and Skoufias et al., J. Biol. Chem., 269:1477-1485 [1994]) or motor regulation (Coy et al., Nat. Cell Biol., 1:288-292 [1999]; Friedman and Vale, Nat. Cell Biol., 1:293-297 [1999]; Hackney and Stock, Nature Cell Biol., 2:257-260 [2000]; and Stock et al., J. Biol. Chem., 274:14617-14623 [1999]). KLC has a conserved N-terminal coiled-coil domain that binds KHC (Diefenbach et al., Biochem., 37:16663-16670 [1998]; and Gauger and Goldstein, [1993], supra), and a C-terminal domain that consists of six imperfect repeats of a 34 amino acid tetra-trico peptide repeat (TPR) module (Gindhart and Goldstein, Trends Biochem. Sci., 21:52-53 [1996]). Although the function of the TPR domain in KLC is unknown, TPR domains are involved in protein-protein interactions in a large group of structurally and functionally diverse proteins (Lamb et al., Trends Biochem. Sci., 20:257-259 [1995]; and Blatch and Lassie, Bioessays 21:932-939 [1999]), and could thus be involved in linking KLC to receptor proteins in vesicular or organellar cargoes. Whether KLC interacts directly with membrane associated receptor proteins in vesicles, and what the identity of such motor-binding receptor proteins might be is unknown.

Previously, a candidate kinesin-I receptor protein called kinectin (Kumar et al., Science 267:1834-1837 [1995]; and Toyoshima et al., J. Cell Biol., 118:1121-1131 [1992]) was described and found to be largely absent from axons (Toyoshima and Sheetz, Neurosci. Lett., 211:171-174 [1996]). Thus, kinectin cannot support transport of APP and other proteins used at nerve termini. Some recent experiments have suggested that "cargo" molecules themselves might interact directly with microtubule-dependent motor proteins (See e.g., Tai et al., Cell 97:877-887 [1999]). However, until the development of the present invention, the association of APP with the KLC subunit of kinesin-I was unknown. The data obtained during the development of the present invention indicate that APP transport from sites of synthesis in the neuronal cell body to sites of utilization or pathogenesis at the axonal terminus, as well as within the axon, is mediated by direct binding of APP to KLC.

Kinesin-I and APP Complexes

As indicated herein, results obtained during the development of the present invention indicate that axonal transport of APP requires the formation of a complex containing kinesin-I and APP by direct binding of APP to the KLC subunit of kinesin-I. This conclusion is supported by several lines of evidence, including co-immunoprecipitation, sucrose gradients, and direct in vitro binding experiments, as described herein. In addition, association of APP with microtubules and axonal transport of APP was found to be greatly diminished in a mouse mutant of KLC1, thus providing compelling evidence for the role of KLC in the microtubule based axonal transport of APP in neurons. Thus, the results obtained during the development of the present invention provides direct molecular evidence about the mechanism of axonal transport of APP, and identifies APP as a likely membrane cargo receptor for kinesin-I. However, an understanding of the mechanism(s) is not necessary in order to use the present invention and it is not intended that the present invention be limited to any particular mechanism(s).

The Role of Kinesin-I in Axonal Transport of APP

The results described herein confirm and extend earlier suggestions that kinesin-I might drive the fast anterograde axonal transport of APP. These early suggestions were based on antisense experiments that found decreased APP transport when the expression of the KHC subunit of kinesin-I was inhibited in rabbit optic nerve or hippocampal neurons (Amaratunga et al., J. Neurochem., 64:2374-2376 [1995]; Ferreira et al., J. Neurosci., 13:3112-3123 [1993]; Kaether et al., Mol. Biol. Cell 11:1213-1224 [2000]; and Yamazaki et al., J. Cell. Biol., 129:431-442 [1995]). However, these earlier studies are difficult to interpret since simultaneous inhibition of the transport of the non-kinesin-I cargo, synaptophysin (Okada et al., Cell 81:769-780 [1995]), was also observed (Amaratunga et al., [1995], supra). Thus, work described herein that shows a direct biochemical interaction between APP and KLC, and the dramatic reduction of axonal transport of APP in a gene-targeted mouse mutant of KLC is the most direct in vivo evidence for the role of kinesin-I in the transport of APP.

