Post-translationally modified neurotrophins

Abstract

The present invention describes that neurotrophins undergo post-translational modifications, and that these post-translational modifications mediate the pro-apoptotic and/or pro-neurite activity of neurotrophins. These post-translational modifications notably include nitration and the formation of conformationally-different dimers, as well as of abnormal oligomers, such as tetramers and octamers. The invention further relates to compounds that compete with such modified neurotrophins, as well as to compounds that binds to said modified neurotrophins. The invention thus provides useful agents for the treatment of the conditions or diseases involving a chronic pain and/or neuron loss.

1 : An inducer and/or stimulator of motor neuron apoptosis and/or of neurite outgrowth, which is:
 a modified neurotrophin in an isolated form, wherein at least one residue selected from Tyr and Trp residues comprises at least one nitro group; or   a conservative fragment of said isolated form of modified neurotrophin, wherein said conservative fragment has retained at least one residue selected from Tyr and Trp residues, and wherein said conservative fragment has retained at least one nitro group on said at least one residue selected from Tyr and Trp residues, and has retained a capacity of inducing and/or stimulating motor neuron apoptosis and/or neurite outgrowth; or   a conservative variant of said isolated form of modified neurotrophin or of said conservative fragment, wherein said conservative variant derives from said modified neurotrophin or from said conservative fragment by at least one amino acid substitution and/or deletion and/or addition, but has retained at least one residue selected from Tyr and Trp residues, and wherein said conservative variant has retained at least one nitro group on said at least one residue selected from Tyr and Trp residues, and has retained a capacity of inducing and/or stimulating motor neuron apoptosis and/or neurite outgrowth.   
     
     
         2 : An inducer and/or stimulator of motor neuron apoptosis and/or neurite outgrowth, which is:
 a modified neurotrophin, wherein said modified neurotrophin is prepared by post-translational nitrative modification of a native mature neurotrophin, by addition on said native mature neurotrophin of at least one nitro group on at least one residue selected from Tyr and Trp residues, and wherein said modified neurotrophin is in an isolated form; or   a conservative fragment of said isolated form of modified neurotrophin, wherein said conservative fragment has retained at least one residue selected from Tyr and Trp residues, and wherein said conservative fragment has retained at least one nitro group on said at least one residue selected from Tyr and Trp residues, and has retained a capacity of inducing and/or stimulating motor neuron apoptosis and/or neurite outgrowth; or   a conservative variant of said isolated form of modified neurotrophin or of said conservative fragment, wherein said conservative variant derives from said modified neurotrophin or from said conservative fragment by at least one amino acid substitution and/or deletion and/or addition, but has retained at least one residue selected from Tyr and Trp residues, and wherein said conservative variant has retained at least one nitro group on said at least one residue selected from Tyr and Trp residues, and has retained a capacity of inducing and/or stimulating motor neuron apoptosis and/or neurite outgrowth.   
     
     
         3 : The inducer and/or stimulator of  claim 1 , wherein said modified neurotrophin is a modified neurotrophin monomer. 
     
     
         4 : The inducer and/or stimulator of  claim 1 , wherein said modified neurotrophin is a modified neurotrophin oligomer. 
     
     
         5 : The inducer and/or stimulator of  claim 4 , wherein said modified neurotrophin is a modified neurotrophin dimer. 
     
     
         6 : The inducer and/or stimulator of  claim 4 , wherein said modified neurotrophin is a modified neurotrophin tetramer, or a modified neurotrophin hexamer, or a modified neurotrophin octamer. 
     
     
         7 : An inducer and/or stimulator of motor neuron apoptosis and/or neurite outgrowth, which is a mixture of:
 at least two modified neurotrophin oligomers selected from a modified neurotrophin dimer a modified neurotrophin tetramer, a modified neurotrophin hexamer and a modified neurotrophin octamer, wherein said at least two oligomers are of different oligomer species, and/or at least one modified neurotrophin monomer of  claim 3 , and at least one modified neurotrophin oligomer of selected from a modified neurotrophin dimer a modified neurotrophin tetramer, a modified neurotrophin hexamer and a modified neurotrophin octamer.   
     
