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WO2002003071A2 - Modulateurs de l'activite de la proteine trk, et compositions et methodes d'utilisation - Google Patents

Modulateurs de l'activite de la proteine trk, et compositions et methodes d'utilisation Download PDF

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WO2002003071A2
WO2002003071A2 PCT/US2001/021472 US0121472W WO0203071A2 WO 2002003071 A2 WO2002003071 A2 WO 2002003071A2 US 0121472 W US0121472 W US 0121472W WO 0203071 A2 WO0203071 A2 WO 0203071A2
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ntf
small molecule
trk protein
mimetic
binding
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WO2002003071A3 (fr
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Alan Thomas Bates
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Pangene Corporation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to neurodegenerative diseases and cancer.
  • Provided herein are compositions and methods for the treatment of such diseases.
  • Neurotrophic factors are proteins that promote the survival, development, and differentiation of neurons. These molecules are found throughout the peripheral nervous system (PNS) and central nervous system (CNS) in mammals.
  • neurons synthesize and release NTFs which then bind to specific receptors on innervating or target neurons to exert their effects.
  • target tissues synthesize and release NTFs which then bind to specific receptors on innervating neurons to exert their effects.
  • NTFs include the regulation of cell survival, cell growth , and cell differentiation.
  • processes of synaptic formation and refinement appear to be modulated by NTFs, which thereby sculpt the synaptic connections that form the integrated nervous system (Barde, Trends Neurosci 11[8]:343 (1988); Cowan, et al., Science 225:1258 (1984); Hefti, et al., Neurobiot Aging 10:515 (1989); Hefti, et al., Neurobiol Aging 10 ⁇ 5]:515 (1989); Roback, et al., Comments Dev. Biol 1:311 (1992).
  • NTFs Due to their activities, NTFs have been considered for some time as possible therapeutics for such neurodegenerative disorders such as Alzheimer's disease, Huntington's disease, and Parkinson's disease (Yuen and Mobley, Annals of Neurology 40[3]: 346 (1996)).
  • NTFs there are two significant obstacles to the use of NTFs as therapeutics for the treatment of CNS disorders, namely the large size of NTFs and the poor diffusion of NTFs in nervous system tissue.
  • the blood brain barrier prevents administration of NTFs orally or intravenously, hence delivery to the central nervous system requires infusion which carries a significant risk of infection and pericannular tissue necrosis.
  • the poor diffusion of NTFs in nervous system tissue means that delivery of NTFs to discrete regions of the diseased nervous system requires multiple local infusions.
  • SMMs small molecule mimetics
  • NTF nerve growth factor
  • BDNF brain-derived growth factor
  • NT-3 neurotrophin-3
  • NT-4/5 neurotrophin4/5
  • CNTF ciliary neurotrophic factor
  • GDNF glial cell line-derived neurotrophic factor
  • bFGF basic fibroblast growth factor
  • NTFs All of the NTFs identified appear to effect their actions on neurons through receptor tyrosine kinases.
  • the binding of an NTF to its cognate receptor activates a cascade of intracellular signaling events that promotes cell survival, growth, and other activities mediated by NTFs (see Segal et al., Ann Rev.
  • TrkA neurotrophins
  • TrkB neurotrophins
  • Trk C neurotrophins
  • Distinct neuronal populations are responsive to these three neurotrophins due to the differential distribution of their cognate Trk proteins.
  • NT-3 is capable of binding to TrkA in addition to TrkB, albeit with lower affinity.
  • ail of the neurotrophins are able to bind to an additional receptor, the "low affinity neurotrophin receptor" or "p75" as it is alternatively known in the art.
  • BDNF is a 120 amino acid polypeptide that has approximately 50% homology with NGF at the primary structure level.
  • Biological responses to BDNF are mediated by the TrkB receptor, which like TrkA, is a tyrosine kinase linked second messenger system.
  • TrkB receptor like TrkA
  • TrkB transcripts encode truncated proteins that have no tyrosine kinase domains, which suggests that the binding of BDNF to TrkB receptors may or may not result in signaling events.
  • TrkB receptor which like TrkA, is a tyrosine kinase linked second messenger system.
  • TrkB transcripts encode truncated proteins that have no tyrosine kinase domains, which suggests that the binding of BDNF to TrkB receptors may or may not result in signaling events.
  • BDNF also binds to the low affinity receptor, p75. The importance of BDNF and TrkB in the developing nervous system
  • TrkB deficient mice there is a loss of spinal cord and facial motor neurons (Yuen and Mobley, Annals of Neurology 40[3]:346 (1996)).
  • NGF nuclear factor-binding protein
  • BDNF is found predominantly in the adult central CNS (Longo, et al., Neurotrophic Factors (ed Fallon & Loughlin), New York, Academic p. 209 (1993)).
  • Messenger RNA for BDNF is distributed in several regions of the CNS, with the highest levels being found in the hippocampus, cortex and cerebellum (Maisonplerre, et al., 5:501 (1990); Kunsel, et al., Proc. Natl. Acad. Sci. USA 88:961 (1991)).
  • In situ hybridization studies have shown that BDNF mRNA is found in pyramidal, hilar and dentate granular neurons of the hippocampus (Holtzman and Mobley, Western J.
  • BDNF mRNA is also found in retinal ganglion neurons, basal forebrain cholinergic neurons, basal forebrain ⁇ -aminobutyric acid (GABA)-ergic neurons and (GABA)-ergic neurons of the ventral mesencephalon.
  • GABA basal forebrain ⁇ -aminobutyric acid
  • GABA basal forebrain ⁇ -aminobutyric acid
  • GABA basal forebrain ⁇ -aminobutyric acid
  • GABA ⁇ -aminobutyric acid
  • BDNF is also found in the peripheral nervous system.
  • PNS BDNF is located in sensory neurons in the nodose ganglion and a sub population of DRG neurons.
  • neurons responsive to BDNF include motorneurons (Holtzman and Mobley, Western J.
  • BDNF mimetics make BDNF mimetics attractive as therapeutic agents for use in the treatment of neuromuscular diseases including amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease). BDNF has also been shown to act on trigeminai mesencephalic neurons (Holtzman and Mobley, Western J. Medicine 161[3]:246 (1994)).
  • NT-3 has a specific tyrosine kinase receptor (TrkC) to which it binds with high affinity.
  • the low affinity receptor for NGF and BDNF, p75 also binds NT-3.
  • NGF and BDNF bind with high affinity to TrkA and TrkB respectively, however, NT-3 will also bind, but with diminished affinity to both TrkA and TrkB. Both NT-3 and TrkC are widely expressed in the CNS and PNS (Barbacid, J. Neurobiology 25[11]:1386 (1994)).
  • TrkC tyrosine kinase receptor
  • Residues implicated in binding to the TrkC receptor include: Arg ⁇ , Glu10, Tyr11 , Asp15, Thr22, 39-48, Tyr51 , Glu54, Arg56, Lys80, Gin83, Arg103 (Ibanez, Trends in Biotechnology 13:217 (1995)).
  • Region I of BDNF differs from the same region of NGF and NT- 3 by having a two amino acid insertion as a result of formation of a turn of an ⁇ -helix (Robinson, et al., Biochemistry 34[13]:4139 (1995)).
  • region III has no sequence insertions or deletions, but does include a stretch of 3 10 helix.
  • NT-3 is structurally similar to NGF.
  • BDNF and NGF there is an rms correlation of 1.22 angstroms (Robinson, et al., Biochemistry 34[13]:4139 (1995)).
  • the 2-fold axis of NGF is for the most part retained.
  • TrkA the tyrosine kinase activity of the latent Trk A protein is believed to be activated when TrkA monomers dimerize upon binding to homodimers of NGF.
  • Tyrosine residues in the intracellular domain of TrkA are transphosphorylated upon binding of the extracellular domain to NGF, and the phosphorylated TrkA is capable of propagating intracellular signals that mediate NGF activity.
  • Phosphorylated tyrosine residues serve as recognition sequences for cell signaling proteins such as SHC, PI-3 kinase, and PLCy.
  • Five tryosines on the intracellular domain of TrkA are phosphorylated in response to NGF, namely Y490, Y785, Y670, Y674, and Y67 (Loeb, D. M., et al., (1994)).
  • SHC binds to Y490
  • PLC binds to Y785
  • PI-3 kinase binds to Y751.
  • Trk Roustopril kinase
  • oncogenes As is recognized in the art, the deregulation of such intracellular pathways is believed to contribute to oncogenesis and cancer (Hunter, Cell 100:113-127, (2000)).
  • altered forms of the Trk gene have been identified as oncogenes and appear to encode protein isoforms that constitutively activate signal transduction pathways and elicit changes in cellular physiology (e.g. see Katzav, et al., Oncogene 4: 1129-1135 (1989)).
  • Trk proteins are also frequently expressed in cancers of nervous tissue (e.g. see Chiappa, et al., Neurosurgery 45-A 148-1155 (1999); Brodeur, et al., J Neurooncol. 31 :49-55 (1997)) and other tissue types (e.g. Walch and Marchetti, Clin Exp Metastasis 17:307-314 (1999)).
  • the level of expression and the particular type of Trk expressed in tumor cells correlates with the prognosis in at least some neural tumor types (e.g. Tanaka, et al., Cancer 83:1626-1633 (1998); Kramer et al, Eur. J. Cancer 33:2098-2100 (1997)).
  • agents that modulate Trk activity may find use in the treatment of cancer as well as in the treatment of neurodegenerative diseases.
  • agents that are capable of modulating the signal transduction elicited by the intracellular domain of Trk proteins are able to inhibit the dysregulated growth induced by Trk protein in cancer cells (e.g. Weeraratna, et al., Prostate 45:140-148 (2000); Ruggeri, et al., Curr. Med. Chem. 6:845-857 (1999)).
  • Alzheimer's disease is a dementia that involves the selective loss of neurons that project to the hippocampus (Kosik, Science 256:780 (1992); Hefti, Neurobiol. 25:11 (1994)).
  • cholinergic neurons of the basal forebrain, and noradrenergic neurons of the locus coeruleus and entorhinal cortex are compromised in Alzheimer's disease (Hefti, Neurobiol. 25:11 (1994)).
  • Significant neuronal degeneration is observed in the basal forebrain cholinergic population as Alzheimer's disease progresses, coincident with the appearance of senile plaques and the onset of cognitive dysfunction.
  • NGF mimetic may be used as a therapeutic agent to treat Alzheimer's patients and prevent neuronal death (Hefti, Neurobiol. 25:11 (1994); Tuszynski and Gage, Alzheimer Disease (ed. Katzman & Bick) Raven Press, New York p. 405 (1994)).
  • NGF mimetics may not be able to promote the survival of noradrenergic and seratonergic neurons that do not express TrkA and are affected in Alzheimer's disease, data suggests that the survival of cholinergic neurons may be sufficient to ameliorate or minimize dementia (Fischer, et al, Nature 329:65 (1987)).
  • NTF mimetics may be employed to promote the survival of non-cholinergic neuronal populations affected by Alzheimer's disease.
  • ascending noradrenergic neurons of the locus coeruleus are responsive to NT-3 and NT-4/5 (Friedman, et al., (1993); Tuszynski and Gage, Alzheimer Disease (ed. Katzman & Bick) Raven Press, New York p. 405 (1994)), while noradrenergic neurons of the entorhinal cortex are protected by the action of bFGF (Cummings, et al., (1992);
  • Parkinson's disease is characterized by a loss of dopaminergic neurons in the substantia nigra (Feany and Bender, Nature 404[6776]:394 (2000); Kubis, et al., Brain 123[2]:366 (2000)). Loss of these dopaminergic neurons produces the bradykinesia and dementia exhibited by Parkinson's patients.
  • NTFs including BDNF, NT-4/5, GDNF and bFGF have been shown to promote the survival of midbrain dopaminergic neurons in vitro (Kunsel et al., J. Neurosci. 110:558 (1990); Alexi, et al., Neuroscience 55:903 (1993); Beck, et al., Neuroscience 52:855 (1993); Lin, et al., Science
  • GDNF is capable of protecting these neurons from degeneration (Mandel, et al., Exp. Neurol. 160[1]:205 (1999); Espejo, et al., Cell Transplant 9[1]:45 (2000); Meyer, et al., Exp. Neurol. 164[1]:82 (2000); Nakao, et al., J. Neurosurg.
  • NTFs such as bFGF and NGF may also be used to promote the growth of dopamine producing cells in fetal cell transplantation therapies for Parkinson's disease (Olson, et al., Arch. Neurol. 48[4]:373 (1991); Matsuda, et al., J. Pharmacol. 59:365 (1992); Hefti, J. Neurobiol. 25:11 (1994)).
  • an NTF mimetic To be effective as a therapeutic agent, an NTF mimetic must be capable of binding to an appropriate receptor and eliciting intracellular signals to promote neuronal survival and other desirable NTF effects.
  • One approach to the discovery of such NTF mimetics involves the development of assays for screening large numbers of candidate NTF mimetics to identify those that are capable of binding to an NTF receptor.
  • An alternative approach is the rational design of therapeutics based on molecular modeling of the structures of NTF receptor and NTF. Rational drug design has been particularly successful in the development of inhibitors of viral proteases useful in the treatment of HIV infection (Wlodawer and Erickson, Annu. Rev. Biochem.
