EP1049715A2 - Antagonisten der rhoprotein familie und ihre verwendung zum blockieren der inhibierung des neuritenwachstum - Google Patents

Antagonisten der rhoprotein familie und ihre verwendung zum blockieren der inhibierung des neuritenwachstum

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EP1049715A2
EP1049715A2 EP98951138A EP98951138A EP1049715A2 EP 1049715 A2 EP1049715 A2 EP 1049715A2 EP 98951138 A EP98951138 A EP 98951138A EP 98951138 A EP98951138 A EP 98951138A EP 1049715 A2 EP1049715 A2 EP 1049715A2
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rho
growth
family members
rho family
cells
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French (fr)
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Lisa Mckerracher
Maxime Lehmann
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Universite de Montreal
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4722—G-proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to the regulation of growth of neurons in the Central Nervous System.
  • CNS central nervous system
  • injuredneurons do otregenerate theirtransected axons.
  • An important barrier to regeneration is the axon growth inhibitory activity that is present in CNS myelin and that is also associatedwith theplasmamembrane of oligodendrocytes, the cells that synthesize myelin in the CNS (see Schwab M.E., etal, (1993) Ann. Rev. Neurosci, 16, 565-595, forreview) .
  • CNS myelin The growth inhibitory properties of CNS myelin have been demonstratedin anumber of differentlaboratories by awide variety of techniques, including plating neurons onmyelin substrates or cryostat sections of white matter, and observations of axon contact with mature oligodendrocytes (Schwab, M.E., et al, (1993) Annu. Rev. Neurosci. 16, 565-595). Therefore, it is well documented that adult neurons cannot extend neurites over CNS myelin in vitro.
  • Myelin inhibits axon growth because it contains atleastseveraldifferentgrowth inhibitory proteins. Ithas been well documentedby us and by others thatmyeHn-associatedglycoprotein (MAG) has potent growth inhibitory activity, both in vitro and in vivo (McKerracher, L., et al, (1994) Neuron 13. 805-811; Mukhopadhyay, G, et al, (1994) Neuron 13, 805-811; Li, M., et al, (1996) /. Neurosci. Res. 46, 404414; Schafer,M., etal, (1996) Neuron l ⁇ , 1107-1113.).
  • MAG myeHn-associatedglycoprotein
  • myelin-derivedinhibitors there are also other growth inhibitory molecules expressed in the d tmammahanCNS.
  • Tenacinisagrowthinljibitoryproteinthatisexpressedinsomeurmyelinated regions of the CNS (Bartsch,U., et al, (1994) J. Neurosci. 14, 4756 -4768) and after lesion tenascin is expressedby astrocytes that border the lesion site (Aj emai and David ( 1994) J. Comp.Neurol 34*0. 233 -242) .
  • growth inhibitory proteins that are proteogly cans are expressedby reactive astrocytes , and these proteins form aba ⁇ ier to regeneration at the glial scar (McKeon and Silver ( 1995) Exp. Neurol 136, 32 - 43).
  • S ome embryonic neurons are not sus ceptible to MAG (Mukhopadhyay, G., etal, ( ⁇ 994) Neuron tt, 805-81 l),butmost embryonic eurons are inhibitedbytheothermyelin inhibitors (Schwab, M.E., etal, (1993) Ann. Rev. Neurosci, 16, 565-595). Therefore, in the cases when axons are able to extend onmyelin, signalingthroughintracellularpathways may play animportantrole in stimulating, or blocking the inhibition of axon growth.
  • laminin is able to stimulate rapid neurite growth (Kuhn, T.B., et al, (1995) Neuron U, 275-285), and we have documented that when laminin is present in sufficient concentration, neurites can extend directly onmyelin substrates . These fmdkgssiiggestthepossibihtyt ⁇ for laminin, is sufficientto allow axon growth onmyelin. Similarly,ithasbeendocumentedthatwhentheadhesionmor culeLl is express ed ectopically on astrocytes, it can partially overcome theirnon-permissive substrate properties (Mohajeri, M.H., et al, (1996) Eur. J.
  • Neurosci _8, 1085-1097 neurons can, under appropriate conditions, grow axons oninhibitory substrates, suggesting that the balance of positive to negative growth cues is a critical determinant for the success or failure of axon regrowth after injury.
  • Growth inhibitory proteins typically cause growth cone collapse, a process that causes dramatic rearrangementstothegrowthconecytoskeleton(Bandtlow,C.E.,etal.,(1993) Science259.80-83; Fan, J., etal, (1993) J. CellBiol U ⁇ , 867-878; Li, M., etal., (1996) J. Neurosci. Res. 46, 404-414).
  • One family of proteins that has been imphcatedin receptor-mediated signaling to the cytoskeleton is the small . GTPases oftheRhofamily(Hall,A. (1996) ⁇ r ⁇ . Rev. CellBiol 10,31-54).
  • Rho family members that include Rho, Rac and cdc42, affect adhesion, actin polymerization, and ⁇ eformation of 1- ⁇ mempodiaandf ⁇ lopoda- which -rre all process importanttomotiUty(Nobes,C.D. andHall,A.R. (1995) Cell , 53-62.).
  • Rho Rho
  • Rac Rho
  • cdc42 cdc42
  • Rho might play arole within the my elin-derived growth inhibitory system has been studied (Jin, Z. andStrittmatter, S.M. (1997) /. Neurosci. 1 7, 6256-6263). It was concluded, however, that the inhibitory effects of myelin are not mediated by Rho family members.