     
         8 : The inducer and/or stimulator of  claim 7 , which comprises at least one modified neurotrophin tetramer, and/or at least one modified neurotrophin hexamer, and/or at least one modified neurotrophin octamer. 
     
     
         9 : The inducer and/or stimulator of  claim 1 , wherein said isolated form of modified neurotrophin does not comprise any non-nitrated pro-neurotrophin. 
     
     
         10 : The inducer and/or stimulator of  claim 1 , wherein said isolated form of modified neurotrophin does not comprise any non-nitrated mature neurotrophin. 
     
     
         11 : The inducer and/or stimulator of  claim 1 , which is in a pure form. 
     
     
         12 : The inducer and/or stimulator of  claim 1 , which is in a molecular configuration that does not impede its pro-apoptotic activity and/or its pro-neurite outgrowth activity. 
     
     
         13 : The inducer and/or stimulator of  claim 1 , wherein said neurotrophin is NGF, BDNF, NT-3 or NT-4. 
     
     
         14 : The inducer and/or stimulator of  claim 13 , wherein said neurotrophin is mature NGF. 
     
     
         15 : The inducer and/or stimulator of  claim 13 , wherein said neurotrophin is mature BDNF, mature NT-3 or mature NT-4. 
     
     
         16 : The inducer and/or stimulator of  claim 1 , wherein said at least one residue selected from Tyr and Trp residues that comprises at least one nitro group is a Tyr residue. 
     
     
         17 : The inducer and/or stimulator of  claim 16 , wherein said Tyr residue is Tyr52 or Tyr79 of murine NGF, or Tyr52 or Tyr79 of human NGF. 
     
     
         18 : The inducer and/or stimulator of  claim 1 , wherein said at least one residue selected from Tyr and Trp residues that comprises at least one nitro group is a Trp residue. 
     
     
         19 : The inducer and/or stimulator of  claim 18 , wherein said Trp residue is Trp99 or Trp21 or Trp76 of mouse NGF, or is Trp99 or Trp21 or Trp76 of human NGF. 
     
     
         20 : The inducer and/or stimulator of  claim 1 , wherein at least two of the Tyr and Trp residues of said neurotrophin bear a nitro group in which at least one nitrogroup is on each of said residues. 
     
     
         21 : The inducer and/or stimulator of  claim 20 , wherein said at least two residues are two Tyr residues, or a Tyr residue and a Trp residue. 
     
     
         22 : The inducer and/or stimulator of  claim 20 , wherein the number of Tyr and Trp residues of said neurotrophin that bear a nitro group is of at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine. 
     
     
         23 : The inducer and/or stimulator of  claim 1 , wherein all of the Tyr and Trp residues of said neurotrophin bear at least one nitrogroup. 
     
     
         24 : The inducer and/or stimulator of  claim 1 , wherein said modified neurotrophin is in a non-glycosylated form or in a glycosylated form. 
     
     
         25 : A non-conservative fragment of an inducer and/or stimulator of  claim 1 , or
 a non-conservative variant of an inducer and/or stimulator of  claim 1 , which derives from said inducer and/or stimulator by at least one amino acid substitution and/or deletion and/or addition,   wherein said non-conservative fragment or variant has lost the capacity of inducing and/or stimulating motor neuron apoptosis and/or neurite outgrowth.   
     
     
         26 : The non-conservative fragment or variant of  claim 25 , for use as an antigen to induce an immune antibody response for the therapy and/or palliation and/or prevention of the apoptosis of motor neurons and/or the outgrowth of neurite. 
     
     
         27 : The non-conservative fragment or variant of  claim 25 , for use as an agent for the treatment and/or palliation and/or prevention of pain and/or of a neurodegenerative disease or condition. 
     
     
         28 : An antibody that binds to at least one inducer and/or stimulator according to  claim 1 , without cross-reacting with the unmodified native neurotrophin, from which said inducer and/or stimulator derives. 
     
     
         29 : The antibody of  claim 28 , which is a monoclonal antibody. 
     
     
         30 : The antibody of  claim 28 , which is an antibody that binds to nitrated NGF. 
     
     
         31 : The antibody of  claim 28 , which is an scFv. 
     
     
         32 : The antibody of  claim 28 , for use as an agent for passive immunization therapy and/or palliation and/or prevention of the apoptosis of motor neurons and/or the outgrowth of neurite. 
     