  • NGF The crystal structure of NGF has been determined (McDonald et al., Nature 354:411 (1991)). Two monomers of NGF assemble to form a dimer that is symmetrical about two planes. The NGF monomers interface at highly hydrophobic regions, promoting a high association constant. It is presumed by analogy to epidermal growth factor and immunogiobulin structures (Campbell, et al., J. Cell Sci 13[suppl]:5 (1990); Chothia, et al., Nature 342:877 (1989)) that the surface loop regions of ' NGF are important for receptor binding. Variable loop regions, which differ between neurotrophins, may play a role in the recognition of specific Trk proteins.
  • the BDNF/NT-3 heterodimer has biological activity with cells that are responsive to BDNF and NT-3. This activity is diminished with respect to that measured for the BDNF and NT-3 homodimers (Robinson, et al., ⁇ /oc ⁇ em sfry 34[13]:4139 (1995)).
  • NGF receptor specificity and activation
  • the first is a motif comprising residues Val-48, Pro-49 and Gln96, situated in a ⁇ -loop.
  • the second motif comprises residues Pro-5 and Phe-7 situated in the proximal part of the N-terminus.
  • the sequences of TrkA with which these motifs interact has not been determined.
  • Neurotrophin residues implicated in Trk binding do not form a spatially distinct cluster on each neurotrophin protomer.
  • variable regions I and V of one protomer are in close proximity to variable region II of the other protomer. Both protomers may contribute to each receptor binding site (Ibanez, et al., Cell 69:329 (1992); Robinson, et al., S/oc ⁇ e/w/sf/y 34[13]:4139 (1995)).
  • the heterodimer presents two non-identical receptor binding sites consisting of different surface residues than those forming the binding sites in the homodimer (Robinson, et al., Biochemistry 34[13]:4139 (1995)).
  • BDNF/NT-3 heterodimer displays a ten-fold lower activity than an equimolar ratio of BDNF and NT-3 homodimers.
  • the heterodimer displays partial Trk binding characteristics of both protomers (Robinson , et al., Biochemistry 34[13]:4139 (1995).
  • Chorionic gonadotrophin transforming growth factor and platelet- derived growth factor AB
  • cysteine knot superfamily members of the cysteine knot superfamily that form heterodimers that have been shown to play major functional roles (Wu, et al., Structure 2:545 (1994), Lapthorn, et al., Nature 369:455 (1994); Robinson, et al., Biochemistry 34[13]:4139 (1995)).
  • These examples of functioning heterodimers suggest that both hetero and homodimers may play complimentary roles in vivo. Formation of the less active heterodimers may regulate the output of neurotrophins. Both heterodimers and homodimers may indeed act upon certain subsets of neurons, eliciting different and distinct actions in the process.
  • TrkA receptor The structural domains of the TrkA receptor include a signal peptide (SP), two cysteine clusters (CC1 and CC2), leucine-rich motifs (LRMs), two immunoglobulin-like regions (Ig), transmembrane region (TM) and catalytic tyrosine kinase domain (TK).
  • SP signal peptide
  • LRMs leucine-rich motifs
  • Ig immunoglobulin-like regions
  • TM transmembrane region
  • TK catalytic tyrosine kinase domain
  • An isoform of TrkA that is expressed in human neurons contains a six amino acid juxtamembrane sequence that is largely absent in TrkA isoforms expressed outside the nervous system.
  • the leucine rich domain has three leucine rich repeats (LRRs) that are short sequence motifs typically consisting of 24 residues.
  • LRRs leucine rich repeats
  • a number of proteins with diverse functions and cellular locations have been shown to contain LRR's. All LRR-containing proteins appear to be involved in protein-protein interactions and/or signal transduction pathways (Kobe and Deisenhofer, TIBS 19:415 (1994)).
  • Trks A, B and C contain LRR's hence may adopt the conformation seen in the N-terminus of RI.
  • many proteins that contain the LRR motif also have homologous regions flanking the LRR domain. These homologous regions are characterized by four similarly spaced cysteines in a stretch of about 20 amino acids in the amino-terminal flanking region and in about 50 amino acids at the carboxyl-terminal flanking region (Rothberg, et al., Genes Dev. 4:2169 (1990), Fischer, et al., J. Biol. Chem. 266:14371 (1991); Kobe and Deisenhofer, TIBS 19:415 (1994)).
  • cysteine rich flanking regions consist of 32 residues N-terminal to the LRR and 52 C-terminal residues.
  • Trk protein extracellular domains also possess two repeats of the immunoglobulin-like C2-type domain. These repeats are similar to repeats found in the neural cell adhesion molecules (NCAMs) and in the platelet-derived growth factor receptor (PDGFR) family ( Schneider and Schweiger, 1991). Highly conserved residues in the Ig-like domain (e.g. 343-365 in TrkA) suggest a functional role for these sequences. A single invariant cysteine residue is found in the second Ig-like domain; mutation to a serine or deletion of this cysteine results in oncogenic activation of the TrkA receptor. It has been suggested that the two Ig-like domains may interact with other membrane bound proteins including the low affinity NGF receptor, p75 (Schneider and Schweiger, 1991).
  • TrkA and TrkB receptors Some studies of the TrkA and TrkB receptors suggest that the leucine-rich regions of the receptors are responsible for neurotrophin binding and that the Ig-like domains do not participate in neurotrophin binding (for example, see Windisch, et al., Biochemistry 270[47]:28133 (1995a); Ninkina, et al., J.
  • TrkA and TrkB A significant number of conclusions relating to the neurotrophin binding regions of the Trk receptors have been derived from recombinant proteins; namely from MBP-fusion proteins of TrkA and TrkB
  • the present application discloses the results of an investigation of Trk-neurotrophin binding which makes use of the highly sensitive lAsys biosensor system (Affinity Sensors).
  • This instrumentation allows for the measurement of the interaction of two or more biomolecules without the need for labels.
  • Previous studies of the binding of neurotrophins to Trk receptors have been made using whole cells expressing Trk receptors on their surfaces and as isolated proteins.
  • the binding of radiolabled neurotrophins was used to establish equilibrium binding constants of the neurotrophins to their receptor. While the equilibrium constant is a reasonable measure of the biological activity of the receptors, their ligand and of the avidity of the interaction, this constant fails to provide detailed information of the interaction at the molecular level.
  • Both the rate of interaction of the ligand binding to (as measured by the binding rate constant, k on ) and dissociating from (as measured by the dissociation rate constant, k off ) the receptor is only readily measurable using biosensors.
  • the molecular interactions between neurotrophins and Trks are determined.
  • the determination of these interactions provides a basis for designing and/or screening for small molecule NTF mimetics capable of binding to Trk proteins, modulating the binding of neurotrophins to Trk proteins, and modulating Trk protein activity.
  • NTF mimetics are currently lacking in the field, and are desirable for use in the treatment of neurodegenerative disorders and cancer.
  • Figure 1 shows a naturally occurring cyclic peptide from the tropical snail shell Conus Pennaceus.
  • the peptide has the amino acid sequence GCCSLPPCALSNPDYC, referred to herein as SEQ ID NO:1.
  • Figure 2 shows the immobilization chemistry used in covalently linking Trk proteins to the biosensor cuvette surface.
  • Figure 3 shows a model of neurotrophin binding to Trk receptors.
  • Figure 4 shows a sequence alignment for rat and human TrkA, TrkB and TrkC proteins. Sequence alignment was accomplished using CLUSTALW (Thompson et al., 1994).
  • HA is human TrkA.
  • RA is rat TrkA.
  • HB is human TrkB.
  • RB is rat TrkB.
  • HC is human TrkC.
  • RC is rat TrkC.
  • Figure 5 shows murine NGF amino acid sequence.
  • Figure 6 shows human BDNF amino acid sequence.
  • Figure 7 shows human NT-3 amino acid sequence.
  • Figure 8 shows rat TrkA amino acid sequence and domains therein.
  • Figure 9 shows human TrkA amino acid sequence, and domains therein.
  • Figure 10 shows rat TrkB amino acid sequence, and domains therein.
  • Figure 11 shows human TrkB amino acid sequence, and domains therein.
  • Figure 12 shows rat TrkC amino acid sequence, and domains therein.
  • Figure 13 shows human TrkC amino acid sequence, and domains therein.
  • Figure 14 shows portions of TrkA, TrkB and TrkC fused to maltose binding protein (MBP) to form fusion proteins.
  • MBP maltose binding protein
  • Figure 15 shows kinetic analysis data. Both association and dissociation data was analyzed with Fastfit software (Affinity Sensors) and Sigmaplot software (SPSS). All data was fitted to monophasic and biphasic kinetics algorithims. The goodness of fit for the data to both mono and biphasic kinetics was assessed by the quality of the fit of the association and association curves to the algorithims and by the F statistic. Quality of the data fit was also determined by the ⁇ 2 value. After accumulation of association and dissociation rates for each neurotrophin concentration, data was plotted to give the linear response in arc sees versus the concentration of neurotrophin. A binding curve was also plotted from the same data. An example of data analysis at one NGF concentration for TrkA MBP-C1 LRR at 15°C is shown below. From an accumulation of such data, the plots of response (in arc sees) versus neurotrophin concentration are produced.
  • Figure 16 shows examples of kinetic binding data.
  • Figure 17 shows a typical set of association and dissociation data generated from the biosensor study of neurotrophin binding to an immobilized Trk protein.
  • Figure 18 shows a table summarizing the interaction of TrkA proteins with NGF.
  • Figure 19 shows a table summarizing the interaction of TrkB proteins with BDNF.
  • Figure 20 shows a table summarizing the interactions of TrkC proteins with NT-3.
  • Figure 21 shows tables summarizing the interactions of NGF with various TrkA domains.
  • Figure 22 shows tables summarizing the interactions of BDNF with various TrkB domains.
  • Figure 23 shows tables summarizing the interactions of NT-3 with various TrkC domains.
  • Figure 24A The plot above shows the lAsys biosensor response to NGF and to the incubate of NGF with the extracellular domain of TrkA.
  • the top curve (blue) represents the baseline instrument response in NGF binding to immobilized TrkA extracellular domain.
  • the lower (red) curve represents the instrument response to the incubate of NGF (of the same concentration used for the baseline response) with the extracellular TrkA domain. All following figures follow this same designation of baseline response and instrument response to incubates of NGF with different TrkA domains.
  • Figure 24B The plot above shows the response from the lAsys biosensor to NGF and to the incubate of NGF with the immunoglobulin-like domain of TrkA.
  • Figure 24C The plot above shows the response from the lAsys biosensor to NGF and to the incubate of NGF with the leucine-rich region of TrkA.
  • Figure 24D The plot above shows the response from the lAsys biosensor to NGF and to the incubate of NGF with the second leucine-rich region of TrkA.
  • Figure 24E The plot above shows the response from the lAsys biosensor to NGF and to the incubate of NGF with an antibody to the C-terminal region of NGF (a gift of Uri Saragovi).
  • Figure 24F The plot above shows the response from the lAsys biosensor to NGF and to the incubate of NGF with an antibody to the N-terminal region of NGF (Santa Cruz antibodies).
  • Figure 24G The plot above shows the response from the lAsys biosensor to NGF and to the incubate of NGF with BSA as a binding control.
  • the slightly lower maximum response of the instrument to the incubate is probably a result of non-specific absorption of NGF to BSA and/or reduction of the sensitivity of the instrument due coating of the biosensor surface with excess BSA.
  • Figure 24H As a control, the antibody to the immunoglobulin-like domain of TrkA was immobilized at a high level ( ⁇ 2000 arc sees) to the surface of a CMD cuvette.
  • the plot above shows the response of the biosensor to the addition of NGF at the highest concentration of NGF used in the kinetics experiments. It is clear that significant binding of NGF to the antibody does occur.
  • Figure 24I The plot above shows the binding of NGF to the extracellular domain of TrkA after the immunoglobulin-like domain had been reacted with the antibody to this domain.
  • Figure 24J The plot above shows the binding curve of NGF to the extracellular domain of TrkA after the immunoglobulin-like domain had been reacted with the antibody to this domain.
  • Figure 24K The plot above shows the binding of NGF to the extracellular domain of TrkA after the immunoglobulin-like domain had been reacted with the antibody to this domain.
  • Figure 24L The plot above shows the binding curve of NGF to the extracellular domain of TrkA after the immunoglobulin-like domain had been reacted with the antibody to this domain.
  • the examples provided herein demonstrate that the mammalian neurotrophins NGF, BDNF and NT-3 as well as their receptors, namely TrkA, TrkB and TrkC, respectively, have bivalent binding properties.
  • the binding of a neurotrophin to a Trk protein involves two distinct and separate molecular interactions, and bound neurotrophins may be in equilibrium in their binding to these two sites.
  • neurotrophins are capable of binding an LRR domain and an Ig-like domain of a Trk protein.
  • NTF mimetics (sometimes referred to herein as NTF mimetics) capable of binding to a Trk protein.
  • methods for screening for small molecule NTF mimetics capable of binding to a Trk protein comprise combining a candidate small molecule NTF mimetic and a fragment of a Trk protein comprising an LRR domain, an Ig-like domain, or both and determining the binding of candidate small molecule NTF mimetic to Trk protein fragment.