  • Thepresentinvention relates to antagonists andinhibitors to members of the Rho family of proteins and diagnostic, therapeutic, andresearchuses for each of these aspects.
  • members oftheRho family of proteins serve as atherapeutic target to foster regrowth of injured or degenerating axons inthe
  • apreferred embodiment relates to antagonists and inhibitors of members of the Rho family of proteins and their use as ameans ofblocking a common signalingpathway usedby the diverse growth inhibitory molecules .
  • the antagonists andinhibitors may be mutated forms of Rho andbiologically active (Rho family-inhibitory) fragments, peptides, C3 andbiologically active (Rho family-inhibitory) fragments, or small molecules such as Y-27632.
  • Rho family member proteins can be used to design small molecules that antagonize and inhibit Rho family proteins, to blockinhibition of neurite outgrowth.
  • Rho family members can beusedto design antagonist agents that suppress the myelin growth inhibitory system. These antagonist agents can be used to promote axon regrowth and recovery from trauma or neurodegenerative disease.
  • This invention provides for the use of Rho, or proteins related to Rho as therapeutic targets for agents designedtoblockgrowth ibitionbymyelinormyehnproteins.
  • the therapeutic agent or antagonist can be small molecules, proteins or peptides, or any agent that binds to Rho or its family - members to inactivate this pathway.
  • Rho regulatory pathway involves the GDP/GTP exchangeproteins(GEPs).
  • Rho has two interconvertible forms, GDP-boundinactive, and GTP-boundactive forms.
  • the GEPs promote the exchange of nucleotides and thereby constitute targets for regulating the activity of Rho.
  • GDP dissociation inhibitors inhibitthe dissociation of GDP fromRho, and thereby prevent the binding of GTP necessary forthe activation of Rho. Therefore, GDIs are targets for agents thatregulate Rho activity.
  • the GTP-bound active Rho can be convertedto the GDP-found inactive form by a GTPase reaction that is facilitated by its specific GTPas e activating protein (GAP) .
  • GAP GTPase activating protein
  • Another embodiment pertains to the use of GAPs as targets forthe regulation of Rho activity. Such inhibitors could block exchange of the GTP/GDP cycle of Rho activation/inactivation.
  • Rho is foundinthe cytoplasm complexedwith a GTPase inhbitingprotein(GDI). Tobecomeactive,Rhobinds GTPandistranslocatedtothemembrane.
  • GDI GTPase inhbitingprotein
  • Yet another embodiment pertains to the observation that abacterialmon-ADP ribosyltransferase, C3 transferase, ribosylates Rho to inactivate the protein.
  • this embodiment pertains to the use of C3 transferaseto inactivate Rho andstimulate axon growth.
  • other bacterial toxins such as toxins A and B , with related Rho-inhibitory activity are considered to be within the scope of this invention.
  • Rho protein can create dominantnegative Rho, which can interfere with the biological activity of endogenous Rho inneurons.
  • yetafiirther embodimentof this invention pertains to the use of dominant negative forms of Rho, used to inactivate Rho, to foster axon growth.
  • an assay methoduseful to identify Rho family member antagonist agents that suppress inhibition of neuron growth comprising the steps of: a) culturingneurons onagrowthpermissivesubsfratethatincorporates a growth-inhibiting amount of a Rho family member; and b) exposingthe culturedneurons of step a) to acandidate Rho family member antagonist agentin an amount and for a period sufficient prospectively to permit growth of the neurons; thereby identifying as Rho family antagonists the candidates of step b) which elicitneurite outgrowth from the cultured neurons of step a).
  • amethod to suppress the inhibition ofneuron comprising the steps of delivering, to the nerve growth environment, aRho family antagonist in an amount effective to reverse said inhibition.
  • kinases activated by Rho are antagonist candidates.
  • compounds such as Y-27632 (U.S. PatentNo.04997834), thatblockRho-associated kinase activity, thereby inactivating the Rho signaling pathway are also embodiments of thi- invention.
  • the use other compounds within this family of compounds as describedinU.S. PatentNo.04997834 that inhibit Rho kinase are also considered within the scope of this invention.
  • kit comprising components necessary to conduct the assay method useful to screen Rho family antagonist agents .
  • Figure 1 shows results oftreatmentwith C3 to stimulate neurite outgrowth on inhibitory MAGsubstrates.
  • Fig ⁇ e2showstheroleofintegrinskoverri ⁇ nggrowthM ⁇ ibiti
  • Theanti- ⁇ l integrin function blocking antibody, 3A3 was used to determine if integrin functionis necessary for laminin to override growminhibitionbymyelin or MAG.
  • Figure3 presents theresults of studies in whichPC12 cells transfectedwith dominantnegativeRhoextend short neurites on MAG substrates.
  • Mock-transfected PC12 cells (a,c,e) or cells transfected with dominant-negative Rho (b,d,f) wereplated on laminin (a,b) or MAG (c-f) .
  • MAG inhibits neurite outgrowth (c), but dominantnegative Rho cells spread on MAG andsome cells extend shortneurites (d).
  • Treatment with C3 further stimulates neurite outgrowth on MAG from both lines of cells (e,f).
  • FIG. 4 shows activation of Rho on MAG substrates .
  • Activated Rho is associated with the plasma membrane.