Recently, yeast two-hybrid analyses identified a protein (PAT1) that binds to the C-terminal basolateral-sorting signal of APP and was suggested to play a role in the transport of APP (Zheng et al., Proc. Natl. Acad. Sci. USA 95:14745-14750 [1998]). Interestingly, PAT1 expressed in transfected cells co-sediments with microtubules, and also possesses TPR repeat domains similar to those found in KLC and other TPR-containing proteins. However, the TPR domains of PAT1 are only 26% identical to the TPR domains of KLCs, and the overall sequence of PAT1 is only 15% identical to KLC. In addition, although PAT1 is reported to associate with a 110 KDa protein, this protein is clearly not KHC (Zheng et al., [1998], supra). Thus it appears that PAT1 is not a true homolog of KLC, but instead may be a microtubule interacting protein that also binds APP. It is possible that PAT1 has a function at the nerve terminus after APP is delivered there by kinesin-I, although whether PAT1 is expressed in neurons is unknown. Indeed, an understanding of the mechanism(s) involved is not necessary in order to use the present invention.

It is intriguing that overexpression of an APP homologue (APPL) in Drosophila neurons leads to axonal blockage and neuronal dysfunction that is synergistic with overexpression of the microtubule-associated protein tau (See, Torroja et al., Curr. Biol., 9:489-492 [1999]). This axonal blockage phenotype is similar to what is observed in Drosophila mutants lacking known kinesin (See, Gindhart et al., J. Cell Biol., 141:443-452 [1998]; Hurd and Saxton, Genetics 144:1075-1085 [1996]; and Hurd et al., Genetics 142:195-204 [1996]) and dynein (Bowman et al., J. Cell. Biol., 146:165-179 [1999]; and Martin et al., Mol. Cell Biol., 10:3717-3728 [1999]) motor subunits or other membrane proteins that bind kinesin-I (Bowman et al., [2000], supra). Besides the phenotypic similarity between APPL overexpression and mutations in microtubule motor subunits, a genetic interaction between APPL and the KHC gene was also demonstrated (Torroja et al., [1999], supra), further supporting a direct functional association of APP and kinesin-I.

Potential Role of APP as a Membrane Cargo Receptor for Kinesin-I

One of the most poorly understood aspects of microtubule-dependent trafficking is the identity of the membranous cargo that each motor carries. Although an understanding of the mechanism(s) is not necessary in order to use the present invention, it is thought that motor-cargo recognition may require three players: the motor proteins, a cargo-bound receptor and accessory components. The results obtained during the development of the present invention indicate that APP may be a membrane cargo receptor for kinesin-I and might link kinesin-I to a particular subset of axonal transport vesicles. This hypothesis is consistent with the finding that APP, a kinesin-I cargo, is enriched in Rab5-positive vesicles whereas there is virtually no APP present in synaptophysin positive vesicles that are most likely cargoes for the UNC104/KIF1A kinesin (See, Ikin et al., J. Biol. Chem., 271:31783-31786 [1996]; Okada et al., Cell 81:769-780 [1995]; Otsuka et al., Neuron 6:113-122 [1991]; and Yonekawa et al., J. Cell Biol., 141:431-441 [1998]). These data indicate that different motors could interact with different membrane cargo receptors on particular subsets of axonal transport vesicles. However, an understanding of the mechanism(s) is not necessary in order to use the present invention.