  • an LRR domain comprises the second LRR domain of a Trk protein, situated to the C-terminal side of the first LRR domain of the Trk protein.
  • an Ig-like domain comprises the C- terminus Ig-domain of the extracellular domain of a Trk protein.
  • methods for screening for small molecule NTF mimetics capable of binding to a Trk protein comprise combining a candidate small molecule NTF mimetic and a fragment of a Trk protein consisting essentially of an LRR domain, an Ig-like domain, or both and determining the binding of candidate small molecule NTF mimetic to Trk protein fragment.
  • an LRR domain comprises the second LRR domain of a Trk protein, situated on the C-terminal side of the first LRR domain of the Trk protein.
  • an Ig-like domain comprises the C-terminus Ig-domain of the extracellular domain of a Trk protein.
  • methods for screening for small molecule NTF mimetics capable of binding to a Trk protein comprise attaching a Trk protein fragment to an lAsys cuvette to form a Trk protein fragment lAsys cuvette.
  • a candidate small molecule NTF mimetic is combined with the Trk protein fragment cuvette in an lAsys biosensor, and the binding of candidate small molecule NTF mimetic to Trk protein fragment is determined by determining an interaction with the Trk protein fragment cuvette using the lAsys biosensor.
  • a mammalian Trk protein fragment is used to screen for a small molecule NTF mimetic capable of binding to a Trk protein.
  • a human Trk protein fragment is used to screen for a small molecule NTF mimetic capable of binding to a Trk protein.
  • Most preferred is the use of a fragment of human TrkA, TrkB, or TrkC protein to screen for a small molecule NTF mimetic capable of binding to a Trk protein.
  • methods for screening for a small molecule NTF mimetic capable of modulating the binding of a neurotrophin to a Trk protein comprise combining a candidate small molecule NTF mimetic, a neurotrophin, and a fragment of a Trk protein comprising an LRR domain, an Ig-like domain, or both, and determining the binding of neurotrophin to Trk protein fragment in the presence and absence of candidate agent.
  • an LRR domain comprises the second
  • an Ig-like domain comprises the C-terminus Ig-domain of the extracellular domain of a Trk protein.
  • methods for screening for a small molecule NTF mimetic capable of modulating the binding of a neurotrophin to a Trk protein comprise combining a candidate small molecule NTF mimetic, a neurotrophin, and a fragment of a Trk protein consisting essentially of an LRR domain, an Ig-like domain, or both, and determining the binding of neurotrophin to Trk protein fragment in the presence and absence of candidate agent.
  • an LRR domain comprises the second LRR domain of a Trk protein, situated to the C-terminal side of the first LRR domain of the
  • an Ig-like domain comprises the C-terminus Ig-domain of the extracellular domain of a Trk protein.
  • methods for screening for small molecule NTF mimetics capable of modulating neurotrophin binding to Trk protein comprise attaching a Trk protein fragment to an lAsys cuvette to form a Trk protein fragment lAsys cuvette.
  • a candidate small molecule NTF mimetic is combined with the Trk protein fragment cuvette and a neurotrophin in an lAsys biosensor, and the binding of neurotrophin to Trk protein fragment is determined by determining an interaction with the Trk protein fragment cuvette using the lAsys biosensor.
  • a mammalian Trk protein fragment is used to screen for a small molecule NTF mimetic capable of modulating the binding of a mammalian neurotrophin to a Trk protein.
  • a human Trk protein fragment is used to screen for a small molecule NTF mimetic capable of modulating the binding of a human neurotrophin to a Trk protein.
  • Most preferred is the use of a fragment of human TrkA, TrkB, or TrkC protein to screen for a small molecule
  • NTF mimetic capable of modulating the binding of human NGF to TrkA, human BDNF to TrkB, human NT-3 to TrkC, or human NT-4/5 to TrkC.
  • methods for screening for a small molecule NTF mimetic capable of modulating Trk protein activity comprise screening for a small molecule NTF mimetic capable of binding to a Trk protein using the methods described herein, and subsequently using the small molecule NTF mimetic in a Trk protein activation assay.
  • methods for screening for a small molecule NTF mimetic capable of modulating Trk protein activity comprise screening for a small molecule NTF mimetic capable of modulating the binding of neurotrophin to Trk protein using the methods described herein, and subsequently using the small molecule NTF mimetic in a Trk protein activation assay.
  • a small molecule NTF mimetics capable of binding to Trk proteins.
  • a small molecule NTF mimetic provided herein is capable of binding to an LRR domain, an Ig-like domain, or both such domains of a Trk protein.
  • an LRR domain comprises the second LRR domain of a Trk protein, situated to the C-terminal side of the first LRR domain of the Trk protein.
  • a small molecule NTF mimetic provided herein is capable of binding to a mammalian Trk protein.
  • a small molecule NTF mimetic provided herein is capable of binding to a human Trk protein.
  • a small molecule NTF mimetic provided herein is capable of binding to a human TrkA, TrkB, or TrkC protein.
  • small molecule NTF mimetics capable of modulating the binding of a neurotrophin to a Trk protein.
  • a small molecule NTF mimetic provided herein is capable of modulating the binding of a mammalian neurotrophin to a mammalian Trk protein.
  • a small molecule NTF mimetic provided herein is capable of modulating the binding of a human neurotrophin to a human Trk protein.
  • a small molecule NTF mimetic provided herein is capable of modulating the binding of NGF to TrkA, BDNF to TrkB, NT-3 to TrkC, or NT-4/5 to TrkC, wherein NGF, BDNF, NT-3, NT-4/5, TrkA, TrkB and TrkC are human proteins.
  • an NTF mimetic provided herein is capable of functioning as an agonist of Trk protein activity.
  • an NTF mimetic provided herein is capable of functioning as an antagonist of Trk protein activity.
  • a small molecule NTF mimetic provided herein is capable of modulating the activity of a mammalian Trk protein.
  • a small molecule NTF mimetic provided herein is capable of modulating the activity of a human Trk protein.
  • a small molecule NTF mimetic provided herein is capable of modulating the activity of a human TrkA, TrkB, or TrkC protein.
  • medicaments which may be used for the treatment of neurodegenerative diseases and which comprise at least one small molecule NTF mimetic identified by the methods provided herein.
  • a small molecule NTF mimetic is capable of binding to an LRR domain, an Ig-like domain, or both such domains of a Trk protein.
  • such medicaments may be used for the treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis.
  • such medicaments comprise agonists of Trk protein activity.
  • medicaments which may be used for the treatment of cancer and which comprise at least one small molecule NTF mimetic identified by the methods provided herein.
  • a small molecule NTF mimetic is capable of binding to an LRR domain, an Ig-like domain, or both such domains of a Trk protein.
  • such medicaments comprise agonists of Trk protein activity.
  • the present invention also provides cyclic peptides capable of binding to an LRR domain, an Ig-like domain, or both such domains of a Trk protein, which cyclic peptides comprise an amino acid sequence having at least about 90%, more preferably at least about 95%, more preferably at least about 98% identity to the amino acid sequence set forth in SEQ ID NO:1.
  • such a cyclic peptide comprises the amino acid sequence set forth in SEQ ID NO:1.
  • an LRR domain comprises the second LRR domain of a Trk protein, situated to the C- terminal side of the first LRR domain of the Trk protein.
  • a cyclic peptide provided herein is capable of binding to a mammalian Trk protein. In a further preferred embodiment, a cyclic peptide provided herein is capable of binding to a human Trk protein. Most preferably, a cyclic peptide provided herein is capable of binding to a human TrkA, TrkB or TrkC protein.
  • cyclic peptides capable of modulating the binding of a neurotrophin to a Trk protein, which cyclic peptides comprise an amino acid sequence having at least about 90%, more preferably at least about 95%, more preferably at least about 98% identity to the amino acid sequence set forth in SEQ ID NO:1.
  • such a cyclic peptide comprises the amino acid sequence set forth in SEQ ID NO:1.
  • a cyclic peptide provided herein is capable of modulating the binding of a mammalian neurotrophin to a mammalian Trk protein. In a further preferred embodiment, a cyclic peptide provided herein is capable of modulating the binding of a human neurotrophin to a human Trk protein. In a further preferred embodiment, a cyclic peptide provided herein is capable of modulating the binding of NGF to TrkA, BDNF to TrkB, NT-3 to TrkC, or NT-4/5 to TrkC, wherein NGF, BDNF, NT-3, NT-4/5, TrkA, TrkB and TrkC are human proteins.
  • cyclic peptides capable of modulating the activity of a Trk protein, which cyclic peptides comprise an amino acid sequence having at least about 90%, more preferably at least about 95%, more preferably at least about 98% identity to the amino acid sequence set forth in SEQ ID NO:1.
  • such a cyclic peptide comprises the amino acid sequence set forth in SEQ ID NO: 1.
  • a cyclic peptide provided herein is capable of modulating the activity of a mammalian Trk protein. In a further preferred embodiment, a cyclic peptide provided herein is capable of modulating the activity of a human Trk protein. Most preferably, a cyclic peptide provided herein is capable of modulating the activity of a human TrkA, TrkB, or TrkC protein.
  • medicaments for the treatment of neurodegenerative diseases comprising at least one cyclic peptide provided herein.
  • a medicament may be used for the treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis.
  • a medicament comprises an agonist of Trk protein activity.
  • medicaments for the treatment of cancer which comprise a cyclic peptide provided herein.
  • a medicament comprises an antagonist of Trk protein activity.
  • medicaments for the treatment of neurodegenerative diseases such as Parkinson's disease, Huntington's disease, and Alzheimer's disease, which comprise a Trk protein agonist or antagonist identified by the methods provided herein.
  • medicaments for the treatment of cancer which comprise a Trk protein agonist or antagonist provided herein or identified by the methods provided herein.
  • Also provided herein are methods for the treatment of neurodegenerative diseases which comprise administering to a patient an effective dose of a medicament provided herein.
  • a method may be used to treat neurodegenerative diseases such as Parkinson's disease, Huntington's disease, Alzheimer's disease and amyotrophic lateral sclerosis.
  • Trk proteins are known. Homologs of Trk proteins have been identified in chick, mouse, rat, dog, bird, zebrafish, Xenopus, and human. Trk proteins comprise an intracellular tyrosine kinase domain, the activity of which is activated upon binding of the extracellular domain to ligand. Mammalian Trk proteins and fragments thereof are preferred for use in the present methods. Particularly preferred for use in the present methods are human Trk proteins and fragments thereof.
  • Trk proteins are TrkA, TrkB, TrkC.
  • the amino acid sequence of a human TrkA protein is disclosed at Genbank Accession No. BAA34355.
  • the amino acid sequence of a human TrkB protein is disclosed at Genbank Accession No. A56853.
  • the amino acid sequence of a human TrkC protein is disclosed at Genbank Accession No. CAA12029.
  • the amino acid sequences of human and rat TrkA, TrkB and TrkC are disclosed in the figures provided herein.
  • methods for screening for small molecule NTF mimetics capable of binding to a Trk protein comprise combining a candidate small molecule NTF mimetic and a fragment of a Trk protein comprising an LRR domain, an Ig-like domain, or both and determining the binding of candidate small molecule NTF mimetic to Trk protein fragment.
  • LRR domains of Trk protein are known. TrkA, TrkB, and TrkC contain multiple LRR domains. Particularly preferred for use in the present invention is the second LRR domain of a Trk receptor, which is situated to the C-terminal side of the first LRR domain of the Trk protein.
  • the amino acid sequence of the second LRR domain of the human TrkA protein is shown in Figure 9.
  • the sequence of the second LRR of human TrkA consists of the amino acid sequence from residues 96- 117 as shown.
  • the amino acid sequences of the second LRR domain of human TrkB and TrkC are also provided in the figures. Also provided in the figures are the amino acid sequences of the second LRR domain of rat TrkA, TrkB and TrkC.
  • the second LRR domains of rat and human Trk proteins are also evident from the alignment of rat and human Trk protein amino acid sequences in Figure 4.
  • Ig-like domains of Trk protein are known. Particularly preferred for use in the present invention are the C-terminus Ig-like domain of Trk proteins. Particularly preferred are the C- terminus Ig-like domains of human Trk proteins.
  • the amino acid sequence of the C-terminus lg-like domain, situated to the C-terminus side of the N-terminus Ig-like domain, of human TrkA protein is given in Figure 9.
  • the sequence of the C-terminus Ig-like domain of human TrkA consists of the amino acid sequence from residues 299-365 as shown.
  • the amino acid sequences of the C-terminus Ig-like domain of human TrkB and TrkC are also provided in the figures.
  • Trk protein variants find use in the present invention if they comprise an intact LRR domain, an intact Ig-like domain, or both.
  • Trk protein comprising an LRR domain, preferably the second LRR domain, and an Ig-like domain, preferably the C-terminus Ig-like domain
  • a full length Trk protein or any fragment thereof comprising an LRR domain and/or an Ig-like domain may be used.
  • Such fragments include but are not limited to Trk protein fragments comprising the extracelluar domain and Trk protein fragments consisting essentially of the extracelluar domain.