  • cells were grown in suspension or plated on MAG or collagen substrates. Two hours later the plasmamembranes were purified, the proteins separatedby SDS PAGE, and the proteins transferedto nitrocellulose and stainedwith Ponceau S .
  • Rho A was detected on the blots by immunoreactivity with anti- RhoA antibody. Immunoreactivity was strongest when cells were grown in suspension or when cells were plated on MAG. Therefore, Rho A is more active when cells are keptin suspension orplated on MAG than when plated on growth-permissive collagen.
  • Figure 5 shows treatment of retinal neurons with C3 stimulates neurite growth onpolylysine andMAG substrates. On nMAG substrates neurite growth is inhibited (a) , but after C3 treatment retinal neurons plated onnMAGsubstrates extendneurites (b). Growth of neurites fromretinalneurons plated onPLL(c). Bar, 50 ⁇ m.
  • Figure 6 demonstrates ADP-ribosylation of Rho by C3 detected in cultured cells.
  • PC12 cells orretinal neurons were cultured in the presence (+) or absence of C3 (-) for two days.
  • the cells were lysed, and 10 ⁇ g of protein from eachsample was separatedona 11% acrylamidegel.
  • Theproteins weretransfered to nitrocellulose, probed withmouse anti-RhoA antibody and anti-mouse-HRP antibody, andrevealed by a chemiluminescent reaction (top panel).
  • Themembranes werethenreprobedwithrabbbit anti-Cdc42 and anti-rabbitalkalinephosphatase andrevealed withNTB/BCTP colorreaction.
  • Treatment of cells with C3 resulats inanADP-ribosylation-inducedde ⁇ -easeinthemobility of RhoA.
  • Themobility of Cdc42 does not change with C3 treatment.
  • Figure 7 illustrates methods usedto study the effect of C3 on injured opticnerve.
  • Figure7a shows the optic nerve was removed from the sheath prior to crushing with 10.0 sutures (top) and C3 was applied in Gelfoam and Elvaxtubes (redbars) immediately following opticnerve crush (middle). Theretinal ganglion cell axons were detected by anterograde labeling with cholera toxin and timmunodetection of the cholera toxininlongitudinal sections of the opticnerve (bottom).
  • Figures 7c, 7d, 7e, and7f showtreatment of crushed optic nerve with C3 stimulates regenerative growth of retinal ganglion cell axons.
  • themaximumnumberof axons observedinasingle 14 ⁇ msection was countedatdifferentdistancesfromthesiteofthecrushEach point represents one animal, but animals with growth past 500 ⁇ m are also represented at the shorter distances. Largenumbers ofregenerating fibers (>10/section)wereobservedto cross the lesion after C3 treatment compared to treatment with PBS.
  • This invention arises frommediscovery atRhofamilymembers are key molecules in regulating inhibition by myelin proteins, andby MAG
  • this invention provides eadvantageofiden inganintracellular target, Rho family members, for all of the multiple inhibitory proteins that must be inactivatedto allow for growth on myelin.
  • This invention provides antagonists of Rho family members, that permit axon regeneration.
  • the method of this invention provides for inactivation of Rho family members , thereby stimulatingneurite growth on growth inhibitory substrates. Therefore, antagonists thatinactivate Rho family members in vivo allow axon regeneration in the injured or diseased CNS.
  • This invention provides for the use of Rho, or proteins related to Rho as therapeutic targets for agents designedto block growthinhibition by myelin or myelin proteins.
  • One embodiment pertains to the use of
  • Rho antagonists that foster axon regeneration in the central nervous system.
  • the therapeutic agent or antagonist can be small molecules, proteins or peptides, or any agent that binds to Rho or its family members to inactivate this pathway.
  • Another embodiment pertains to the use of the Rho regulatory pathway as atargetforRho antagonists. This pathway involves the GDP/GTP exchangeproteins(GEPs). Rho has two interconvertible forms , GDP-bound inactive, and GTP-bound active forms . The GEPs promote the exchange ofnucleotides and thereby constitute targets for regulating the activity of Rho.
  • GDP dissociation inhibitors inhibit the dissociation of GDP fromRho, and thereby prevent the binding of GTP necessary forthe activation of Rho. Therefore, GDIs are targets for agents thatregulate Rho activity.
  • the GTP-boundactiveRho canbe convertedto the GDP-boundinactive form by a GTPase reaction that is facilitated by its specific GTPase activating protein (GAP).
  • GAP GTPase activating protein
  • Another embodiment pertains to the us e of GAPs as targets for the regulation of Rho activity .
  • Another embodiment pertains to the factthat Rho is found inthe cytoplasm complexedwith a GTPase inhibiting protein (GDI).
  • Rho binds GTP andis translocated to the membrane.
  • agents that promote Rho bindingto GDI, andblockRho bindingto theplasmamembrane are also considered within the scope of this invention.
  • Yet another embodiment pertains to the observation that abacterial ADP ribosylfransferase,C3lransferase J ribosylates Rho to inactivate totheuseofC3transferasetoinactivateRhoandstimulateaxongrowth.
  • otherbacterialtoxins such as toxins A andB , withrelatedRho-inhibitory activity are considered to be within the scope of this invention.
  • Rho protein can create dominantnegative Rho, which can mterfere with me biologic-d activity of endogenous Rhoinneurons.
  • etafurtherembodimentofthis invention pertains to me use of dominantnegative forms of Rho, used to inactivate Rho, to foster axon growth.
