Ligands that control the interaction between GAG and its effector molecules and uses thereof
阅读说明：本技术 控制gag与其效应分子之间相互作用的配体及其用途 (Ligands that control the interaction between GAG and its effector molecules and uses thereof ) 是由 J-M·马莱 S·拉维亚勒 R·马康 A·普罗奇安茨 A·迪纳尔多 D·泰斯塔 于 2019-06-17 设计创作，主要内容包括：本发明涉及新化合物,所述化合物模拟糖胺聚糖,并且能够控制糖胺聚糖与其效应分子之间的相互作用。本发明的化合物为肽,并且能够阻止或减少至少一种效应分子与至少一种糖胺聚糖的结合。根据本发明的化合物可以用作药物,特别是用于刺激神经发生的药物,更一般地是用于治疗神经系统相关病理的药物。(The present invention relates to novel compounds which mimic glycosaminoglycans and are capable of controlling the interaction between a glycosaminoglycan and its effector molecules. The compounds of the invention are peptides and are capable of preventing or reducing the binding of at least one effector molecule to at least one glycosaminoglycan. The compounds according to the invention can be used as medicaments, in particular for stimulating neurogenesis, more generally for treating pathologies associated with the nervous system.)
1. A ligand comprising or consisting of a polypeptide of general formula (I), or any pharmaceutically acceptable salt thereof:
(I) [X]n
wherein
n is a number from 3 to 50,
x is a peptide comprising 4 to 6 amino acids,
x comprises an amino acid selected from the group consisting of glutamic acid and aspartic acid,
x comprises one or two cysteines, preferably two cysteines,
x comprises at least one neutral amino acid other than cysteine,
wherein the ligand is capable of binding interaction with an effector molecule and at least one glycosaminoglycan (GAG).
2. Ligand according to claim 1, characterized in that n is 3 to 35, more particularly 3 to 15, preferably 3 to 6.
3. Ligand according to claim 1 or 2, characterized in that X comprises two cysteines.
4. Ligand according to any one of claims 1 to 3, characterized in that X comprises two homocysteines.
5. Ligand according to any one of claims 1 to 4, characterized in that the "at least one neutral amino acid other than cysteine" is selected from the group consisting of alanine, asparagine, glutamine, histidine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine and 2-aminoisobutyric acid.
6. Ligand according to any one of claims 1 to 5, characterized in that the neutral amino acid other than cysteine is alanine.
7. Ligand according to any one of claims 1 to 6, characterized in that the amino acid selected from the group consisting of glutamic acid and aspartic acid is located at the first position of peptide X from the C-terminus.
8. Ligand according to any one of claims 1 to 7, characterized by a polypeptide [ X ]]nWherein X is selected from the group consisting of EACC, ECCA and ECAC, wherein C is cysteic acid, A is alanine, and E is glutamic acid.
9. Ligand according to any one of claims 1 to 8, characterized in that the ligand is capable of interacting with a glycosaminoglycan (GAG) binding site.
10. Ligand according to claim 9, characterized in that said glycosaminoglycan GAG is selected from the group consisting of heparan sulphate, heparin and chondroitin sulphate.
11. Ligand according to any one of claims 1 to 8, characterized in that the ligand of the invention is capable of preventing the binding of at least one effector molecule to at least one glycosaminoglycan (GAG).
12. Ligand according to claim 11, characterized in that the effector molecule is selected from the group consisting of transcription factors, growth factors, signal transduction factors and coagulation cascade proteins, in particular from the group consisting of axon guidance factors and the family of homologous proteins, preferably Otx2 or axon guidance factor 3A.
13. A ligand according to any preceding claim, for use as a medicament.
14. A ligand according to any one of claims 1 to 12, for use in the treatment of a disease and/or a neurological condition and/or a neurological injury, wherein the disease and/or neurological condition and/or neurological injury is selected from:
-nervous system injury, ischemic injury, hemorrhagic injury, neoplastic injury, degenerative injury, traumatic injury and/or neurodevelopmental injury following a cerebrovascular accident;
nervous system injury after an event, such as a stroke or nervous system injury after an injury;
-neurodegenerative diseases, such as multiple sclerosis, amyotrophic lateral sclerosis, subacute sclerosing panencephalitis, parkinson's disease, huntington's disease, muscular dystrophy, alzheimer's disease, idiopathic dystonia, spinal muscular atrophy or wilson's disease;
-nutritional deficiencies or toxin-induced disorders;
-neurodevelopmental diseases, such as autism or dyslexia;
-neuropsychiatric diseases such as schizophrenia and bipolar disorder;
-depression, epilepsy;
degenerative diseases affecting the eye or ear, such as glaucoma or amblyopia.