     
         33 : The antibody of  claim 28 , for use as an agent for the treatment and/or palliation and/or prevention of pain. 
     
     
         34 : The antibody of  claim 28 , for use as an agent for the treatment and/or palliation and/or prevention of a neurodegenerative disease or condition. 
     
     
         35 : A ribosomal display system, which consists of at least one ribosome, to which at least one scFv of  claim 31 , as well as at least one mRNA encoding such a scFv, are attached. 
     
     
         36 : A method of screening for compounds that are capable of inhibiting and/or blocking the apoptotic activity that a inducer and/or stimulator of  claim 1  may have on motor neurons, and/or the neurite outgrowth effect that a inducer and/or stimulator of  claim 1  may have on sensory ganglia, wherein candidate compounds are screened for their capacity of binding to at least one antibody of that binds to at least one inducer and/or stimulator according to  claim 1 , without cross-reacting with the unmodified native neurotrophin, from which said inducer and/or stimulator derives, or to at least one ribosomal display system consisting of at least one ribosome, to which at least one scFv, as well as at least one mRNA encoding such a scFv, are attached, whereby such a binding capacity is indicative of a potential to inhibit and/or block said apoptotic activity and/or said neurite outgrowth effect. 
     
     
         37 : A composition which comprises at least one non-conservative fragment of an inducer and/or stimulator of  claim 1  or a non-conservative variant of an inducer and/or stimulator of  claim 1 , and/or at least one antibody that binds to at least one inducer and/or stimulator according to  claim 1 , without cross-reacting with the unmodified native neurotrophin, from which said inducer and/or stimulator derives. 
     
     
         38 : The non-conservative fragment or variant according to  claim 25 , for use in the treatment and/or prevention and/or palliation of a neurodegenerative condition or disease, any disease or condition involving a memory deficit and/or a concentration disorder, as well as neuroinflammatory conditions or diseases. 
     
     
         39 : The non-conservative fragment or variant according to  claim 25 , for use in the treatment and/or prevention and/or palliation of:
 neuropathic pain,   articular pain,   inflammatory pain, or   cancer pain.   
     
     
         40 : The antibody according to  claim 28 , for use in the treatment and/or prevention and/or palliation of a neurodegenerative condition or disease, any disease or condition involving a memory deficit and/or a concentration disorder, as well as neuroinflammatory conditions or diseases. 
     
     
         41 : The antibody according to  claim 28 , for use in the treatment and/or prevention and/or palliation of:
 neuropathic pain,   articular pain,   inflammatory pain, or   cancer pain.   
     
     
         42 : The composition of  claim 37 , for use in the treatment and/or prevention and/or palliation of a neurodegenerative condition or disease, any disease or condition involving a memory deficit and/or a concentration disorder, as well as neuroinflammatory conditions or diseases. 
     
     
         43 : The composition of  claim 37 , for use in the treatment and/or prevention and/or palliation of:
 neuropathic pain,   articular pain,   inflammatory pain, or   cancer pain.   
     
     
         44 : The inducer and/or stimulator of  claim 1 , for use as an agent useful for the induction and/or the stimulation of motor neuron apoptosis and/or for the induction and/or the stimulation of neurite outgrowth. 
     
     
         45 : The inducer and/or stimulator of  claim 1 , for use in the treatment and/or prevention and/or palliation of a disease, condition or trauma selected from the group comprising neuropathy, nerve injury, stroke, spinal cord injury, and neurodegenerative illness. 
     
     
         46 : A composition, which comprises at least one inducer and/or stimulator according to  claim 1 . 
     
     
         47 : The composition of  claim 46 , for use in the treatment and/or prevention and/or palliation of a disease, condition or trauma selected from the group comprising neuropathy, nerve injury, stroke, spinal cord injury, and neurodegenerative illness. 
     
     
         48 : An in vitro method for the diagnosis of a neurodegenerative condition or disease, and/or of a pain state or condition or feeling, which comprises:
 contacting a biological sample with at least one antibody according to  claim 28 , and   determining whether said at least one antibody binds to a ligand contained in said biological sample,   whereby such a binding is indicative or predictive of said disease, state, condition or feeling.