  • Antagonists whichmay beused in accordance with thepresentinvention include without limitation, one or more Rho family members fragment, a derivative of Rho family members or of a Rho family members fragment, an analog of Rho family members or of a Rho family members fragment or of said derivative, and apharmaceutical agent, and is further characterizedby the property of suppressing Rho family members-mediated inhibition of neurite outgrowth.
  • Preferred antagonists include: mutated forms ofRho, such as Rho wherein the effector domain, A-37, has been mutatedto prevent GTP .
  • GDIs GDP dissociation inhibitors
  • RhoGDIfromHomo sapiens Rho GDP-dissociationinhibitor 1
  • Rho family members in accordance with thepresent invention is not limitedto Rho family members or its derivatives, but also includes the therapeutic application of all agents, referredherein as pharmaceutical agents, which alter the biological activity of the Rho family members protein such that inhibition of neurons or their axon is suppressed.
  • effective amount or “growth-promoting amount” refers to the amount of pharmaceutical agent required to produce a desired antagonist effect of the Rho family members biological activity. The precise effective amountwill vary with the nature of pharmaceutical agentused andmay be determined by one or ordinary skill in the art with only routine experimentation.
  • the Rho family of proteins comprises, but is not limited to rho, rac, cdc42 and their isotypes, such as RhoA, RhoB, RhoC, as well as Rho-associatedkinase that are expressedin neural tissue.
  • Othermembers ofthe Rho family that are determined andwhose inhibition of activity allows forneurite outgrowth are comtemplated to be part of this invention.
  • Katoh, H., et al., J. Biol. Chem, 273:2489-2492, 1998 vanLeeuwen, F., etal, J. CellBiol, 139:797-807, 1997; Matsui etal., EMBOJ. 15:2208-2216, 1996; Amanoetal., Science, 275:1308; Ishizak ⁇ ,T. etal,(1997)FEBSLett, 404: 118-124).
  • Rho family member biological activity refers to cellular events triggeredby, beingofeitherbiochemicalorbiophysicalnatui-e.
  • Thefollowingj ⁇ tisprovided,withoutHmitation discloses some ofthe known activities associated with contact-mediated growth inhibition of neurite outgrowth, adhesion to neuronal cells, andpromotion of neurite outgrowth fromnewbom dorsalroot ganglion neurons.
  • biologically active refers to apolypeptide that is able to produce one ofthe functional characteristics exhibitedby Rho family members or its receptors describedherein.
  • biologically activeproteins are those that demonstrate inhibitory growth activities central nervous system neurons. Such activity may be assayed by any method known to those of skill in the art.
  • C3 refers to C3 ADP-ribosyltransferase, a specific Rho inactivator .
  • a pref erredrepres entative example is C3 ADP-ribosyltransferase, a23 KDaexoenzyme secreted from certainstrains oftypes Cand D fromClostiidiumbotulinum, which specifically ADP-ribosylates therho family of these GTP-binding proteins.
  • This ADP-ribosylation occurs at aspecific asparagine residue in their putative effector domain, andpresumably interferes with their interactionwithaputative effector molecule downstream insignal transduction.
  • Rho family members can affect growth inhibitory protein signals in myelin
  • Rho family member antagonists will be inhibitors of GTPase activity.
  • the GTP/GDP cycle of Rho family members activation/inactivation is regulated by a number of exchange factors .
  • Compounds that block exchange, thereby inactivatingRho family members are preferred embodiments of this invention.
  • Rho family member antagonist candidates comprising fragments, analogs andderivatives of ho family members. Sequences for Rho family members are known, such as those described: Chardin,P., etal., (1988) Nucleic Acids Research, 16:2717; Yeramian, etal., (1987) Nucleic Acids Research, 15: 1869). Such candidates mayinterferewithl ofam ⁇ ymembers- mediated growth inhibition as competitive butnon-functional mimics of endogenous Rho family members.
  • Rho family members fragments can be produced either by peptide synthesis or by recombinantDNA expression of either atruncated domain ofRho family members , or of intact Rho family members couldbe preparedusing standard recombinantprocedures, that can then be digested enzymically in either arandom or a site-selective manner.
  • Analogs of ho family members or Rho family members fragments can be generated als o by recombinant DNA techniques or by peptide synthesis , and will incorporate one or more, e.g. l-5,L-orD-aminoacidsubstitutions.
  • Rho family members, Rho family members fragments and Rho family members analogs can be generated by chemical reaction ofthe parent substance to incorporate the desired derivatizing group, such as N-teiminal, C-terminal and intra-residue modifying groups that have the effect of masking or stabilizing the substance or target amino acids within it.
  • desired derivatizing group such as N-teiminal, C-terminal and intra-residue modifying groups that have the effect of masking or stabilizing the substance or target amino acids within it.
  • candidate Rho family member antagonists include thosethat are derivedfromadetermination ofthe functionally active region(s) of aRho family member.
  • Antibodies ca be preparedusing techniques known inthe against epitopes in Rho family members, which, when found to be function-blocking in z ' n vitro assays, canbeusedtomaptheactiveregions ofthe polypeptide as has been reported for other proteins (for example, seeFahrig, et al, (1993) Europ. J. Neurosci, 5, 1118- 1126; Tropak, et al, (1994) J. Neurochem., 62, 854-862).
  • Rho familymembersGTPases recognized by substrate molecules that are involvedminhibitionofneurite outgrowth.
  • synthetic peptides can be prepared to be assayed as candidate antagonists ofthe Rho family members effect.