As indicated above, one previously reported potential receptor for kinesin-I was kinectin, an integral membrane protein that is localized to the endoplasmic reticulum (Kumar et al., Science 267:1834-1837 [1995]; and Toyoshima et al., J. Cell Biol., 118:1121-1131 [1992]). However, proteins other than kinectin might be important for axonal transport since kinectin has been reported to be absent from axons (Toyoshima and Sheetz, Neurosci. Lett., 211:171-174 [1996]). In addition, no direct connection between kinectin and either subunit of kinesin-I has been demonstrated, and kinectin is not found in C. elegans or Drosophila (Rubin et al., Science 287:2204-2215 [2000]).

Recently, analysis of an axonal transport mutant in Drosophila led to the identification of a novel membrane associated protein, Sunday-driver (SYD), which may also be a membrane receptor for kinesin-I (Bowman et al., [2000], supra). GFP-tagged mammalian SYD localized to tubular and vesicular elements that co-stained with kinesin-I and Golgi markers, suggesting that SYD might function as a membrane associated receptor for the axonal transport of post-Golgi vesicles. Thus, it is contemplated that both APP and SYD could be membrane cargo receptors for kinesin-I in axonal transport and post-Golgi transport. However, an understanding of the mechanism(s) involved is not necessary in order to use the present invention.

The Role of KLC in the Interaction of Kinesin-I With Cargo

Results obtained during the development of the present invention indicate that KLC interacts with membrane associated proteins through one or more of its TPR repeat domains. The binding stoichiometry observed of two APP molecules per KLC fits well with the atomic structure of other TPR domains, which indicates that three TPR repeats fold together to bind one ligand (Blatch and Lassie, Bioessays 21:932-939 [1999]; and Scheufler et al., Cell 101:199-210 [2000]). The KLC construct used during the development of the present invention has six TPR repeats so the observed binding stoichiometry fits the theoretical binding saturation that is predicted from the atomic structure. It is intriguing that the observation that APP directly binds to the TPR domain of KLC and that APP binding to KLC is inhibited by the KLC-A11 antibody, which binds specifically to the TPR domain of KLC (Stenoien and Brady, [1997], supra), is similar to recent work on the SYD protein. The SYD protein directly interacts with the TPR domain of KLC by yeast two-hybrid analyses and the KLC-A11 antibody also inhibits binding of SYD to KLC in GST pulldown experiments (Bowman et al., [2000], supra). Strikingly, in an in vitro organelle motility system, the KLC-A11 antibody inhibits the binding of kinesin-I to membranes and blocks fast axonal transport, while no such effects were seen with the 63-90 antibody, which recognizes the N-terminal domain of KLC (Stenoien and Brady, [1997], supra). Together, these results demonstrate that the TPR domains of KLC directly interact with membrane-associated proteins of vesicular cargo.

Although taken in isolation, the data discussed herein are most consistent with the simple suggestion that the function of the KLC subunit of kinesin-I is to directly bind cargo receptor proteins such as APP, previous studies of the relative roles of KLC and KHC in cargo-attachment and motor regulation have yielded apparently contradictory results. For example, while antibody inhibition studies suggest that KLC is needed for interaction of kinesin-I with membranes (Stenoien and Brady, [1997], supra), another study showed that KHC alone is sufficient to bind membranes (Skoufias et al., J. Biol. Chem., 269:1477-1485 [1994]). This latter finding is consistent with recent work on a null mouse mutant of KLC1 that found KHC accumulation in the absence of KLC at the Golgi apparatus, a presumed site of cargo transport initiation (Rahman et al., [1999], supra). This apparent binding of KHC to potential cargoes in vivo, in the absence of KLC, is also consistent with work on fungal kinesin-I, which has no KLC subunit, yet appears to be capable of cargobinding (Steinberg and Schwila, Mol. Cell Biol., 6:1605-1618 [1995]). There has also been conflicting evidence about whether KLC, or the tail of KHC, or both repress kinesin-I motor activity in the absence of membrane or cargo binding (See, Coy et al., Nat. Cell Biol., 1:288-292 [1999]; Friedman and Vale, Nat. Cell Biol., 1:293-297 [1999]; Stock et al., [1999], supra; and Verhey et al., J. Cell Biol., 143:1053-1066 [1998]). The inconsistencies among these studies could be attributable to the various experimental systems used. However, based upon the results obtained during the development of the present invention, it is contemplated that both KLC and the tail of KHC combine to fully repress motor activity, that the tail of KHC binds relatively indiscriminately to membrane cargoes, and that KLC interaction with specific membrane proteins (such as APP or SYD) relieves motor repression and activates transport. Thus, it is contemplated that the role of KLC is to provide specificity for cargo binding and transport, perhaps via an activation function. However, an understanding of the mechanism(s) is not necessary in order to use the present invention.