  • Preferred Trk protein fragments include protein fragments comprising both the second LRR domain and the C-terminal Ig-like domain of Trk proteins. These fragments may include other domains, such as the first LRR domain.
  • a preferred fragments for use in the methods disclosed herein is a fragments comprising amino acids 72-365, which comprises the first LRR domain, the second LRR domain, and the N- and C-terminal Ig-like domains.
  • Trk fusion proteins comprising full length Trk proteins or fragments thereof may also be used, and are sometimes referred to herein as Trk fusion proteins.
  • amino acid sequence comprising a fusion partner is linked to Trk protein amino acid sequence.
  • linkage may be done recombinantly, using nucleic acid sequences encoding the desired amino acid sequences and methods known in the art.
  • the Trk protein portion of the fusion protein is the portion to which binding of a candidate small molecule NTF mimetic is sought.
  • the Trk protein portion of the fusion protein therefore preferably comprises an LRR domain, an Ig-like domain, or both.
  • the fusion partner portion of the fusion protein serves to facilitate the method, but does not bind to small molecule NTF mimetic.
  • Functions served by the fusion partner may include protein targeting, provision of detectable label, and provision of epitope for manipulation of the fusion protein including immobilization and recovery from an extract.
  • Fusion partners may comprise any portion of a Trk fusion protein, but preferably do not disrupt the contiguous amino acid sequence that constitutes an LRR domain or an Ig-like domain.
  • Trk fusion proteins are able to bind to neurotrophins via their LRR domain, their Ig-like domain, or both.
  • Trk fusion proteins comprising a C-terminus or N- terminus poly-histidine tag, preferably a 6-histidine tag.
  • Preferred fusion proteins also include MBP- fusion proteins (see Windisch et al, 1995a, 1995b, 1995c).
  • tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field, et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7,
  • Tag polypeptides include the Flag-peptide [Hopp, et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin, et al., Science, 255:192-194 (1992)]; tubulin epitope peptide [Skinner, et al., J. Biol.
  • Trk proteins, Trk protein fragments, and Trk fusion proteins may be recombinant.
  • a "recombinant protein” is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid encoding a Trk protein, a Trk protein fragment, or a Trk fusion protein.
  • a recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics. For example, the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure.
  • an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight of the total protein in a given sample.
  • a substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred.
  • the definition includes the production of Trk proteins, Trk protein fragments, and Trk fusion proteins from one organism in a different organism or host cell.
  • the protein may be made at a significantly higher concentration than is normally seen, through the use of a inducible promoter or high expression promoter, such that the protein is made at increased concentration levels. ⁇
  • candidate small molecule NTF mimetic or “candidate bioactive agent” or “candidate agents” or “agents” or grammatical equivalents thereof as used herein describes any molecule, e.g., protein, small organic molecule, carbohydrates (including polysaccharides), polynucleotide, lipids, etc.
  • assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations.
  • one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
  • positive controls i.e. the use of agents known to modulate neurotrophin binding to Trk protein and/or known to modulate Trk protein activity may be used.
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than about 100 daltons and less than about 2,500 daltons, more preferably less than about 2000 daltons, more preferably less than about 1750 daltons, more preferably less than about 1500 daltons, more preferably less than about 1250 daltons, more preferably less than about 1000 daltons.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • Candidate agents also often comprise functional groups for electrostatic interactions with proteins, and often include chemical groups that are charged in physiological media.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.
  • the candidate bioactive agents are proteins.
  • protein herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
  • the protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptiddmimetic structures.
  • amino acid or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention.
  • Amino acid also includes imino acid residues such as praline and hydroxyproline.
  • the side chains may be in either the (R) or the (S) configuration.
  • the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations. Chemical blocking groups or other chemical substituents may also be added.
  • the candidate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins.
  • cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts may be used.
  • libraries of procaryotic and eukaryotic proteins may be made for screening in the systems described herein.
  • Particularly preferred in this embodiment are libraries of bacterial, invertebrate, fungal, viral, and mammalian proteins of molecular weight 100 to 2500 daltons.
  • the candidate bioactive agents are organic chemical moieties or small molecule chemical compositions, a wide variety of which are available in the literature.
  • the candidate bioactive agents are linked to a fusion partner.
  • fusion partner or “functional group” herein is meant a sequence that is associated with the candidate bioactive agent, that confers upon all members of the library in that class a common function or ability.
  • Fusion partners can be heterologous (i.e. not native to the host cell), or synthetic (not native to any cell). Suitable fusion partners include, but are not limited to: a) presentation structures, which provide the candidate bioactive agents in a conformationally restricted or stable form; b) targeting sequences, which allow the localization of the candidate bioactive agent into a subcellular or extracellular compartment; c) tagged (or "rescue") sequences which allow the purification or isolation of either the candidate bioactive agents or the nucleic acids encoding them; d) stability sequences, which confer stability or protection from degradation to the candidate bioactive agent or the nucleic acid encoding it, for example resistance to proteolytic degradation; e) dimerization sequences, to allow for peptide dimerization; or f) any combination of a), b), c), d), and e), as well as linker sequences as needed.
  • the Trk protein fragment or the candidate small molecule NTF mimetic is non-diffusibly bound to an insoluble support (e.g. a microtiter plate, an array, etc.).
  • the insoluble support may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening.
  • the surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads.
  • Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. In some cases magnetic beads and the like are included.
  • the particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable.
  • Preferred methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or agent, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety. Also included in this invention are screening assays wherein solid supports are not used; examples of such are described below.
  • the insoluble support is a biosensor cuvette, for example an lAsys biosensor cuvette, described in detail below.
  • the Trk protein fragment is bound to the support, and a candidate small molecule NTF mimetic is added to the assay.
  • the candidate small molecule NTF mimetic is bound to the support and the Trk protein fragment is added.
  • Novel small molecule NTF mimetics include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, centrifugation assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.
  • the determination of the binding of the candidate small molecule NTF mimetic to the Trk protein fragment may be done in a number of ways.
  • the candidate bioactive agent is labeled, and binding determined directly. For example, this may be done by attaching all or a portion of the Trk protein fragment to a solid support, adding a labeled candidate small molecule NTF mimetic (for example a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support.
  • a labeled candidate small molecule NTF mimetic for example a fluorescent label
  • washing off excess reagent for example a fluorescent label
  • binding interaction is monitored directly by biosensor assays.
  • label herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g. radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc.
  • Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc.
  • the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above.
  • the label can directly or indirectly provide a detectable signal.
  • the proteins may be labeled at tyrosine positions using 125 l, or with fluorophores.
  • more than one component may be labeled with different labels; using 125 l for the proteins, for example, and a fluorophor for the candidate agents.
  • neither the Trk protein fragment nor the candidate small molecule NTF mimetic are labeled.
  • the ability of a candidate small molecule NTF mimetic to bind to a Trk protein is determined using an lAsys biosensor machine.
  • a Trk protein fragment is affixed to an lAsys cuvette forming a Trk fragment lAsys cuvette.
  • the Trk protein fragment comprises an Ig-like domain, an LRR domain, or both.
  • a Trk protein fragment comprising a Trk protein C-terminus Ig-like domain, a Trk protein second LRR domain, or both.
  • the Trk fragment cuvette is combined with a candidate small molecule NTF mimetic in an lAsys biosensor machine. An interaction with the Trk fragment cuvette is then determined in the presence and absence of candidate small molecule NTF mimetic. In a preferred embodiment, interaction with a Trk protein fragment cuvette is determined in the presence of a number of different concentrations of candidate small molecule NTF mimetic.
  • an lAsys biosensor machine and while the description herein refers to the use of an lAsys biosensor, it will be appreciated that an lAsys biosensor and lAsys biosensor cuvettes are only one example of a biosensor and biosensor cuvettes. It will be appreciated that other biosensor machines and biosensor cuvettes may be used in the present invention.
  • methods for screening for small molecule NTF mimetics capable of binding to a Trk protein comprise combining a candidate small molecule NTF mimetic and a fragment of a Trk protein consisting essentially of an LRR domain, an Ig-like domain, or both and determining the binding of candidate small molecule NTF mimetic to Trk protein fragment.
  • an LRR domain comprises the second LRR domain of a Trk protein, situated to the C-terminal side of the first LRR domain of the Trk protein, and the Ig-like domain comprises the C-terminal Ig-like domain of a Trk protein.
  • Trk protein fragments are human protein fragments.
  • methods for screening for small molecule NTF mimetics capable of binding to a Trk protein comprise attaching a Trk protein fragment to an lAsys cuvette to form a Trk protein fragment lAsys cuvette.
  • a candidate small molecule NTF mimetic is combined with the Trk protein fragment cuvette in an lAsys biosensor, and the binding of candidate small molecule NTF mimetic to Trk protein fragment is determined by determining an interaction with the Trk protein fragment cuvette using the lAsys biosensor.
  • Trk fusion proteins may be linked to CMD via a tag and an antibody that specifically binds to the tag.
  • Trk fusion proteins comprising Trk protein fragments which comprise an LRR domain, an Ig-like domain, or both, as well as a poly-histidine tag, preferably a 6-histidine tag, may be immobilized to the CMD surface by first immobilizing an that specifically binds to a poly-histidine tag, preferably a 6-histidine tag sequence
  • This antibody has a very slow dissociation rate and does not bind neurotrophin. This antibody is quite suitable for kinetics studies of neurotrophin binding.
  • Trk protein fragments may be directly chemically linked to an lAsys cuvette.
  • Protein amino groups have a number of features that make them suitable for immobilizing proteins, in particular e-amino groups are both charged and hydrophilic. These groups are usually found projecting from the surface of proteins and are able to be covalently linked to the CMD surface with little possibility of denaturation, thus preserving the native conformation of the immobilized protein. Lysine residues occur with an average frequency of 5.7% in proteins
  • MBP maltose binding protein
  • Immobilization of the Trk protein fragments to the CMD surface may be accomplished by activation of the CMD surface with the EDC/NHS chemistry (see Figure 2). After activation, 20 mM ammonium acetate pH 4.2 buffer is added to the reaction cuvette (the choice of immobilization buffer was dictated by the pi of the proteins). Trk protein fragment is then added to the cuvette and allowed to form covalent bonds with the activated carboxymethyl dextran. When an adequate level of immobilization is achieved, the remaining activated CMD groups may be blocked by the addition of 1 M Tris/HCI pH 8.
  • lAsys biosensor Affinity Sensors
  • This instrumentation allows for the measurement of the interaction of two or more biomolecules without the need for labels.
  • the lAsys biosensor uses the ability to couple one of the reactant molecules to a surface and the use of an evanescent field to monitor changes in refractive index, caused by molecular interactions occurring within a few hundred nm of the sensor surface (Cush et al. 1993). Electromagnetic waves undergo refraction upon crossing the boundary between two media (a consequence of different speed of propagation within the two media).
  • the angle of the exiting wave ⁇ 2 is 90° and the refracted wave is directed along the interface between the two media.
  • the incident wave is no longer transmitted through the second media and is completely reflected (total internal reflection).
  • the electric field associated with the electromagnetic radiation penetrates into the low refractive index media, where it dies away exponentially.
  • the distance (Z 0 ) that this "evanescent field" penetrates depends upon n.,, n 2 and the incident angle.
  • Optical biosensors such as the lAsys, utilize the properties of the evanescent field to measure changes at the interface surface due to molecular interaction events.
  • the lAsys utilizes a waveguide (a region of high refractive index bounded on either side by a low refractive index media), to constrain the incident electromagnetic wave to total internal reflections at the low refractive index boundaries. Wave guidance only occurs when the "round trip" reflection is an integral multiple of 2 ⁇ and for a thin waveguide, this condition is met by only one angle between ⁇ c , and 90°. Light propagation within the waveguide occurs at one reflection angle
  • n 1 may for example result from a biomolecular interaction event.
  • Prism coupling is used to introduce laser light into the thin waveguide and to measure changes in ⁇ gu ⁇ de .
  • the lAsys biosensor uses a high refractive index prism to introduce light into the waveguide. Provided the prism is situated close to the waveguide surface, a light ray is totally reflected off the prism face parallel to the waveguide surface.
  • the prism face and the waveguide is similar to sin ⁇ gu ⁇ de , then while most of the light is reflected, some is able to continue uninterrupted in the direction of the original light beam.
  • a plot of the intensity of the light propagated within the waveguide versus the prism angle ⁇ p shows a sharp peak corresponding to the propagation angle ⁇ g ui de - ' n order to use the resonant mirror principle for measurement of biomolecular interactions, the prism is coated with a low refractive index coupling layer ( ⁇ 100 nm thick), forming a gap between the prism and waveguide.
  • the prism forms the base of a stirred cuvette used to contain the reactants.
  • light reflected from the inside of the resonant mirror is measured as a function of the incident angle.
  • the incident light propagates down the waveguide and within the coupling layer through the evanescent field.
  • the angle of excitation at resonance is sensitive to changes at the sensing surface, interactions at the surface may be monitored by changes in the excitation angle. Changes in the excitation angle are measured by arbitrary units, referred to as reaction units (RU's) and are measured as "arc sees".