  • Derivatives ofthese canbe prepared, includingthosewith selected amino acid substitutions to provide desirable properties to enhance their effectiveness as antagonists of the Rho family members candidate functionalregions of Rho family members can also be determinedby the preparation of altered forms ofthe Rho family members domains using recombinant DNA technologies to produce deletion or insertionmutants that canbe expressed in various cell types as chimericproteins.
  • the Rho family member antagonist is formulated as a pharmaceutical composition which contains the Rho family member antagonists an amount effective to suppress Rho family member-mediated inhibition of nerve growth, in combination with a suitable pharmaceutical carrier.
  • a suitable pharmaceutical carrier Such compositions are useful, in accordance with another aspect ofthe * invention, to suppress Rho family member-inhibitednerve growth inpatients diagnosedwith avariety of neurological disorder, conditions andailments of thePNS and the CNS where treatmentto increase neurite extension, growth, or regeneration is desired, e.g., inpatients with nervous system damage.
  • Rho familymembers antagonists include but are notlimitedto Strokes, Alzheimer's disease, Down's syndrome, Creutzfeldt- Jacob disease,kura, Gerstrnan-Sfra ⁇ sslersyndrome,SCTapie.
  • Rho family members antagonists may be usedto promote the regeneration of CNS pathways, fiber systems andtracts. In aparticular embodiment ofthe invention, the Rho family members antagonist is usedto promote the regeneration of nerve fibers over long distances following spinal cord damage.
  • the invention provides an assay method adapted to identify Rho family member antagonists, that is agents that block or suppress the growth-inhibiting action of Rho family members.
  • the assay is a tissue culture assay that measures neurite out-growth as a convenient end-point, and accordingly uses nerve cells that extendneurites when grown on apermissive substrate.
  • Nerve cells suitable in this regard include neuroblastoma cells ofthe NG108 lineage, such as NG108-15, as well as otherneuronal cell lines such as PC12 cells (American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 USA, ATCC Accession No.
  • the nerve cells for instance about 10 3 cells-microwell or equivalent, are cultured on a growth permissive substrate, such as polylysine or laminin, that is over-layed wimagrow -inhibiting amount ofRho family members .
  • a growth permissive substrate such as polylysine or laminin
  • the Rho family members incorporatedin the culture is suitably my elin- extractedRho family members, although forms ofRho family members otherthan endogenous forms can be usedprovided they exhibit the Rho family members property of inhibiting neuron growth when added to a substrate that is otherwise growth permissive.
  • candidate Rho family member antagonists i. e. , compounds thatblockthe growth-inhibiting effect ofRho fatnilymembers, are addedto the Rho f-imily membei'-containing tissue culture preferably in amount s ufficientto neutralize the Rho family member growth-inhibiting activity, that is between 1.5 and 15 ⁇ g of Rho family members antagonist per well containing a density of 1000NG108-15 cells/well cultured for 24 hr. inDulbecco' s minimal essential medium. After culturing for aperiod sufficient for neurite outgrowth, e.g.
  • Rho family member antagonists are those which elicit neurite outgrowth to astatistically significant extent, e.g. , in at least 50%, more desirably at least 60%, e.g. 70%, per 1,000 cultured neurons.
  • Rhofamilymemberantagonists have uses in diagnostics. Suchmolecules canbeusedinassaystodetect, prognose, diagnose, or monitor various conditions, diseases, and disorders affecting neurite growth extension, invasiveness, andregeneration. Alternatively, the Rho family member antagonists may be used tomonitortherapies for diseases andconditions which ultimatelyresultinneive damage; such diseases and conditions include but are not limited to CNS trauma, (e.g.
  • such moleculess may be usedto detect anincreaseinneurite outgrowth as an indicator of CNS fiberregeneration.
  • alteredlevels ofRho family members activity in apatient sample containing CNS myelin canbe adiagnosticmarkerforthepresence of amalignancy, including butnot limitedto ghoblastoma, neuroblastoma, andmelanoma, or a condition involvingnerve growth, invasiveness , or regeneration in a patient.
  • Rho family member antagonists include fragments ofRho family members that incorporate the ectodomain, including the ectodomainper se and other N- and/or C-terminally truncated fragments ofRho family members orthe ectodomain, as well as analogs thereof in which amino acids, e.g. from 1 to 10 residues, are substituted, particularly conservatively, and derivatives ofRho familymembers or Rho family members fragments in which theN- and/or C-terminal residues are derivatizedby chemicalstabilizing groups.
  • mutated forms ofRho family members are used as antagonists.
  • Rho with amutated effector domain, A-37 which prevents GTP exchange.
  • Rho protein that create dominate negative Rho which can interfere with the biological activity of endogenous Rho inneurons are consideredas antagonists within the scope of this invention to inactivate Rho, thereby fostering growth of neurons.
  • GTPase activating protein which f acilitates the conversion ofthe GTP-bound active Rho to the GDP-bound inactive form forms the target for regulation ofRho activity.
  • GAP GTPase activating protein
  • Rho antagonists of this invention compounds that affect Rho bindingto the plasmamembrane, thereby decreasing the activity of ho are also considered Rho antagonists of this invention.
  • thetarget design is basedontiieknowledge that RhoisfoundinthecytoplasmcomplexedwithaGTPaseinhibiting protein (GOT). To become active, Rho binds GTP andis translocatedto the membrane.