Significance of APP Interaction With Kinesin-I in Alzheimer's Disease

There are numerous suggestions that aberrant trafficking or transport of APP may contribute to the development of AD (reviewed in Checler, J. Neurochem., 65:1431-1444 [1995]; Selkoe, Nature 39 Suppl.:A23-A30 [1998]; and Sinha and Lieberburg, Proc. Natl. Acad. Sci. USA 96:11049-11053 [1999]). The finding of a direct interaction of APP and the microtubule transport machinery described herein, leads to the intriguing indication that abnormal interactions of APP and kinesin-I play a role in the pathogenesis of AD, perhaps by blocking or otherwise interfering with normal axonal transport. However, an understanding of the mechanism(s) is not necessary in order to use the present invention.

Indeed, the methods and compositions of the present invention provide means to identify compounds that are effective in alleviating the signs and symptoms of Alzheimer's disease. In particular, the present invention provides assay systems to detect normal APP or C-terminal fragments of APP. In some embodiments, these assays are conducted in solution, while in other embodiments, these assays involve solid-phase binding. In still further embodiments, the present invention provides competition assays. In some embodiments, complete APP is used, while in other embodiments, proteolytic fragments or recombinantly produced fragments of APP are utilized.

Assay Systems for Detection of APP Stimulation of ATPase of Kinesin-I

As indicated above, the present invention further provides assay systems for the detection of APP stimulation of the ATPase of kinesin-I. In some embodiments, the assay involves direct stimulation of kinesin-I (i.e., in solution), while in other embodiments, native kinesin-I is captured from solution and the ATPase reaction is observed. In preferred embodiments, APP binding to kinesin-I is assayed based on its ability to bind ATPase or stimulate ATPase activity. In still further embodiments, the presence or absence of APP or kinesin-I in a sample is detected. In additional embodiments, in situ immunocytochemistry methods are used to detect the binding of APP and kinesin-I in tissues and/or organ samples. In particularly preferred embodiments, the antibodies and other reagents described herein are utilized in these assay systems.

In some binding assays, the ability of a test agent or compound to specifically bind to APP and/or kinesin-I alone, or in a complex is determined. In a particularly preferred embodiment, the ability of a test compound to bind to the light chain of kinesin-I is assessed. There are a wide variety of formats available for appropriate binding assays. In one embodiment, APP or kinesin-I is immobilized on a surface and exposed to the test compound, while in other embodiments, test compounds are immobilized on a surface and the specific binding of APP and/or kinesin-I is assayed. Binding is often easier to detect in systems in which the test compound, APP, and/or kinesin-I are labeled (e.g., with fluorescence, radioactivity, an enzyme, etc., as known in the art). After exposing the components to each other and washing off unbound reagents, the presence of the labeled moiety (i.e., bound to the unlabelled component of the test system).

Solution phase binding assays are also known to those in the art. For example, in one embodiment, the binding assay is a cosedimentation assay. In this assay system, when the test compound binds to APP and/or kinesin-I, the bound test compound and APP and/or kinesin-I cosediment when centrifuged. Unbound APP and/or kinesin-I and test compound either sediment at a different rate or remain in solution.