  • Interactions with Trk receptor fragments may be analyzed by plotting the measured on-rate constant, k on obtained from association analysis at a number of neurotrophin concentrations, versus neurotrophin concentration. From such plots, the equilibrium dissociation constant, K D may be determined for each Trk protein-neurotrophin interaction.
  • k ass is the derived association rate constant obtained from the slope of the plot of k on versus concentration of sample.
  • k dlss is the derived dissociation constant obtained from the intercept of the plot of k on versus concentration of sample.
  • R t is the instrument response at time t
  • R eq is the maximal instrument response
  • R 0 is the initial instrument response value.
  • Equation (3) is in the form of a straight line and hence a plot of the values of k on derived from a complete (6-10 measurements) interaction experiment, against the ligate concentration at which they were conducted, allows k ass and k dlss to be estimated.
  • the gradient of the line gives k ass while the y-axis intercept allows the value of k dlss to be estimated.
  • R t A[1 - exp(-k on(1) t >)] + B[1 - exp(-k on(2) ⁇ )] + R 0 (4)
  • a and B represent the two phases of the association and k on(1) and k on(2) are the respective, apparent association rates.
  • association curves obtained for each ligate concentration are fitted to both theoretical monophasic and biphasic association curves. From this analysis, the best fit of the association data, to either mono or biphasic kinetics, is determined. Such data analysis is shown in the appendices.
  • the phrase "modulating the binding of means increasing the binding of, decreasing the binding of, or altering in some other way the binding of one entity to another, particularly the binding of neurotrophin to Trk protein.
  • alterations in binding include change in the kinetics of neurotrophin binding to Trk protein characterized by a change in the association rate, the dissociation rate, or both, for the binding of neurotrophin to Trk protein.
  • changes in both the association rate and dissociation rate may not alter the overall affinity of neurotrophin for Trk protein.
  • the mechanism of binding of neurotrophin to Trk protein may be altered by a small molecule NTF mimetic.
  • methods for screening for a small molecule NTF mimetic capable of modulating the binding of a neurotrophin to a Trk protein comprise combining a candidate small molecule NTF mimetic, a neurotrophin, and a fragment of a Trk protein comprising an LRR domain, an Ig-like domain, or both, and determining the binding of neurotrophin to Trk protein fragment in the presence and absence of candidate agent.
  • an LRR domain comprises the second LRR domain of a Trk protein, situated to the C-terminal side of the first LRR domain of the Trk protein.
  • an Ig-like domain comprises the C-terminus Ig-domain of the extracellular domain of a Trk protein.
  • Trk protein fragments used are human protein fragments and neurotrophins used are human neurotrophins.
  • the ability of a candidate small molecule NTF mimetic to modulate the binding of neurotrophin to Trk protein is determined using an lAsys biosensor machine.
  • a Trk protein fragment is affixed to an lAsys cuvette forming a Trk fragment lAsys cuvette.
  • the Trk protein fragment comprises an Ig-like domain, an LRR domain, or both.
  • an LRR domain is the second LRR domain of a Trk protein situated to the carboxy-terminus side of the first LRR domain.
  • an LRR domain is the second LRR domain of a Trk protein situated to the carboxy-terminus side of the first LRR domain.
  • Ig-like domain is the C-terminal lg-like domain of a Trk protein situated to the C-terminal side of an N- terminus Ig-like domain in the extracellular domain of a Trk protein.
  • the Trk fragment cuvette is combined with a neurotrophin and a candidate small molecule NTF mimetic in an lAsys biosensor machine. An interaction with the Trk fragment cuvette is then determined in the presence and absence of candidate small molecule NTF mimetic while in the presence of neurotrophin.
  • the neurotrophin is known to bind to the Trk protein fragment.
  • interaction with a Trk protein fragment cuvette is determined in the presence of a number of different concentrations of candidate small molecule NTF mimetic while in the presence of a constant concentration of neurotrophin.
  • Trk protein fragments and neurotrophins are human proteins.
  • the candidate small molecule NTF mimetic is labeled. Either the candidate small molecule NTF mimetic, or neurotrophin known to bind to the Trk protein fragment, is added first to the Trk protein fragment for a time sufficient to allow binding, if present. Alternatively, the neurotrophin and the candidate small molecule NTF mimetic can be added at the same time.
  • Incubations may be performed at any temperature which facilitates optimal activity, typically between 4°C and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high throughput screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
  • the neurotrophin is added first, followed by the candidate small molecule NTF mimetic.
  • Displacement of the neurotrophin is an indication that the candidate small molecule NTF mimetic is capable of modulating the binding of neurotrophin to Trk protein.
  • either component can be labeled.
  • the neurotrophin is labeled, the presence of label in the wash solution indicates displacement by the small molecule NTF mimetic.
  • the candidate small molecule NTF mimetic indicates displacement of the neurotrophin.
  • the candidate small molecule NTF mimetic is added first, with incubation and washing, followed by the neurotrophin.
  • the absence of binding by the neurotrophin may indicate that the small molecule NTF mimetic is capable of binding the Trk protein with a higher affinity than the neurotrophin, and that the small molecule NTF mimetic is capable of modulating the binding of neurotrophin to Trk protein by competitively binding to Trk protein.
  • the candidate small molecule NTF mimetic is labeled, the.presence of the label on the support, coupled with a lack of neurotrophin binding, may indicate that the candidate agent is capable of binding to the Trk protein and modulating the binding of neurotrophin to Trk protein.
  • a Trk protein may be recombinant full length protein produced and collected by means known in the art, or may be a recombinant Trk protein segment, preferably a native extracellular segment, more preferably a segment comprising an LRR domain, an Ig-like domain, or both. More preferably, a Trk protein segment comprises a second Trk LRR domain (situated C-terminus side of the first LRR domain), a Trk C-terminus Ig-like domain, or both.
  • a Trk protein may be a full length protein or a fragment thereof from a cell lysate from a cell comprising a Trk protein or fragments thereof, or a recombinant Trk protein or fragments thereof.
  • the Trk proteins and segments thereof comprise amino acid sequences of human Trk proteins.
  • Positive controls and negative controls may be used in the assays.
  • Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.
  • reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein- protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.
  • a mammalian Trk protein fragment is used to screen for a small molecule NTF mimetic capable of modulating the binding of a mammalian neurotrophin to a Trk protein.
  • a human Trk protein fragment is used to screen for a small molecule NTF mimetic capable of modulating the binding of a human neurotrophin to a Trk protein.
  • Most preferred is the use of a fragment of human TrkA, TrkB, or TrkC protein to screen for a small molecule NTF mimetic capable of modulating the binding of human NGF to TrkA, human BDNF to TrkB, human NT-3 to TrkC, or human NT-4/5 to TrkC.
  • neurotrophins for use in the present invention are preferably in their functional form and capable of binding to Trk protein.
  • Neurotrophin activity may be determined in control experiments done using portions of samples of neurotrophin in order to ensure that the samples used in the methods provided herein contain functional forms of neurotrophin.
  • small molecule NTF mimetics capable of binding to Trk proteins.
  • a small molecule NTF mimetic provided herein is capable of binding to TrkA, TrkB, or TrkC.
  • small molecule NTF mimetics capable of modulating the activity of a Trk protein.
  • a small molecule NTF mimetic provided herein is capable of modulating the activity of TrkA, TrkB, or TrkC.
  • small molecule NTF mimetics provided herein may be capable of oligomerization, preferably dimerization. Such oligomers, preferably dimers, may cause the clustering of Trk proteins.
  • NGF neurotrophin
  • TrkA tyrosine kinase activity
  • p75 plays in the binding of NGF to TrkA; in one model, NGF first binds to p75 which acts as an initial recruitment molecule for NGF. Following NGF binding, a complex is formed consisting of NGF, p75 and TrkA (Huber and Chao, J. Neurosci. Res. 40:557 (1995); Ross, et al., Eur. J. Neurosci.
  • TrkA/p75 heterodimer that is the high affinity receptor observed in a number of binding studies with whole cells (Sutter, et al., J. Biol. Chem. 254:5972 (1979); Rodriguez-Tabar, et al., J.
  • the heterodimer complex then dissociates, resulting in a complex consisting of an NGF dimer bound to a TrkA homodimer.
  • TrkA NGF complex transphosphorylation on tyrosine residues occurs (Jing, et al., J. Mol. Biol. 226:851 (1992)) with maximum tyrosine kinase activity 5 to 10 minutes after NGF binding (Martin-Zanca, 1991).
  • TrkA transphosphorylation on tyrosine residues occurs
  • maximum tyrosine kinase activity 5 to 10 minutes after NGF binding
  • There appear to be three transphosphorylation sites in the kinase domain of TrkA that are required for the efficient transphosphorylation of other tyrosines necessary for propagation of NGF signals (Kaplan, et al., J. Neurobiol. 25[11]:1404 (1994)).
  • Transphosphorylation provides recognition or docking sites for cellular signalling proteins.
  • Five tyrosines are phosphorylated on the intracellular domain of TrkA, namely, Y490, Y785, Y670, Y 674 and Y675 (Loeb, et al., 1994).
  • Cytoplasmic proteins such as PI-3 kinase may utilize receptor binding to cross the membrane where their substrates (phosphatidylinositol phospholipids) are found. It is possible that neurotrophin binding to its receptor may in itself stimulate the activity of the signaling proteins (Kaplan, et al., J. Neurobiol. 25[11]:1404 (1994), as has been shown for PI-3 kinase (Carpenter, et al., J. Biol. Chem. 268:9478 (1993)). In response to NGF, the activities of PLC ⁇ -1 and PI-3 kinase are stimulated (Raffioni, et al., Proc. Natl. Acad. Sci.
  • Diacylglycerol activates protein kinase C and IP-3 transiently increases the levels of intracellular calcium (Rhee and Choi, J. Biol. Chem. 267:12392 (1992)).
  • NGF treatment of PC12 cells that result from increases in PLCy- 1 activity
  • changes in ion fluxes, pH changes, cytoskeleton rearrangements and induction of cellular genes Heasley and Johnson, J. Biol. Chem. 264:8646 ((1989).
  • TrkA mutation of the site responsible for inducing the tyrosine phosphorylation and receptor association of PLC ⁇ -1 , results in a selective loss of the NGF mediated increase in the transcription of peripherin, a gene that encodes an intermediate filament protein (Loeb, et al., 1994).
  • PI-3 kinase catalyses the formation of phosphoinositides with phosphate at the D-3 position of the inositol ring (Whitman, et al., 1987).
  • PI-3 kinase derived second messengers The actions of PI-3 kinase derived second messengers is unknown, however, the sequence of the catalytic subunit of this protein is similar to a yeast protein (Vps34p) responsible for vacuolar sorting and protein trafficking (Schu, et al., Science 260:88 (1993)); hence PI-3 may be involved in targeting activated receptor tyrosine kinases to specific intracellular sites. Retrograde transport of NGF activated Trk receptors in neurons may be mediated by PI-3. SHC proteins are major substrates of NGF induced tyrosine phosphorylation activity in PC12 cells (Ohmichi, et al., J. Biol. Chem. 269:1143 (1994)).
  • Tyrosine phosphorylated SHC associates with a small adaptor molecule Grb2, which in turn binds the SOS Ras guanine nucleotide exchange factors in NGF treated PCI2 cells (Ohmichi, et al., J. Bio.
  • Trk and PLC ⁇ -1 Serine phosphorylation of Trk and PLC ⁇ -1 is stimulated by NGF (Vetter, et al., Proc. Natl. Acad. Sci. USA 88:5650 (1991).
  • a possible action of Erk1 is to alter, by phosphorylation, the activity of Trk associated proteins.
  • Ras In signal transduction pathways, downstream of molecules such as PLC ⁇ -1 , SHC, and PI-3 kinase, are Ras, regulators of Ras and serine/threonine kinases that are activated directly or indirectly by Ras activity (Li, et al., (1992); Woo, et al., (1992)).
  • Antibodies to Ras or introduction of a dominant inhibitory Ras allele into PC12 cells inhibit NGF induced neuronal outgrowth (Kremer, et al., J. Cell Biol. 115:809
  • Ras plays a central role in the transmission of NGF induced signals from TrkA to TrkA activated proteins, that in turn sequentially activate Ras and a series of serine/threonine kinases.
  • Ras must directly interact with Raf-I in order to activate the biological and enzymatic activity of Raf-1 (Fabian, et al., Proc. Natl. Acad. Sci. USA 91 :5982 (1994)). It appears that Raf kinases are the most important mediators of the Ras signaling pathway. Ras may act to coordinate the proteins involved in the signaling pathways by binding to cytoplasmic Raf- 1 and inducing it to locate to the plasma membrane a site of potential activators of Raf activity (Leevers, et al., Nature 369:411 (1994)).
  • Raf-1 Activated Raf-1 phosphorylates and stimulates the activity of the Erk activator molecule MEK, which in turn activates and induces Erk to phosphorylate and activate Rsk (Crews, et al., Cell 74:215 (1993)). Transcription factors such as RSF are regulated by Rsk, when after activation, it translocates to the nucleus (Chen, et al., Mol. Cell Biol. 12:915 (1992)). A number of activation and translocation events have been reported in PCI2 cells (Ohmichi.et al., (1992); Traverse, et al., (1992); Nguyen, et al., J. Biol. Chem. 268:9803 (1993).