  • GTP RhoisfoundinthecytoplasmcomplexedwithaGTPaseinhibiting protein
  • agents that promote GDI activity andblockRho bindingto the plasmamembrane would decrease Rho activity, thereby serving as Rho antagonists that would permit neuron growth.
  • candidate Rho family members antagonists includespecificregions ofthe Rho family members molecule, and analogs or derivatives of these. These canbe identified by using the same technologies described above for identification ofRho family members regions that serve as inhibitors of neurite outgrowth.
  • Rho family members related derivatives, analogs, and fragments ofthe invention can be producedby various methods known in theart. Themanipulationswhichresultin their production can occur at the gene orproteinlevel.
  • Rho family members-encoding DNA canbemodifiedby any of numerous strategies known inthe art (Maniatis et al. , Molecular Cloning, ALaboratoiy Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. , 1982), such as by cleavage at appropriate sites with restriction endonuclease(s), subjected to enzymatic modifications if desired, isolated, and ligated in-vitro.
  • Rho family members-encoding gene can be mutated in-vitro or in-vivo for instance in the manner applied for production ofthe ectodomain, to create and/or destroy translation, initiation, and/or termination sequences, orto create variations in codingregions and or formnew restriction endonuclease sites or destroy preexisting ones, to facilitate further in-vitro modification.
  • Any technique formutagenesis jmownintheartcabbeuseo ⁇ includingbutnotUmitedto, in-vitro site directedmutagenesis Hutchinson, et al, (1978) J. Biol. Chem. 253, 6551
  • TABTM linkers Pharmacia
  • Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, andintranasal routes, and transfusion into ventricles or asite of operation (e.g. for spinal cordlesions) ortumorremoval.
  • cells secreting Rho family members antagonist vity for example, andnot by way oflimitation,hybridoma cells encapsulatedinasuitablebiologicalmembranemay be implanted in a patient so as to provide a continuous source of Rho family members inhibitor.
  • antagonists, derivatives, analogs, inhibitors ofRho familymembers can be used in regimens where anincreaseinneurite extension, growth, or regeneration is desired, e.g., inpatients with nervous system damage.
  • Patients suffering from traumaticdis orders including butnot limitedto spinal cord injuries, spinal cordlesions, or other CNS pathway lesions), surgicalnerve lesions, damage secondary to - infarction, infection, exposure to toxic agents, malignancy, paraneoplasticsyndromes, orpatients with various types of degenerative disorders ofthe central nervous system can be treated with such inhibitory protein antagonists.
  • Such disorders include but are not limited to Alzheimer's Disease, Parkinsons' Disease, Huntington's Chorea, amyotrophiclateralsclerosis,progressivesupranuclearpalsy and other dementias.
  • Such antagonists may be usedto promote the regeneration of CNS pathways, fiber systems and tracts .
  • Administration of antibodies directedto an epitope of, (or the binding portion thereof, or cells secreting such as antibodies) can also beusedtoinMbitRhofamtiymembersprotem function in patients .
  • antibodies directed to Rho family members may be usedto promote the regeneration of nerve fibers over long distances following spinal cord damage.
  • Various delivery systems are known andcanbeusedfor delivery of antagonists or irM ⁇ tor ofTho family members andrelatedmolecules, e.g., encapsulationinliposomes orsemipeimeablemembranes, expression by bacteria, etc.
  • Linkage to ligands such as antibodies can be used to target myelin associated protein-relatedmolecules to therapeutically desirable sites in vivo.
  • Methods of introduction include but are not mitedto intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, andintranasal routes, and infusion into ventricles or a site of operation (e.g. for spinal cord lesions) or tumor removal.
  • any method which results in decreased synthesis ofRho family members may" be usedto diminish theirbiological function.
  • agents toxicto the cells which synthesize Rho family members and/or its receptors e.g. oligodendrocytes
  • This example demonstrates in vitro evidence that Rho family members are respons ible for regulating the . neuronal response to MAG.
  • this demonstrates thatthesmallGTPaseRhoregulatesthe responsetoMAG PC12 cells were plated onpolylysine (PLL), 1- ⁇ minin, or MAG substrates and exposed toNGFtostimulateneurite growth.
  • PC12 cells differentiatedneurites onPLLandlamininsubstrates,but on MAG substrates the cells remained rounded and did not grow neurites.
  • N19 RhoAPC12 cells were ableto extend neurites when plated on MAG substrates . Therefore, the inactivation ofRho stimulates neurite outgrowth and allows neurite extension on MAG substrates . These results imphcate Rho in signaling growth inhibition by MAG
  • PC 12 cells from three different sources: from Dr. Phil Barker (Montreal Neurological Institute); from the ATCC (obtained from W. Mushinsky, McGill), andfromGaborTigyi, (University of Tennessee) andwe found that all lines of cells were inhibitedby bothmyelin andMAG.
  • PC12 cells were grown in Dulbecco'smodifiedeagle's medium (DMEM) with 10 % horse serum and 5 % fetal bovine serum.
  • DMEM Dulbecco'smodifiedeagle's medium
  • PC 12 cells stably transfectedwith constitutively active and dominant negative RhoA constructs were kindly providedby Dr. G. Tigyi (University of Tennessee, Memphis, USA) .
  • TransfectedPC12 cell lines weremaintainedin the growthmedium containing 400 mg/L G418.
  • cells were plated on appropriate substrates in DMEM with 1 % fetal bovine serum and 100 ng/ml nerve growth factor.