Methods for performing various binding assays are known in the art, including but not limited to the assay systems such as those described in U.S. patent application Ser. No. 60/057,895 and related PCT application US98/18368. Various references provide general descriptions of various formats for protein binding assays, including competitive binding assays and direct binding assays, (See e.g., Stites and Terr, Basic and Clinical Immunology, 7 th ed. [1991]; Maggio, Enzyme Immunoassay CRC Press, Boca Raton, Fla. (1980); and Tijssen, Practice and Theory of Enzyme Immunoassays, in Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, B. V. Amsterdam, [1985]). In some particularly preferred embodiments, high-throughput assays are contemplated for use in the present invention to identify compounds that inhibit or enhance binding of APP and kinesin-I. It is not intended that the present invention be limited to any particular high-throughput method, as it is contemplated that various methods will find use in the present invention.

Solid-Phase Assay Systems

Thus, in some embodiments, immunoassays are provided in which APP is bound to a solid support (e.g., the well of a microtiter plate, a microcard, or any other suitable format), a sample suspected of containing kinesin-I, and observing the binding of the kinesin-I to the bound APP (i.e., present in a complex of APP and kinesin-I). Thus, in these embodiments, one or more of the assay components are attached to a solid surface. In some embodiments, an ATPase assay system is then used (as known in the art) to detect the stimulation of ATPase activity due to the binding of APP to kinesin-I. In alternative embodiments, an indirect immunoassay system is used in which the complex is detected by the addition of antibodies directed against kinesin-I to the test mixture, as known in the art.

Virtually any solid surface is suitable, as long as the surface material is compatible with the assay reagents and it is possible to attach the component to the surface without unduly altering the reactivity of the assay components. Those of skill in the art recognize that some components exhibit reduced activity in solid phase assays, but this is generally acceptable, as long as the activity is sufficient to be detected and/or quantified.

Solid supports include, but are not limited to any solid surface (e.g., glass beads, planar glasses, controlled pore glasses, plastic porous plastic metals, or resins to which the molecule may be adhered, etc.). Those of skill in the art recognize that in some embodiments, the solid supports of the present invention are derivatized with functional groups (e.g., hydroxyls, amines, carboxyls, esters, and sulfhydryls) to provide reactive sites for the attachment of linkers or the direct attachment of the test compound or other assay component.

Adhesion of the assay component (e.g., APP or kinesin-I) to the solid support can be direct (i.e., the component directly contacts the solid surface) or indirect (i.e., a particular compound or compounds [e.g., APP or kinesin-I] is/are bound to the support, and the other assay component (e.g., kinesin-1 or APP) binds to this compound or compounds rather than to the solid support). In some embodiments, the compound is covalently immobilized (e.g., utilizing single reactive thiol groups of cysteine for anchoring protein components [See e.g., Colliuod et al., Bioconjug. Chem., 4:528-536 (1993)], or non-covalently, but specifically (e.g., via immobilized antibodies or other specific binding proteins [See e.g., Schuhmann, Adv. Mater., 3:388-391 (1991); Lu et al., Anal. Chem., 67:83-87 (1995)]), the biotin/streptavidin system (See e.g., Iwane et al., Biophys. Biochem. Res. Commun., 230:76-80 [1997]), or metal-chelating Langmuir-Blodgett films (See e.g., Ng et al., Langmuir 11:4048-4055 [1995]; Schmitt et al., Angew. Chem. Int. Ed. Engl., 35:317-320 [1996]; Frey et al., Proc. Natl. Acad. Sci. USA 93:4937-4941 [1996]; and Kubalek et al., J. Struct. Biol., 113:117-123 [1994]), and metal-chelating self-assembled monolayers (See e.g., Sigal et al., Anal. Chem., 68:490-497 [1996]), for binding of polyhistidine fusion proteins.