  • Trk associating proteins in NGF mediated differentiation responses has been made by the use of mutant TrkA receptors expressed in PCI2 derived cells. These proteins encode mutations in the SHC (Y490) and PLC ⁇ -1 (Y785) association sites (Kaplan and Stephens, J. Neurobiol.
  • TrkA with a mutation in either the SHC or PLC ⁇ -1 binding sites respond to NGF with normal neurite outgrowth and Erk1 activation (Loeb, et al., 1994).
  • Cells expressing TrkA with mutations at both Y490 and Y785 when treated with NGF fail to extend stable neurites and do not efficiently induce Erk activity or Raf-1 phosphorylation (Stephens, et al., Neuron 12:691 (1994)).
  • NGF neurotrophic Factors (ed. Fallon & Loughlin) New York, Academic p. 209 (1993)) and as a consequence, NGF is available to bind to and activate receptors only on distal axons.
  • a signal created by receptor activation on distal axons must be communicated to the cell soma, since NGF present only at the tips of neurites has been shown to be sufficient to maintain the viability of cell bodies (Campenot, Proc. Natl. Acad. Sci. USA 74:4516 (1977)). Retrograde transport of NGF takes place at ⁇ 2500 mm/hr (Claude, et al., 1982) through a distance greater than 1000 times the diameter of the cell body for those neurons with long axons. Since this rate of transport is greater than is possible through simple diffusion, an active transport system for neurotrophins within the axon of neurons is implied. Microtubules may serve as the active transport system (Hendry and Bonyhady, Brain Res.
  • NGF signaling has recently been advanced by Grimes et al. (Grimes, et al., J. Neurosci 16[24]:7950 (1996)).
  • endocytosed activated TrkA receptors serve as the retrograde messenger.
  • NGF induces rapid internalization of TrkA in PCI2 cells and NGF and TrkA are both found in intracellular vesicles.
  • NGF remains bound to TrkA in the vesicles and the TrkA receptors are active as assessed by tyrosine phosphorylation and association with PLC ⁇ -1. It is possible that signaling endosomes containing activated TrkA, convey the NGF signal to the cell body.
  • Retrograde transport has been observed for p75 in NGF responsive neurons in the PNS and CNS (Raivich, et al., EMBO J 4:637 (1991); Kiss, et al., Neuroscience 57:297 (1993)). Internalization and endocytosis have not been observed for p75 in NGF-responsive PCI2 cells (Hosang and Shooter, J. Biol. Chem. 260:655 (1987)) and disruption of p75 function has little effect on retrograde transport of NGF, implying that the NGF signal is not carried by p75 (Curtis, et al., Neuron 14:1201 (1995)).
  • TrkA binding and signaling through TrkA have been examined over variable intervals of NGF treatment (Zhou, et al., J. Neurochem. 65[3]:1146 (1995)). This study showed that rapid down regulation of cell surface and total cellular TrkA occurred after induction of tyrosine phosphorylation of TrkA. Increased TrkA mRNA and TrkA protein expression were observed to follow the down regulation events. Delayed responses to NGF include expression of a number of genes whose products identify differentiated neurons (Longo, et al., Neurotrophic Factors (ed. Fallon & Loughlin) New York, Academic, p. 209 (1993)).
  • TrkA receptors does not appear to be a simple function of the level of protein present, but rather reflects the possibility that a number of processes including synthesis, membrane insertion, internalization, recycling and degradation influence the number of surface NGF receptors (Zhou, et al., J. Neurochem. 65[3]:1146 (1995)).
  • NGF For the maintenance of neurites in PCI2 cells, NGF has to be continuously present in the culture media suggesting that continuous signaling through TrkA is required to induce morphological differentiation.
  • TrkA receptors appear, by confocal microscopy, to be intact and contained in organelles near the cell surface after NGF treatment (Grimes, et al., J. Neurosci 16[24]:7950 (1996)). Some TrkA positive organelles colocalized with staining for the clathrin heavy chain and adaptin, indicating that TrkA internalization is at least partially mediated through clathrin coated pit endocytosis. Hence, NGF activation of TrkA may result in the recruitment of the receptor into clathrin-coated pits as has been shown for other receptor tyrosine kinases (Lamaze and Schmidt, J. Cell Biol. 129:47 (1995)).
  • TrkA staining Confocal microscopy and TrkA staining, indicate that the internalized receptor appears to be colocalized with a marker for lysosomes. Other studies have shown that surface down regulation of receptor tyrosine kinases results in the receptor being sequestered in lysosomes where low pH (5.4), results in degradation (van der Geer, et al., Annu. Rev. Cell Biol. 10:251 (1994)). If the TrkA receptor is to act in signal transduction, it must first be internalized at the axon tip and then undergo retrograde transport to the cell body without experiencing degradation in the process.
  • Late endosomes and lysosomes are prominently located in the cell body and proximal dendrites (Nixon and Cataldo, TINS 18:489 (1995)) and there is no evidence that these organelles are present in axons or presynaptic terminals of cultured hippocampal neurons (Parton, et al., J. Cell Biol. 119:123 (1992); Grimes, et al., J. Neurosci 16[24]:7950 (1996)).
  • Trk protein binding to neurotrophin many binding assays for Trk protein binding to neurotrophin are known. It will also be appreciated that many activity assays for determining Trk protein activity, particularly in response to ligand, preferably in response to neurotrophin, are known. These include bioassays including but not limited to neurite extension assays in primary neuronal cultures and PC12 cell line cultures, as well as biochemical assays including but not limited to phosphorylation assays
  • Such activity assays may be used herein as a matter of routine screening to determine the activity of small molecule NTF mimetics provided by the methods herein.
  • the present invention provides methods for screening for small molecule NTF mimetics capable of modulating Trk protein activity in which activity assays known in the art are combined with steps for determining the ability of a small molecule NTF mimetic to bind to neurotrophin binding sites in Trk protein described herein.
  • candidate small molecule NTF mimetics are screened as described herein for their ability to bind to Trk protein.
  • Activity assays known in the art are subsequently used for routine screening to determine the activity of candidate small molecule NTF mimetics determined to be capable of binding to Trk protein. As will be recognized, such assays do not constitute undue experimentation.
  • the present invention provides for the modification of known competitive binding assays as described above to determine the binding of a neurotrophin or a small molecule NTF mimetic to a Trk protein fragment comprising an LRR domain (preferably the second LRR domain), an Ig-like domain (preferably the C-terminal Ig-like domain) or both.
  • LRR domain preferably the second LRR domain
  • Ig-like domain preferably the C-terminal Ig-like domain
  • the Trk protein fragment or the neurotrophin is non-diffusibly bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate, an array, etc.) as described above.
  • isolated sample receiving areas e.g. a microtiter plate, an array, etc.
  • the neurotrophin is bound to the insoluble support. Trk protein fragment is then added to permit binding to neurotrophin, if present.
  • Candidate small molecule NTF mimetic is then added to permit modulation of the Trk protein fragment-neurotrophin interaction, if present.
  • the Trk protein and small molecule NTF mimetic can be pre-incubated prior to addition to the immobilized neurotrophin.
  • the Trk protein fragment and the small molecule NTF mimetic can be added simultaneously to the immobilized neurotrophin.
  • Trk protein fragment is bound to the insoluble support.
  • Neurotrophin is then added to permit binding to Trk protein fragment, if present.
  • Candidate small molecule NTF mimetic is then added to permit modulation of the Trk protein fragment- neurotrophin interaction, if present.
  • the neurotrophin and small molecule NTF mimetic can be pre-incubated prior to addition to the immobilized Trk protein fragment.
  • the neurotrophin and the small molecule NTF mimetic can be added simultaneously to the immobilized Trk protein fragment.
  • the present invention also provides methods for screening for a small molecule NTF mimetic that is capable of modulating the activity of a Trk protein.
  • the methods involve determining Trk protein activity using Trk protein activity assays. Such assays can be done with cells comprising Trk protein or functional fragments or variants thereof.
  • the methods for screening for a small molecule mimetic capable of modulating Trk protein activity comprise a first series of steps for screening for a candidate small molecule NTF mimetic capable of modulating the binding of neurotrophin to a Trk protein.
  • a candidate small molecule NTF mimetic is isolated, optionally identified, and subsequently used in a Trk protein activity assay.
  • the methods for screening for a small molecule mimetic capable of modulating Trk protein activity comprise a first series of steps for screening for a candidate small molecule NTF mimetic capable of binding to a Trk protein.
  • a candidate small molecule NTF mimetic is isolated, optionally identified, and subsequently used in a Trk protein activity assay.
  • Trk protein activity assay can be done in a number of ways. In addition, there are multiple measurable Trk protein activities.
  • a small molecule NTF mimetic provided herein is capable of modulating the activity of a Trk protein.
  • Modulating the activity of a Trk protein includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present.
  • the candidate small molecule NTF mimetic may bind to Trk protein (although this may not be necessary), and should alter its biological or biochemical activity as described herein or as otherwise known in the art.
  • Trk protein activity assays may comprise combining a Trk protein sample and a candidate small molecule NTF mimetic, and evaluating the effect of the small molecule NTF mimetic on the activity of the Trk protein.
  • Trk protein activity or grammatical equivalents herein is meant at least one of the Trk protein's biological and/or biochemical activities, including, but not limited to neurite outgrowth assay, neuronal survival assay, apoptosis assay, tyrosine phosphorylation assay, AKT activation assay, SNT activation assay, PI3-K activation assay, PLC-gamma activation assay.
  • the activity of the Trk protein is decreased; in another preferred embodiment, the activity of the Trk protein is increased.
  • small molecule NTF mimetics that are antagonists are preferred in some embodiments, and small molecule NTF mimetics that are agonists are preferred in other embodiments.
  • a Trk protein sample comprises a cell which comprises a recombinant nucleic acid that encodes Trk protein that is expressed in the cell.
  • Preferred cell types include PC12 cells, NIH 3T3 cells, 293 cells, MAH cells, mammalian sympathetic neurons, mammalian sensory neurons, mammalian central neurons, mammalian neural stem cells, vertebrate neural crest stem cells, neuroblastoma cells, glioblastoma cells and astrocytoma cells.
  • the present invention provides small molecule NTF mimetics capable of binding to Trk proteins and/or modulating their activity, including and preferably small molecule chemical compositions as discussed herein, which are useful in the treatment of neurodegenerative disease.
  • compositions provided herein are useful for administration to patients with neurodegenerative diseases or predisposition to such diseases including Parkinson's disease, Huntington's disease, Alzheimer's disease and amyotrophic lateral sclereosis.
  • the present invention provides compositions for the treatment of other diseases which are generally characterized by the loss of a population of neurons due to decreased or eliminated Trk protein activity and/or loss of a population that may be sustained by Trk protein activity, including ectopic Trk protein activity. A reduction or loss of Trk protein activity may occur due to a loss or reduction in the amount of a functional neurotrophin provided to a neuronal population.
  • a reduction or loss of Trk protein activity may also occur due to a reduction in the level expression of a particular Trk protein isoform or an increase in the level of expression of particular dominant negative Trk protein isoform or a change in the localization of particular Trk protein isoforms.
  • the proteins and nucleic acids provided herein can also be used for screening purposes wherein the protein-protein interactions of the Trk proteins can be identified. Genetic systems have been described to detect protein-protein interactions. The first work was done in yeast systems, namely the "yeast two-hybrid" system. The basic system requires a protein-protein interaction in order to turn on transcription of a reporter gene. Subsequent work was done in mammalian cells. See Fields, et al.,
  • small molecule NTF mimetics are identified. These small molecule mimetics may be used to produce compounds with pharmacological activity which are able to modulate Trk protein activity.
  • the compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as further described below.
  • the present invention provides compositions that may be used for the treatment of diseases involving dysfunction or dysregulation of Trk protein activity and/or signaling pathways downstream of Trk protein.
  • compositions also find use in the treatment of diseases involving dysfunction or dysregulation of neurotrophins.
  • compositions find additional use in the treatment of cells that express a Trk protein endogenously or cells that are made to express an exogenous Trk protein and possess signal transduction pathways capable of mediating Trk-induced signals.
  • the present invention provides methods comprising administering to a cell or individual in need thereof, a small molecule NTF mimetic in a therapeutic amount.
  • a small molecule NTF mimetic provided herein is capable of binding to a Trk protein
  • Trk proteins are human proteins.
  • a small molecule NTF mimetic administered functions as an agonist of Trk protein activity.
  • a small molecule NTF mimetic administered functions as a Trk protein antagonist.
  • a small molecule NTF mimetic which functions as an agonist of Trk protein activity is used to treat a neurodegenerative disease which may include but is not limited to Parkinson's disease, Huntington's disease, Alzheimer's disease and amyotrophic lateral sclerosis.
  • a small molecule NTF mimetic which functions as an antagonist of Trk protein activity is used to treat cancer.
  • a therapeutically effective dose of a small molecule NTF mimetic is administered to a patient.
  • therapeutically effective dose herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art, adjustments for NTF mimetic degradation, systemic versus localized delivery, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
  • a "patient” for the purposes of the present invention includes both humans and other animals, particularly mammals, and organisms. Thus the methods are applicable to both human therapy and veterinary applications.