  • PC12 For experiments on mixed subsfrat ⁇ (lamininMAGorlamimn myehn),PC12were ⁇ latedinDMEMwit ⁇ in the presence or the abs ence of 50 ⁇ g/ml of an irrelevant antibody or of a purified function blocking antibody (clone 3 A3) against the rat ⁇ 1 ⁇ 1 integrin (a gift of S .Carbonetto) .
  • PC12 cell differentiation experiments were done in 96-well plates in duplicate, andeachexperimentwasrepeatedaminimumof three times.
  • the cell suspension was placed on an iso-osmotic percoll gradientwith 60% and35% percoll, centrifugedfor 15' at2300 ⁇ m, and the interface between the 60% and35% ⁇ ercollwas collected.
  • Cells werewashedonce, andresuspendedinDMEMwitii 10% FBS, vitamins, andpenicillin/streptomycinin the presence or absence of 20 ⁇ g/ml C3 transferase.
  • Cells were placed in 4-chamber, chamber slides coated withpoly-1-lysine or laminin andtreatedwith spots of MAG ormyelin. 200,000 cells per chamber were plated.
  • Poly-1-lysine was obtained from Sigma (St. Louis, Mo). Laminin was prepared from EHS tumors (Paulsson and Lindblom ( 1994). Cell biology: A laboratory handbook, Academic Press, pp589-594) and collagen fromrattails (Greene, etal. (1987) Meth. Enzymology 147,207-216). Myelinwas made from bovine brain corpus callosum, and native MAG was purified from myelin after extraction in 1% octylglucoside and separation by ion exchange chrOmatography (McKerracher, L , et al. , ( 1994) Neuron 13, 805-811).
  • This native MAG has some additional proteins, including some tenascin (Xiao, Z., et al., (1997) Neurosci. Abstr. 23, 1994). Recombinant MAG was made in baculovirus as described (McKerracher, L., et al, (1994) Neuron 13. 805-811).
  • Test substrate were prepared as uniform substrates in96-well plates or4-chambered slides, or as spots on 18 mm glass coverslips .
  • poly-L-ly sine was coated by incubation of 100 ⁇ g/ml for 3 hours at 37°C, andthewells or coverslips were washedwith water anddried.
  • Laminin substrates were prepared by incubating 25 ⁇ g/ml laminin on poly-L-lysine coated dishes for 3 hours at 37°C.
  • SolidMAG ormyelin substrates werepreparedbydrymgdownMAGovemight ⁇ orincubatingalOmg/ ⁇ jimyelinsolutionfor 3 hours onpolylysine coatedsubstrates.
  • PC12 cells were visualized by phase contrast microscopy, or following labelling with the lipophilic fluorescent dye, Dil (McKerracher, L. , etal, ( 1994) Neuron 13, 805-811).
  • Granule cells were visualized by immunocytochemistry. Following 12-24hours in culture, cellswerefixedfor30'atroomtemperature in4%paraformaldehyde,0.5% glutaraldehyde, 0.1 M phosphate buffer. Following fixation, cellswere washed3 X5' withPBS andthen blocked for 1 hour at room temperature in 3 %BS A, 0.1%Triton-X 100. Granulecellculhireswei-emcubatedovernightwithapolyclonalanti-rM ⁇ to label MAG spots. The MAG antibody was detected using anFITCconjugatedsecondaryantibody. Rhodamineconjugatedphalloidin as diluted 1:200 with thesecondary antibody to label granide cell -ictin filaments.
  • TheplasmidpGEX2T-C3 coding forthe GST-C3 fusion protein was obtained from A. Hall (London). Recombinant C3 was purified as described by Dillon and Feig (Met. Enzymology, ( 1994), 256, pp 174- 184). After fusionprotein cleavage by thrombin,thrombin was removedbyincubatingtheproteinsolution 1 hour onicewith lOO ⁇ lof p-aminobenzamidine agarose-beads (Sigma). The C3 solution was desalted onPDIO column (Pharmacia)withPBS, andsterilizedti ⁇ iOughaO.22 ⁇ m filter.
  • C3 concentration was evaluatedby Lowiy assay (DC protein assay, Bio-Rad) and toxin purity was controlledby SDS-PAGE analysis.
  • C3 transferase was scrape loadedinto the cells before plating on appropriate substrates.
  • Cells were grown to confluenceinse mcontainingmediain ⁇ well plates.
  • Cells werewashedonce withscraping buffer (114mMKCl, 15mMNaCl, 5.5mMMgCl 2 , 1 OmM Tris-HCl). Cells were then scrapedwith arubber policeman into 0.5 ml scraping buffer in the presence or absence of 20 ⁇ g/ml C3 fransferase.
  • the cells were pelleted, and resuspended in 2 ml DMEM, l%FBS, and50ng/mlnervegrowthfactorbefore ⁇ latrng. 10 ⁇ g/ml C3 was addedto scrape loaded cells. Cells were differentiated for 48 hours then fixed in 4 % paraformaldehyde, 0.5 % glutaraldehyde, 0.1 M P0 4 buffer.
  • PC12 cells were collected andresuspendedinDMEM, 0.1 %BSA, 50ng/mlNGF, then plated on 100 mm dishes coated with collagen or MAG, or left in suspension. Two hours later, cells were washed with ice cold PBS + protease inhibitors (1 ⁇ g/ml aprotinin, 1 ⁇ g/ml leupeptin, 1 ⁇ g/ml antipain, 1 ⁇ g/ml pepstatin). Cells were then scraped into SmlPBS+proteaseinhibitors, and the cells were pelleted and washedwith PBS ⁇ protease inhibitors.