In some embodiments, standard direct or indirect ELISA, IFA, or RIA methods as known in the art are used to detect the binding of APP or kinesin-I. In some embodiments, APP is detected in a sample, while in other embodiments, kinesin-I is detected. Thus, it is clear that the methods of the present invention are adaptable to the detection, identification, and characterization of multiple elements (e.g., APP, kinesin-I, the light chain of kinesin-I, fragments of either APP or kinesin-I, etc.).

Thus, in some embodiments of the methods of the present invention, a sandwich ELISA (enzyme-linked immunosorbent assay) with a monoclonal or polyclonal antibody for capture (i.e., a capture antibody) and a secondary antibody (i.e., a reporter antibody) for detection of bound monoclonal antibody-antigen complex (i.e., APP bound to anti-APP antibody or kinesin-I bound to anti-kinesin-I antibody) is used. In some embodiments, measures are included in order to reduce background noise, as discussed below.

In some ELISA embodiments, alkaline phosphatase conjugates are used, while in other embodiments, horseradish peroxidase conjugates are used. In addition, avidin-biotin systems are contemplated, for assay systems in which increased signal is desired. Thus, in one method of the present invention, 100 .mu.l biotinylated antibody (e.g., directed against either APP or kinesin-I) appropriately diluted in blocking buffer is added to each well of avidin-precoated ELISA plates (e.g., the neutravidin plates commercially available from Pierce). After 2 hr, the plate is washed with wash buffer (e.g., TBS/Tween 20 0.1%, with or without a blocking agent). Further nonspecific binding is inhibited by adding blocking buffer (e.g., by adding 300 .mu.l SuperBlock (Pierce) twice, as per the manufacturer's recommendations). Following incubation to allow binding of the biotinylated antibody to the surfaces of the wells, the plate is washed (e.g., 3 times) as known in the art, to remove any unbound antibody present in the wells.

Samples suspected of containing either APP or kinesin-I are diluted with an appropriate buffer and added to the wells of the ELISA plate, as well as standards and controls. The diluted standards, controls, and samples, are added to the wells of the ELISA plate) (e.g., 100 hundred .mu.l/well). Standards, controls, and samples are tested in duplicate. The plate is incubated overnight or for another appropriate length of time, typically on a rocking table at 5 RPM in a humidor. The plate is washed (e.g., 3 times) with washing buffer as known in the art. Then, 100 .mu.l of appropriately diluted monoclonal or polyclonal reporter antibody (preferably preabsorbed with the biotinylated antibody used to coat the wells of the plate, e.g., using an avidin column), is added and allowed to incubate at room temperature overnight (i.e., 18-20 hours) or for another incubation period as appropriate. The plate is washed again, as described above, and 100 .mu.l alkaline phosphatase-conjugated anti-rabbit Ig (commercially available from Pierce) appropriately diluted in blocking buffer (e.g., BSA Blocker in TBS) are added, and allowed to incubate for 2 hours with rocking as described above. The plate is then washed again as described above. The enzyme substrate is added to the wells and the reaction allowed to occur for an appropriate length of time, at the end of which the reaction is stopped as known in the art, and the optical densities of the solutions within the wells determined as known in the art.

Because background signal is often the limiting factor in amplified assays, in some embodiments, measures are undertaken to reduce background signal in these assays. First, the antiserum used for detection of the APP or kinesin-I is reabsorbed by passing it over a streptavidin column to which biotinylated antibody (i.e., the primary antibody) has been coupled. This is done to remove nonspecific reactivity against mouse IgG (i.e., the reporter antibody). Second, in some embodiments, conjugated anti-rabbit-Ig is selected which is been depleted of reactivity with murine and human IgG. Third, biotin/avidin is used to fix the capture antibody to the plate; this in turn allows for more vigorous washing protocols with a detergent-containing buffer. Fourth, efficient blocking reagents (e.g., BSA Blocker and SuperBlock, Pierce) are used.