15. A pharmaceutical composition comprising a ligand according to any one of claims 1 to 12 and a pharmaceutically acceptable excipient.
Background
Glycosaminoglycans (GAGs) are naturally occurring carbohydrate-based molecules involved in the regulation of many cellular processes, including blood coagulation, angiogenesis, tumor growth, neuronal cell development, smooth muscle cell proliferation and gene expression, most often through interaction with effector molecules such as cytokines, growth factors, serine protease inhibitors and the like.
GAGs are linear, non-branched chains of two repeating saccharide (disaccharide) units, which can be up to 150 units in length, and are well known and described in detail in the art. GAGs are often, but not always, found covalently bound to the protein core in a structure called proteoglycan (proteoglycan). Proteoglycan structures are abundantly present on the cell surface and are associated with the extracellular matrix surrounding the cell.
Glycosaminoglycans can be divided into four major classes based on the repeating disaccharide unit in the backbone. Typically, one sugar is uronic acid and the other is N-acetylglucosamine or N-acetylgalactosamine. These categories are exemplified by the following four GAGs: (1) heparan sulfate (D-glucuronic acid/N-acetyl-D-glucosamine or N-sulfo-D-glucosamine); (2) chondroitin sulfate/dermatan sulfate (D-glucuronic acid or L-iduronic acid/N-acetyl-D-glucosamine); (3) keratan sulfate (D-galactose/N-acetyl-D-glucosamine); and (4) hyaluronic acid. With the exception of hyaluronic acid, all GAGs contain sulfate groups, which are esterified in various ways with the cyclic hydroxyl groups of the sugars. These negatively charged groups are believed to play an important role in the biological properties of glycosaminoglycans. In fact, the naturally occurring forms of GAGs (in particular heparin, heparan sulfate, chondroitin sulfate and dermatan sulfate) are complex heterooligosaccharides, which are composed of a mixture of differently sulfated sugar residues. For example, Chondroitin Sulfate (CS) is the most abundant glycosaminoglycan (GAG) in Central Nervous System (CNS) matrices (Djerbal et al, 2017, glycoconj.34,363-376), which consists of a protein backbone to which chains containing hundreds of disaccharide repeating units are attached.
An attractive feature of GAGs is that their polysaccharide units are modified by epimerization, N-and O-sulfation and deacetylation and can therefore be fine-tuned for the interactions.
For example, it has been demonstrated that during postnatal brain development, specific patterns of sulfation are critical for timing of critical periods during which specific neural circuits are highly plastic. As used herein, the term "critical phase" refers to a phase during the development of an organism in which the nervous system of the organism is able to acquire a particular functional capacity and/or structural form, typically at least partially affected by an external environmental stimulus. The timing and duration of the critical period may depend on the environmental stimulus experienced. For example, the lack of certain environmental stimuli will extend the critical period. These critical phases will remodel neuronal connections and are critical for adaptation to the environment and for learning and behavior.
These critical periods are closely related to nervous system plasticity. As used herein, the term "plasticity" refers to the ability of a nervous system or a portion of a nervous system to alter (e.g., recombine) its structure and/or function, typically affected by environmental conditions, injury, experience, or sustained nervous system activity. Plasticity may involve proliferation, growth, or movement of neurons or glial cells. Plasticity may involve the formation of new synaptic connections between neurons and/or the enhancement or attenuation of existing synaptic connections. Plasticity may be involved in neurogenesis.
These key periods are known to exist in the sensory system, such as binocular vision in the visual cortex or tonal topological refinement in the auditory cortex (tonotopic map refinement). They are also present in the motor system, even in higher cognitive regions of the brain, such as human speech acquisition.