  • the patient is a mammal, and in the most preferred embodiment the patient is human.
  • the administration of the small molecule NTF mimetic of the present invention can be done in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.
  • the composition may be directly applied as a solution or spray.
  • the compounds may be formulated in a variety of ways.
  • the concentration of therapeutically active compound in the formulation may vary from about 0.1-100 % wt.
  • compositions (or medicaments) of the present invention comprise at least one small molecule NTF mimetic in a form suitable for administration to a patient.
  • a small molecule NTF mimetic is capable of binding to a Trk protein LRR domain, a Trk protein Ig-like domain, or both.
  • an LRR domain is the second LRR domain situated to the C-terminal side of the first LRR domain.
  • an Ig-like domain is the C-termal Ig-like domain situated to the C-terminal side of the N-terminal Ig-like domain.
  • Trk proteins are human proteins.
  • the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
  • carrier proteins such as serum albumin
  • buffers such as buffers
  • fillers such as microcrystalline cellulose, lactose, corn and other starches
  • binding agents such as microcrystalline cellulose, lactose, corn
  • compositions may be administered. Moreover, the compositions may be administered in combination with other therapeutics, including growth factors or chemotherapeutics and/or radiation.
  • Targeting agents i.e. ligands for receptors on cancer cells
  • cyclic peptides comprise an amino acid sequence having at least about 90%, more preferably at least about 95%, more preferably at least about 98% identity to the amino acid sequence set forth in SEQ ID NO:1.
  • such cyclic peptides comprise the amino acid sequence set forth in SEQ ID NO:1.
  • small molecule NTF mimetics comprising cyclic peptides provided herein are capable of binding to a Trk protein LRR domain, a Trk protein Ig-like domain, or both.
  • an LRR domain is the second LRR domain situated to the C-terminal side of the first LRR domain.
  • an Ig- like domain is the C-termal Ig-like domain situated to the C-terminal side of the N-terminal Ig-like domain.
  • Trk proteins are human proteins.
  • small molecule NTF mimetics comprising cyclic peptides provided herein are capable of modulating the binding of a neurotrophin to a Trk protein.
  • such small molecule NTF mimetics are capable of modulating the binding of a mammalian neurotrophin to a mammalian Trk protein.
  • such small molecule NTF mimetics are capable of modulating the binding of a human neurotrophin to a human Trk protein.
  • such small molecule NTF mimetics are capable of modulating the binding of human NGF to human TrkA, human BDNF to human TrkB, human NT-3 to human TrkC, or human NT-4/5 to human TrkC.
  • small molecule NTF mimetics comprising cyclic peptides provided herein are capable of modulating the activity of a Trk protein.
  • such small molecule NTF mimetics are capable of modulating the activity of a mammalian Trk protein.
  • such small molecule NTF mimetics are capable of modulating the activity of a human Trk protein.
  • such small molecule NTF mimetics are capable of modulating the activity of human TrkA, TrkB, or TrkC.
  • small molecule NTF mimetics comprising cyclic peptides provided herein comprise agonists of Trk protein activity.
  • Trk protein activity is mammalian Trk protein activity, more preferably human Trk protein activity, more preferably human TrkA, TrkB, or TrkC protein activity.
  • small molecule NTF mimetics comprising cyclic peptides provided herein comprise antagonists of Trk protein activity.
  • Trk protein activity is mammalian Trk protein activity, more preferably human Trk protein activity, more preferably human TrkA, TrkB, or TrkC protein activity.
  • cyclic peptide provided herein may be identified based on sequence percent sequence homology or percent sequence identity to the amino acid sequence set forth in SEQ ID NO:1.
  • Sequence identity and/or similarity is determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith, et al., Adv. Appl. Math. 2:482 (1981), by the sequence identity alignment algorithm of Needleman, et al., J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson, et al., PNAS USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
  • percent identity is calculated by FastDB based upon the following parameters: mismatch penalty of 1 ; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng, et al., J. Mol. Evol., 35:351-360 (1987); the method is similar to that described by Higgins, et al., CABIOS, 5:151-153 (1989).
  • Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
  • BLAST BLAST program
  • WU-BLAST-2 WU-BLAST-2 uses several search parameters, most of which are set to the default values.
  • the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
  • Gapped BLAST uses BLOSUM-62 substitution scores; threshold T parameter set to 9; the two-hit method to trigger ungapped extensions; charges gap lengths of k a cost of 10+k; X u set to 16, and X g set to 40 for database search stage and to 67 for the output stage of the algorithms.
  • Gapped alignments are triggered by a score corresponding to ⁇ 22 bits.
  • a % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region.
  • the "longer” sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-
  • percent (%) nucleic acid sequence identity with respect to the coding sequence of the polypeptides identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the coding sequence of the cell cycle protein.
  • a preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
  • the alignment may include the introduction of gaps in the sequences to be aligned.
  • sequences which contain either more or fewer amino acids than the protein encoded by the sequences in the SEQ ID NO:1 it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical amino acids in relation to the total number of amino acids. Thus, for example, sequence identity of sequences shorter than that shown in SEQ
  • ID NO:1 will be determined using the number of amino acids in the shorter sequence, in one embodiment. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as, insertions, deletions, substitutions, etc.
  • identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of "0", which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations.
  • Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. The "longer" sequence is the one having the most actual residues in the aligned region.
  • sequences of the present invention may contain sequencing errors. That is, there may be incorrect nucleosides, frameshifts, unknown nucleosides, or other types of sequencing errors in any of the sequences; however, the correct sequences will fall within the homology and stringency definitions herein.
  • lAsys biosensor Affinity Sensors. This instrumentation allows for the measurement of the interaction of two or more biomolecules without the need for labels.
  • the lAsys biosensor uses the ability to couple one of the reactant molecules to a surface and the use of an evanescent field to monitor changes in refractive index, caused by molecular interactions occurring within a few hundred nm of the sensor surface (Cush.et al., Biosensors & Bioelectronics 8:347 (1993)). Electromagnetic waves undergo refraction upon crossing the boundary between two media (a consequence of different speed of propagation within the two media).
  • ⁇ 0 critical angle
  • the angle of the exiting wave ⁇ 2 is 90° and the refracted wave is directed along the interface between the two media.
  • the incident wave is no longer transmitted through the second media and is completely reflected (total internal reflection).
  • the electric field associated with the electromagnetic radiation penetrates into the low refractive index media, where it dies away exponentially.
  • the distance (Z 0 ) that this "evanescent field" penetrates depends upon n,, n 2 and the incident angle.
  • Optical biosensors such as the lAsys, utilize the properties of the evanescent field to measure changes at the interface surface due to molecular interaction events. When the incident wave is totally internally reflected, the evanescent field is sensitive to changes that occur only within the distance Z 0 of the interface; bulk media changes that occur beyond Z 0 are not detectable.
  • the lAsys utilizes a waveguide (a region of high refractive index bounded on either side by a low refractive index media), to constrain the incident electromagnetic wave to total internal reflections at the low refractive index boundaries.
  • Wave guidance only occurs when the "round trip" reflection is an integral multiple of 2 ⁇ and for a thin waveguide, this condition is met by only one angle between ⁇ c , and 90°.
  • ⁇ guide Light propagation within the waveguide occurs at one reflection angle ( ⁇ guide ) and this angle is extraordinarly sensitive to changes in the refractive index Small changes in n., result in large changes in ⁇ guide . Changes in n., may for example result from a biomolecular interaction event.
  • Prism coupling is used to introduce laser light into the thin waveguide and to measure changes in ⁇ guide .
  • the lAsys biosensor uses a high refractive index prism to introduce light into the waveguide. Provided the prism is situated close to the waveguide surface, a light ray is totally reflected off the prism face parallel to the waveguide surface.
  • the distance between the prism face and the waveguide is similar to sin ⁇ guide , then while most of the light is reflected, some is able to continue uninterrupted in the direction of the original light beam.
  • the prism In order to use the resonant mirror principle for measurement of biomolecular interactions, the prism is coated with a low refractive index coupling layer ( ⁇ 100 nm thick), forming a gap between the prism and waveguide.
  • the prism forms the base of a stirred cuvette used to contain the reactants.
  • light reflected from the inside of the resonant mirror is measured as a function of the incident angle. With the exception of light incident at ⁇ guide light is totally reflected at the prism, coupling-layer interface. At resonance, the incident light propagates down the waveguide and within the coupling layer through the evanescent field.
  • Trk Proteins Coupling of Trk Proteins to the lAsys Surface
  • NGF and the antibody was detectable, negating the suitability of MBP antibodies for immobilizing MBP-fusion proteins.
  • Amylose resin Pierce
  • MBP-fusion protein was immobilized to the resin.
  • MBP expressed in the same expression vector as the fusion proteins was immobilized to amylose. No binding of NGF was measured binding to the MBP control.
  • amylose surface permitted an interaction between NGF and TrkA protein to be assessed, however the quantity of Trk protein immobilized by this method proved inadequate ( ⁇ 100 RU's immobilized) to accurately measure the interaction between the Trk receptor proteins and the neurotrophins.
  • amylose was biotinylated and bound with streptavidin to the biotin surface of an lAsys cuvette. This method of immobilizing amylose is attractive because the affinity of biotin for streptavidin is high ( ⁇ 10 "14 M) and hence capture is essentially irreversible in non-denaturing buffers.
  • biotinylated dextran which is able to bind MBP
  • biotinylated dextran which is able to bind MBP
  • the biotinylated dextran (average MW 3000 daltons) was immobilized to the biotin/streptavidin surface by the same methods as used for the biotinylated amylose.
  • Immobilization of the MBP-fusion proteins was achieved through the carboxylate groups of the carboxymethyl dextran (CMD) surface of an lAsys cuvette and the primary amino groups (N-terminal and Lysine residues) of the fusion proteins, using 1-ethyl-3-(3- dimethylaminopropyl) carbodimide (EDC) and N-hydroxysuccinimide " (NHS) chemistry and protocols (Affinity sensors).
  • CMD carboxymethyl dextran
  • EDC 1-ethyl-3-(3- dimethylaminopropyl) carbodimide
  • NHS N-hydroxysuccinimide
  • Lysine residues occur with an average frequency of 5.7% in proteins (MaCaldon, 1988) and thus represent a relatively high proportion of the total amino acids of any protein. Lysine residues are generally found to be well distributed over the surface of proteins and as such, immobilization via these groups is less likely to introduce steric hindrance which may occur with immobilization through less abundant amino acids.
  • MBP was immobilized on one cell surface (150 arc sees immobilized) and TrkA MBP-LRR (295 arc sees immobilized) was immobilized to the second cell of the double cell cuvette.
  • Immobilization of the proteins, to the CMD surface was accomplished by activation of the CMD surface with the EDC/NHS chemistry (illustrated below). After activation, 20 mM ammonium acetate pH 4.2 buffer was added to the reaction cell (the choice of immobilization buffer was dictated by the pi of the proteins, all of which have pi's ⁇ 5). Protein was then added to the cell and allowed to form covalent bonds with the activated carboxymethyl dextran. When an adequate level of immobilization was achieved, the remaining activated CMD groups were blocked by the addition of 1 M Tris/HCI pH 8.
  • Trk protein fragments were covalently coupled to an lAsys cuvette using the steps schematically depicted in Figure 2.
  • Fig 17 An example of the binding and dissociation response from the lAsys biosensor during the collection of a set of binding/dissociation data is shown in Fig 17 (TrkA MBP-C1 LRR at 15°C). Rising curves result from the binding of neurotrophin to immobilized Trk protein. Neurotrophin of a known concentration was added as a 5 ⁇ l aliquot with a 10 ⁇ l Hamilton syringe, to 50 ⁇ l of buffer (10 mM Hepes pH 7.4, 150 mM NaCl, 0.01% Tween 20) in the cuvette. Descending curves result from dissociation of bound neurotrophin from the Trk protein after the buffer containing neurotrophin was removed from the reaction cuvette and replaced with fresh buffer.
  • the almost vertical response from the instrument results from the removal of the buffer from the reaction cuvette and replacement with 10 mM HCI. This ensured complete removal of any neurotrophin that remained bound to the receptor after the dissociation event and prior to the addition of a new neurotrophin concentration. Following equilibration in fresh buffer, a new association/dissociation reaction data set was collected. Typically, for the reaction of Trk-neurotrophin interactions, association/dissociation reaction rates were measured for 10 different neurotrophin concentrations.
  • Neurotrophin concentrations in the reaction cuvette varied from ⁇ 1200 nM to ⁇ 5500 nM (for NGF interactions with TrkA proteins) after dilution from stock solutions. Intermediate concentrations were prepared by serial dilution from an initial stock solution. Each concentration was further analyzed by UV spectroscopy and the concentration determined by the absorbance at 280 nm and the extinction coefficient for each neurotrophin at this wavelength. The neurotrophin concentrations used, ensured a response of >50 arc sees for the lowest concentration, thus increasing the accuracy of the data analysis. Stirrer speed was maintained at 80% of maximum throughout all experiments to eliminate mass transport effects. The reaction temperature in the cuvette varied by ⁇ 0.3°C of the set temperature throughout each experiment.