The assay system outlined above also finds use in the detection of complexes comprising kinesin-I and APP. In these embodiments, the complexes are detected by using either antibodies or ATPase test methods. Thus, in some embodiments, the presence of kinesin-I/APP complexes in a biological sample (e.g., tissues or bodily fluids) is detected. In addition, these methods are useful for determining the ability of test compounds to inhibit or prevent the ATPase reaction from occurring in the sample. Thus, the present invention provides various methods to assess the binding of APP and kinesin-I and the effects of inhibitors on this binding and/or the consequences of this binding.

In addition to standard indirect and direct immunoassay methods, competitive assays are provided by the present invention. Such competitive assays find use in the detection of compounds that inhibit the interaction (i.e., binding) of APP and kinesin-I. Thus, in these assays, a known concentration of APP or kinesin-I is used to coat the wells of an ELISA plate, and a test compound is added to the wells at about the same time as a known concentration of kinesin-I or APP is added to the wells. The same washing procedures are used as described above. Binding of APP to kinesin-I is then detected using an antibody directed against either kinesin-I or APP, whichever compound is being detected in the test sample (i.e., is different from the compound used to coat the wells of the plate). In preferred embodiments, the tests are run using dilution series of the various reagents (i.e., a titration checkerboard is used). The test compound is then analyzed for its ability to modulate the binding of APP to kinesin-I. Test compounds identified as inhibiting the binding of APP to kinesin-I are then further analyzed. Test compounds that enhance the binding of APP to kinesin-I are are also further analyzed. In some cases, test compounds that have no effect on the binding of APP to kinesin-I are also further analyzed. Thus, the screening methods of the present invention provide means to identify compounds that modulate the binding of APP to kinesin-I and are useful in the treatment and/or prevention of Alzheimer's disease.

Solution-Based Assay Systems

In addition to the assay systems in which a solid support is utilized, the present invention provides methods in which the assay components remain suspended in solution. In these embodiments, the presence of APP binding to kinesin-I is detected by an increase in ATPase activity. Such ATPase methods are also known in the art (See e.g., Huang and Hackney, J. Biol. Chem., 269:16493 [1994]; as well as Sakowicz et al., Science 280:292-295 [1998]). In some embodiments of these methods, basal ATPase activity is stimulated, while in other embodiments, microtubule-stimulated ATPase activity is involved. Indeed, it is not intended that the present invention be limited to any particular assay system or test conditions.

As with the solid phase assay systems, competitive assays are provided by the solution-based methods of the present invention. Such competitive assays find use in the detection of compounds that inhibit the interaction (i.e., binding) of APP and kinesin-I, as indicated by a reduction in ATPase stimulation. Thus, in these assays, known concentrations of APP and kinesin-I are mixed together in the presence of a test compound. Binding of APP to kinesin-I is then detected based on the stimulation of ATPase activity. In preferred embodiments, the tests are run using dilution series of the various reagents (i.e., a titration checkerboard is used). The test compound is then analyzed for its ability to modulate the binding of APP to kinesin-I. Test compounds identified as inhibiting the binding of APP to kinesin-I are then further analyzed. Test compounds that enhance the binding of APP to kinesin-I are are also further analyzed. In some cases, test compounds that have no effect on the binding of APP to kinesin-I are also further analyzed. Thus, the screening methods of the present invention provide means to identify compounds that modulate the binding of APP to kinesin-I and are useful in the treatment and/or prevention of Alzheimer's disease.

Claim 1 of 13 Claims

We claim:

1. A method for identifying modulators of transport of amyloid precursor protein comprising the steps of:

a) providing:

i) kinesin-I,

ii) amyloid precursor protein, and

iii) a test compound suspected of having modulating activity;

b) combining said kinesin-1, said amyloid precursor protein, and said test compound under conditions such that said kinesin-I and said amyloid precursor protein will bind to produce a kinesin-I/amyloid precursor protein complex; and

c) detecting modulation in the binding of said kinesin-I to said amyloid precursor protein in the presence of said test compound compared to in the absence of said test compound, thereby identifying said test compound as a modulator of transport of amyloid precursor protein.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]