These key phases arise, for example, from the maturation of cortical inhibitory gabaergic microalbumin neurons (PV cells), which is driven by orthodentical homeobox 2(Orthodenticle homobox protein 2, Otx2) homeoprotein transcription factors. Otx2 is synthesized extracortically, transported in the extracellular environment, and specifically internalized by PV cells. This internalization specificity is mediated by the disulfated chondroitin sulfate (type D (CS-D) or type E (CS-E)) contained in a specific extracellular matrix called the perineuronal network (PNN).
After this critical period has elapsed, the PV cells remain mature and the intrinsic potential for plasticity is actively inhibited, resulting in stabilization of the brain circuits, which is accompanied by the formation of a neuronal peripheral network (PNN) around the mature PV cells.
It has further been demonstrated that critical phases (during which specific neural circuits are highly plastic) appear to change in a variety of neurological and psychiatric diseases, including, for example, epilepsy, schizophrenia, depression, autism, and alzheimer's disease.
Furthermore, GAG binding motifs (RKQRRER) were found in the primary sequence of Otx2, revealing the interaction between Otx2 and glycosaminoglycans contained in PNN surrounding PV cells (e.g. disulfated chondroitin sulfate). Beuredeley et al (2012, j. neurosci.,32,9429-37) have further demonstrated that cortical infusion of peptides containing this "RK" motif will compete for GAG binding with Otx2, deplete the Otx2 content of mature PV cells, restore visual cortical plasticity in mature mice, and rescue cortical acuity in amblyopic mice.
Lee et al (2017, mol. psychiatry,22, 680-) 688) have demonstrated that Otx2 relates not only to visual plasticity, but also to auditory plasticity.
Winter et al (2016, Neural plant, 3679545) have demonstrated that controlled neutralization of Sema3A in PNN may be an important approach to enhance neuronal plasticity and functional repair after injury.
Miyata et al (2012,. Nat Neurosci.,15,414-422) and Dick et al (2013, J Biol Chem,288,27384-95) have demonstrated that both Otx2 and axon-guiding factor 3A (Semaphorin-3A, Sema-3A) are key roles in visual cortical plasticity and have similar motifs (RKQRRER and RKQRRRQR, respectively) for binding to chondroitin disulfate E-type (CS-E).
The inventors therefore concluded that the key time period and associated plasticity timing is controlled in part by the interaction of GAGs with their effector molecules (e.g. Otx2 or axonal guidance factor) and proposed to interfere with said interaction to provide a method for modifying the timing of key time periods for research and therapeutic applications, more particularly modifying the plasticity of the nervous system. Therefore, there is a need to provide compounds that are capable of controlling the interaction between GAGs and effector molecules.
The extraction of pure compounds from natural sources, as well as the chemical or enzymatic synthesis of GAG fragments, remains difficult and inefficient. Efforts have been made to reduce the complexity of GAG structure and synthesis, for example to demonstrate that the interaction between protein and GAG is predominantly electrostatic and that the most critical aspect is the correct charge spatial distribution, but these results are still at the research level and no compounds capable of controlling the interaction between GAG and effector molecules can be identified.
Similarly, modified natural and synthetic polymers have been proposed as GAG substitutes, such as polyacrylates, poly-2-acrylamido-2-methylpropanesulfonic acid, poly (sodium 4-styrenesulfonate) (PSS), poly (vinylsulfonate) (pVS), sulfated lignin derivatives, polyphenols, polyglycidyl copolymers, poly (ethylene oxide) -bl-poly (propylene oxide) -bl-poly (ethylene oxide) with sulfate groups (Pluronic F-127). Although these strategies show some success, they lack sequence regulation and only show non-specific electrostatic interactions with proteins. Alternatively, specific small molecules have been used as GAG mimetics: sulfated aminoglycosides, N-heteroaroylaminose, b-cyclodextrin sulfates and aptamers.
Therefore, there remains a great need in the art to develop new compounds and methods capable of controlling the interaction between GAGs and their effector molecules. More particularly, there remains a need for alternative methods for altering the plasticity of the nervous system, and more particularly for altering the timing of critical periods of nervous system plasticity.