  • the cell pellets were mechanically homogenizedby 25 strokes in aglass-teflonhomogenizer,thehomogenatecenmTugedfor20minat8,000 ⁇ m,andthecell debris in the pellet was discarded. The supernatant was centrifugedfor 1 hour at 100,000 xg to separate membrane and cytosolic fractions. Membrane pellets were washed 1 X with PBS + protease inhibitors and resuspended in PBS with0.5%SDS, and 50-100 ⁇ gofmembrane protein was analyzedby SDS-PAGE on 12 % gels . Gels were transferred to Protran nitrocellulose membrane and stained with Ponceau S .
  • Rho A antibody was detected by using an alkaline phosphatase conjugated secondary antibody and an alkaline phosphatase detection kit (Gibco-BRL).
  • PC12 cells typically extend neurites in respons e to NGF, but when plated on myelin substrates the cells remainroundanddonotextendneurites(Moskowitz,P.F.,etal.,(1997) J. NeurosciRes. 34, 129-134.) (Fig.2).
  • PC12 cells plated on substrates ofMAG alsoremainedrounded(Fig.1), a finding in contrastto areportthatPC12 cells are not responsive to MAG (Bartschj U., et al, (1995) Neuron ⁇ 5_, 1375-1381).
  • Laminin is known to override completely, growth inhibition ofNGl 08 cells by myelin (David, S . , et al. , (1995) J. Neurosci Res. 42, 594-602). Similarly, we found that PC12 cells areableto extendneurites onmixedmyelinandlamininsubstrates or on mixed laminin/MAG substrates (Fig.2). To determine if signalling t-iroughintegrins is responsible for overriding growth inhibition by myelin, weusedtheintegrin function blocking antibody 3A3raisedagainstthe ⁇ l subunit extracellular domain.
  • V 14GRhoA constitutively active RhoA
  • PC 12 cells transfectedwith dominantnegativeRhoA(N19TRhoA) wereexaminedfortheir ability to extendneurites on differenttestsubstrates.
  • Cellswiththe constitutively activemutation V14GRhoA cells, differentiatedpoorly on all substrates, including poly-L-lysine andlaminin.
  • Thetreatment of the V 14GRhoA cells with C3 allowed the growth of some shortneurites on all ofthe test substrates, including MAG.
  • the growth substrate influences the cellular location ofRho
  • Rho is associated with the plasmamembrane when it is in an activatedstate, andit moves into the cytosolic fraction when it is in the GDP-bound inactive state.
  • the growth substrate influences the cellular localization ofRho, cells were either left in suspension or platedonMAGor collagensubstrates, and preparedmembranes from the cells twohours later. It was shown that Rho was principally localized in the cytosolic fraction when cells wereplated on collagen, agrowthpermissive substrate. However, Rho was associated witiitheplasmamembranewhen cells where grown in suspension andwhen cells were plated on MAG (Fig. 4).
  • EXAMPLE H IN VIVO DEMONSTRATIONS 1. Effect of C3 on cultured retinal neurons
  • the dissociated cells were plated on test substrates in the presence of 50 ⁇ g/ml BDNF in DMEM with 10% FBS, vitamins, andpenicillin/sfreptomycin in the presence or abs ence of 50 ⁇ g/ml C3 transferase. Neurons were visualized by fluorescent microscopy with anti- ⁇ lH tubulin antibody.
  • Retrograde labeling of RGCs from the superior colliculus (not shown), as well as anterograde labeling techniques (eg., Fig.7a) verified that RGC axons were effectively axotomized.
  • Fig.7a Gelfoam soaked with 2 mg/ml C3 was wrapped around the left optic nerve atthe crush site, andtwoElvaxtubes, each loadedwith20 ⁇ gofC3 were positioned for sustained slow release (Fig.7a) . Twelve animals were treated with C3 , and a further 8 animals were treated with PBS as controls .
  • Rho family members Not all ofthe myelin-derived inhibitory molecules are known to date, and less is known aboutthe neuronal receptors for growth inhibitory molecules.
  • MAG receptors Several different MAG receptors have been identified (Collins et al. 1997; Yang etal. 1996), and additional neuronal receptors to myelin inhibitors are likely to exist.
  • Targeting infracellular signaling mechanisms convergingto Rho rather than individual receptors may be the most practical way to overcome growth inhibition in vivo .
  • the advantage of inactivating Rho to stimulate regeneration is that axons can regenerate directly on the n ative terrain ofthe CNS , and thus may be more likely to find their natural targets.
  • Rho protein is concentrated atthe filopodial tips of growth cones in adhesion structures calledpoint contacts (Renaudin etal. 1998). Therefore, our in vitro results suggest the Rho signaling pathway is a key target for regulating growth cone motility and stimulating regeneration.

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US7442686B2 (en) 2001-04-12 2008-10-28 Bioaxone Therapeutique Inc. Treatment of macular degeneration with ADP-ribosyl transferase fusion protein therapeutic compositions
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US7795218B2 (en) 2001-04-12 2010-09-14 Bioaxone Therapeutique Inc. ADP-ribosyl transferase fusion variant proteins
US7829566B2 (en) 2001-09-17 2010-11-09 Werner Mederski 4-amino-quinazolines
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