  • the interactions between neurotrophin and Trk receptor were analyzed by plotting the measured on-rate constant, k on obtained from association analysis at a number of neurotrophin concentrations, versus neurotrophin concentration. From such plots, the equilibrium dissociation constant, K D may be determined for each Trk protein-neurotrophin interaction.
  • K D the equilibrium dissociation constant
  • a single example of this type of analysis is given in the appendices. Because of limitations in the quantity of neurotrophins available for this study, only one K D for each Trk protein, at three different temperatures, could be determined. This limited number of measurements is adequate to determine the interactions of neurotrophin with any Trk protein domain at a specific temperature, although inadequate to accurately determine the K D of the interaction.
  • variation in the K D between the leucine rich TrkA domain proteins for example should not be interpreted as changes in neurotrophin affinity, brought about by the presence or absence of flanking cysteines that are N and C-terminal to the full-length leucine rich domain.
  • the absolute effects of other modifications on neurotrophin affinity, such as the presence or absence of one or more regions of the full-length leucine rich domain should similarly not be interpreted from the measured K D 's.
  • the number of binding curves obtained, namely 10 for each Trk protein, is adequate to show neurotrophin binding and to estimate an error in each equilibrium dissociation constant.
  • k ass is the derived association rate constant obtained from the slope of the plot of k on versus concentration of sample.
  • k d ⁇ ss is the derived dissociation constant obtained from the intercept of the plot of k on versus concentration of sample.
  • R t is the instrument response at time t
  • R eq is the maximal instrument response
  • R 0 is the initial instrument response value.
  • Equation (3) is in the form of a straight line and hence a plot of the values of k on derived from a complete
  • K D The error in K D was determined from the sum of the percentage errors associated.
  • the errors associated with K D k ass and k d ⁇ ss are too large to give more than a general model of the variation of these constants with the different domains of the Trk receptors. Results of this analysis are shown in Figures 21-23.
  • a and B represent the two phases of the association and k on(1) and k on(2) are the respective, apparent association rates.
  • association curves obtained for each ligate concentration are fitted to both theoretical monophasic and biphasic association curves. From this analysis, the best fit of the association data, to either mono or biphasic kinetics, is determined. Such data analysis is shown in the appendices.
  • biphasic dissociation having two phases A and B, is described by two dissociation constants k d ⁇ ss(1) and k d ⁇ ss(2) such that:
  • Rt Aexp(-k d ⁇ ss(1) ') + B(-k dlss(2) l ) (6)
  • the equilibrium dissociation constant for each recombinant Trk protein was calculated from 10 association curves (one data set) for each construct at each of three temperatures. Equilibrium dissociation constants and associated errors, are calculated from only one data set, at each temperature. Association rates and the error associated with each K D was determined by the fitting of a binding curve (through the origin) to the k on rates, at each neurotrophin concentration. These results are tabulated in Figures 18-20. Kinetics data for the interactions of all Trk proteins available for this study, was analyzed using FASTFIT software (Affinity Sensors). Equilibrium dissociation constants for each Trk receptor interaction with its appropriate neurotrophin, were determined from plots of the equilibrium binding response at different ligand concentrations.
  • Figure 23 shows TrkC constructs, dissociation and association rates, at 25°C.
  • Trk proteins With most recombinant Trk proteins, there appears to be considerable variation of the dissociation constant with temperature. These variations may be intrinsic to the different Trk protein domains. Equally possible is some complex biosensor surface effects, although the latter possibility seems less likely, given that no such effects have been reported for biosensors. In general, biosensor measurement of the kinetic parameters of biomolecular interactions is conducted at 20-25°C (Eric Hnath, private communication) and not at lower or higher temperatures as used in this study. It is quite noticeable that the errors in the K D 's for the majority of the proteins studied, is lowest at 25°C and ' increase considerably for the measurements at 15°C and 25°C.
  • Trk protein-neurotrophin kinetics were made at the instrument default temperature of 25°C immediately after immobilization of each Trk protein on the biosensor surface. Before kinetics measurements at 15 and 37°C, the biosensor cuvette was stored at 4°C for periods of 24-48 hours and this may have contributed to some degradation of the protein surface, resulting in loss in protein activity and variability in the observed kinetics. Degradation of the protein surface, especially at 37°C, throughout the long period of data collection, may also have occurred, with obvious consequences for accuracy in the kinetics data.
  • K D lies in the large error often associated with the estimation of k diss .
  • Equilibrium constants, k ass and k diss generated from the plots of k on versus ligand concentration for each protein are shown tabulated above.
  • the large errors in the estimates of the equilibrium constants for many constructs, a consequence in the difficulty of an accurate measurement of k diss is quite noticeable.
  • the most consistent and accurate estimate for the equilibrium dissociation constants for each Trk protein result from fitting a binding-curve through plots of k on versus ligand concentration.
  • TrkA the rate of association is initially high, resulting in a low K D1 A second, considerably lower rate of association results in a K D2 ⁇ considerably greater than K D1
  • electrostatic attraction between the TrkA protein and NGF is initially significant.
  • a structural rearrangement of the receptor protein may occur, resulting in lowered charge attraction between receptor and neurotrophin. The consequence of this rearrangement, may be a reduced rate of association and biphasic kinetics.
  • TrkA the protein expressed in E.
  • coli shows a faster association rate for NGF, when compared with the same domain expressed in Pichia pastoris and in insect cells. This difference may be attributed to steric effects on NGF binding, from the high level of glycosylation on the Pichia pastoris and insect cell expressed extracellular domain. Conceivably, if receptor structural changes occur as NGF binds, then high levels of glycosylation may slow such changes, resulting a lowered rate of NGF binding.
  • TrkB and TrkC proteins show an association rate for their respective neurotrophin, significantly greater than that exhibited by the TrkA proteins. This may be a consequence of high electrostatic attraction between receptor and neurotrophin, without structural rearrangement of the receptorupon ligand binding. Unlike the TrkA extracellular domain, the TrkB glycosylated extracellular domain, resulting from expression of the protein in Pichia pastoris, does not show a lower BDNF association rate, when compared with the unglycosylated, E. coli expressed protein. This suggests that glycosylation plays no role in BDNF binding to TrkB. A possible consequence of no structural rearrangement of the receptor upon ligand binding and of no steric hindrance effects on structural rearrangements resulting from attached sugar chains. Structural changes in the neurotrophins upon binding to their respective receptor,
  • TrkA LRR2 and TrkB LRR2 domain binding to NGF and BDNF respectively, it was clear that the TrkA peptide binds with biphasic kinetics to NGF while the TrkB peptide bings with monophasic kinetics to BDNF.
  • Charge distributions on the surface residues of NGF, BDNF and NT-3, produced with GRASP software show considerable differences in charge distribution on the surfaces of the proteins. From an NMR study of the two peptides (collected by Vladimir Basus, Dept.
  • the TrkA peptide is all random coil, while the TrkB peptide has significant ⁇ -helical secondary structure. It is possible that the dissimilar kinetics displayed by the two peptides arises from the difference in charge distribution on the surfaces of the neurotrophins as well as the solution structure of the peptides.
  • Example 2 Introduction In order to provide additional evidence for the binding of NGF to the leucine rich region and the immunoglobulin-like domains of TrkA, measurement of the binding of NGF to the immobilized extracellular domain of TrkA in the presence of competing molecules was undertaken.
  • Solution decoys to the binding of neurotrophin to the immobilized TrkA receptor include, MBP-fusion proteins representing the LRR of TrkA, the extracellular domain of TrkA (a gift from Uri Saragovi) and the Ig-like protein of TrkA (a gift from Uri Saragovi).
  • NGF was also incubated with bovine serum albumin (BSA) as a non-NGF binding control. The binding of NGF/decoy to the immobilized TrkA extracellular domain was studied after NGF had been first incubated with a molar excess ( ⁇ 20:1) of the solution decoy.
  • BSA bovine serum albumin
  • the baseline response from the lAsys was determined from the binding of NGF of the same quantity incubated with the solution decoys in a total volume of 55 ⁇ l. Following regeneration of the immobilized TrkA and the generation of a new baseline in buffer, the reaction cuvette was evacuated, incubate was added and the new instrument response determined.
  • Trk protein concentration needs to be as high as ⁇ 0.1 M in the incubate to eliminate all free NGF from solution.
  • TrkA rat
  • TrkB TrkB
  • Pichia pastoris Reasonable expression yields (> 3 mg/L) were obtained for both proteins.
  • Pichia pastoris Both TrkA (human) and TrkC (rat) expressed at negligible levels and production of either protein in Pichia pastoris seems unreasonable.
  • a final simple purification scheme was established for the purification of the His-tagged TrkA and TrkB proteins produced in Pichia pastoris; however, the initial expectation that a chelating column alone would be sufficient for a one step purification method, proved to be unfounded.
  • TrkA MBP-lg Only one domain (TrkA MBP-lg) expressed well in the E. coli strain used by Rainer Schneider. A different E. coli species was used in this study to obtain usable yields of protein for kinetic studies. The expression levels of all proteins appears to be an order of magnitude lower than that obtained by Rainer
  • Protein was obtainable at levels necessary for the completion of the proposed studies and this dictated the criterion on which further trials of the optimization of protein expression levels ceased. Attempts. to obtain highly purified protein undoubtedly resulted in low yield, but quality rather than quantity was more important for the proposed studies.
  • TrkA, TrkB and TrkC proteins produced as MBP-fusion proteins, was attempted in E. coli with an expression vector that resulted in a His-tag at the C-terminus of the recombinant protein. No reasonable levels of soluble protein was obtained for any of the His-tagged proteins. It appears that the presence of either a large soluble protein such as MBP, in the case of the Trk
  • MBP-fusion proteins or a high level of glycosylation on the 13 potential glycosylation sites of the Trk proteins produced in Pichia pastoris, is required for soluble protein production. Removal of the MBP from the Trk fusion proteins, results in loss of solubility (Rainer Schneider, private communication). Furthermore, additional attempts to produce the MBP-fusion constructs as His-tagged proteins, resulted in negligible or no soluble protein production (Rainer Schneider, private communication) 2 .
  • Nerve Growth Factor for the studies of the interaction of NGF with Trk receptors, was purified from mouse submaxillary glands (Pel-freeze).
  • Trk receptors full length and domains
  • association and dissociation data was analyzed with Fastfit software (Affinity Sensors) and Sigmaplot software (SPSS). All data was fitted to monophasic and biphasic kinetics algorithims. The goodness of fit for the data to both mono and biphasic kinetics was assessed by the quality of the fit of the association and association curves to the algorithims and by the F statistic. Quality of the data fit was also determined by the 2 value.
  • After accumulation of association and dissociation rates for each neurotrophin concentration data was plotted to give the linear response in arc sees versus the concentration of neurotrophin. A binding curve was also plotted from the same data. An example of data analysis at one NGF concentration for TrkA MBP-C1LRR at 15°C is shown below. From an accumulation of such data, the plots of response (in arc sees) versus neurotrophin concentration are produced. Calculations are shown below. Graphs are shown in Figure 15.
  • Probability of second phase being insignificant is 0 Additional association single phase, association double phase, dissociation single phase, dissociation double phase position curves and error curves not shown.
  • the F Test (the variance ratio distribution) is defined as the defined as the ratio of two independent chi-squared random variables, each divided by it number of degrees of freedom.
  • the chi-squared values for the data fitted to monophasic and biphasic kinetics are compared.
  • the F test is used to assess the monophasic or biphasic nature of the measured K on rates.

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Abstract

L'invention porte sur des tyrosine kinases réceptrices et les ligands à cet effet modulant la croissance, la différentiation et la survie des cellules. Elle porte particulièrement sur la famille Trk des tyrosine kinases réceptrices connues pour médier les activités des neurotrophines, les protéines Trk étant également des proto-oncogènes connus; sur des compositions de petites molécules modulant la fixation et l'activité de la protéine Trk; et sur des procédés de criblage de petites molécules pouvant se fixer à la protéine Trk en se fixant à un ou plusieurs de ses déterminants de fixation à la neurotrophine.
PCT/US2001/021472 2000-07-05 2001-07-05 Modulateurs de l'activite de la proteine trk, et compositions et methodes d'utilisation WO2002003071A2 (fr)

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WO2005025514A3 (fr) * 2003-09-10 2005-08-11 King S College London Composes qui modulent la croissance neuronale et leurs utilisations
US20060239966A1 (en) * 2003-10-20 2006-10-26 Tornoee Jens In vivo gene therapy of parkinson's disease
WO2008015942A1 (fr) * 2006-07-31 2008-02-07 Hisamitsu Pharmaceutical Co., Inc. Agent thérapeutique contre le neuroblastome, procédé de criblage à la recherche de l'agent thérapeutique et procédé de détermination du pronostic d'un neuroblastome
WO2019108983A1 (fr) * 2017-11-30 2019-06-06 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus trkb humanisé
WO2022009992A1 (fr) * 2020-07-10 2022-01-13 国立大学法人東京大学 Peptide cyclique, complexe peptidique et composition médicamenteuse contenant ledit peptide cyclique et/ou ledit complexe peptidique

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WO1999033482A1 (fr) * 1997-12-31 1999-07-08 University Of Utah Research Foundation Utilisation de peptides alpha-conotoxines

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