Therefore, there is a great need in the art to develop compounds and methods that can alter the timing of critical periods of the nervous system, and more particularly, can alter the plasticity of the nervous system.
Similarly, there is a need in the art for improved treatments that will enhance recovery following CNS injury and/or help improve CNS and cognitive function in neuropsychiatric and neurodevelopmental disorders. More particularly, there is a need for new compounds and methods that play a role in key nervous system properties, such as plasticity, and that can be modulated to provide therapeutic benefits.
According to the present invention, the inventors aimed at developing new compounds capable of controlling the interaction between GAGs and their effector molecules, more particularly new ligands that do not have the drawbacks of the previously developed polymers.
Disclosure of Invention
According to a first embodiment, the present invention provides a ligand comprising or consisting of a polypeptide of general formula (I), or any pharmaceutically acceptable salt thereof:
(I)[X]n
wherein
n is a number from 3 to 50,
x is a peptide comprising 4 to 6 amino acids,
x comprises an amino acid selected from the group consisting of glutamic acid and aspartic acid,
x comprises one or two cysteines (cysteine acids), preferably two cysteines,
x comprises at least one neutral amino acid other than cysteine,
wherein the ligand is capable of binding interaction with an effector molecule and at least one glycosaminoglycan (GAG).
In one embodiment, the ligand is characterized in that n is from 3 to 35, more particularly from 3 to 15, preferably from 3 to 6.
The compounds of formula (I) may be in the form of their pharmaceutically acceptable salts. In particular, the counter-ion of the salt may be selected from metal cations such as sodium, potassium, magnesium, calcium, ammonium or alkylammonium.
Cysteic acid is a sulfamic acid, a sulfonic acid analog of cysteine; that is, it is an amino acid having a C-terminal sulfonic acid group. The synthesis thereof is widely disclosed in the art, and the compound is commercially available.
Cysteine is a natural alpha-amino acid characterized by the presence of thiol-forming mercapto-SH groups. Cysteine is present in small amounts in most proteins. Its presence in proteins is very important because it is capable of forming disulfide bonds. The thiol group is very fragile because it is easily oxidized. Its oxidation produces cystine, which consists of two cysteine molecules linked by disulfide bonds. The more energetic oxidizing agent may oxidize cysteine to produce cysteic acid.
Homocysteine is a non-proteinogenic amino acid, a result of methionine or cystathionine metabolism. The name homocysteine derives from its similarity to cysteine. In fact, homocysteine differs from cysteine in that the side chain of homocysteine contains two methyl groups, whereas the side chain of cysteine contains only one methyl group. In cysteine and homocysteine, the reactivity of the sulfhydryl groups is similar.
According to one embodiment, cysteic acid may be replaced by homocysteic acid, so X may comprise one or two amino acids selected from cysteic acid and homocysteic acid, preferably two identical or different amino acids selected from cysteic acid and homocysteic acid.
In another embodiment, the ligand is characterized in that X comprises two cysteines.
In another embodiment, the ligand is characterized in that X comprises two homocysteines.
In another embodiment, the ligand is characterized in that the "at least one neutral amino acid other than cysteine" is selected from the group consisting of alanine, asparagine, glutamine, histidine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and 2-aminoisobutyric acid.
Preferably, the "at least one neutral amino acid other than cysteine" is alanine, serine, threonine, more preferably alanine.
A further preferred embodiment is a ligand, characterized in that the neutral amino acid other than cysteine is alanine.
Preferably, X comprises at least one and at most 2, preferably at most 3, more preferably at most 4 neutral amino acids other than cysteine.
In another embodiment, the ligand of the invention is characterized in that the amino acid selected from the group consisting of glutamic acid and aspartic acid is located at the first position of peptide X from the C-terminus.
In another preferred embodiment, the ligand of the invention is characterized in that the amino acid selected from the group consisting of glutamic acid and aspartic acid is glutamic acid.
According to a particular embodiment, the amino acid residues of the ligands of the invention may be D or L or a mixture of D and L.
According to a particular embodiment, the ligands of the invention are characterized in that X are identical or different. This means that the "n" repeat of motif "X" may comprise a plurality of repeats of the same peptide motif or a plurality of repeats of different peptide motifs, or a mixture thereof. By different is meant that at least one motif X may be different from the other motifs X. By different is meant that the nature and number of amino acids are different.
According to another embodiment, the ligands are characterized in that X are identical. This means that the ligand comprises 3 to 50, more particularly 3 to 15, preferably 3 to 6 repeats of the same peptide motif X.
In a preferred embodiment, the ligand according to the invention is characterized in that X is a peptide comprising 4 amino acids.
In a particular embodiment, the ligand is characterized in that it is synthetic and non-natural.
According to a preferred embodiment, the polypeptide [ X ]]nWherein X is selected from the group consisting of EACC, ECCA and ECAC, wherein C is cysteic acid, A is alanine, and E is glutamic acid.
According to a preferred embodiment, the amino acid is in the L form.
In a preferred embodiment, the ligand according to the invention comprises a polypeptide [ X ]]nOr from the polypeptide [ X ]]nComposition of said polypeptide [ X]nSelected from (EACC)n、(ECCA)nAnd (ECAC)n。
According to a preferred embodiment, the amino acid is in the L form.
According to a preferred embodiment, n is from 3 to 6, more preferably n is 5 or 6, even more preferably n is 6.
In an even more preferred embodiment, the ligand according to the invention comprises a polypeptide [ X ]]nOr from the polypeptide [ X ]]nComposition of said polypeptide [ X]nSelected from (ECCA)nOr (ECAC)n。
In an even more preferred embodiment, the ligand according to the invention comprises a polypeptide [ X ]]nOr from the polypeptide [ X ]]nComposition of said polypeptide [ X]nSelected from (ECCA)nOr (ECAC)nWherein n is 3 to 6, in particular n is 5 or 6, even more preferably n is 6.
According to the invention, the polypeptide [ X ]]nMay be linear or branched.
According to a particular embodiment, the polypeptide [ X ] according to the invention]nAre linear. By linear is meant that the polypeptide comprises a succession [ X ] that repeats according to successive amino acid chains connected by peptide bonds]And (4) partial.
According to another particular embodiment, the polypeptide [ X ] according to the invention]nIs dendritic and [ X]Moieties, more particularly (ECCA) or (ECAC) moieties, are grafted to polylysine core chain K as described belowpThe above.
Peptide dendrimers (peptide dendrimers) are high molecular weight radial or wedge-shaped molecules comprising basic amino acids linked by peptide or amide bonds, both present inside and on the outer surface of a branched core. This gives a "hair-like" presentation of ligands that favors multivalent interactions (e.g., two ligand chains, one protein), which may be more correlated with affinity and specificity in vivo.
Thus, the polypeptide may be in the form of a comb, in which the main branch core is linked to the polypeptide [ X ]]nAnd (4) grafting. Preferably, the branched core is a polylysine core chain (K)p(ii) a Wherein K is Lys and p is 3 to 8, preferably 3 to 5, especially 4; and the branched core is linked to a polypeptide [ X ]]nAnd (4) grafting.
As used herein, the term "ligand capable of binding interaction with an effector molecule and at least one glycosaminoglycan (GAG)" means that the ligand of the invention is capable of preventing or reducing binding of at least one effector molecule to at least one glycosaminoglycan (GAG).
According to a preferred embodiment, the ligand of the invention is capable of preventing the binding of at least one effector molecule to at least one glycosaminoglycan (GAG).
According to another preferred embodiment, the ligand of the invention is capable of reducing the binding of at least one effector molecule to at least one glycosaminoglycan (GAG).
According to a preferred embodiment, the ligand of the invention is capable of binding at least one effector molecule.
According to a preferred embodiment, the effector molecule is a protein comprising a glycosaminoglycan binding site. In the present application, the expression "glycosaminoglycan binding site" refers to the region of the protein that interacts strongly with glycosaminoglycan GAGs, in particular GAGs consisting of heparan sulphate, heparin or chondroitin sulphate. The binding pocket is typically docked (mated) from a basic side chain of the protein and comprises a BBXB motif, wherein X is a hydrophilic residue and B is selected from arginine and lysine. The determination of the glycosaminoglycan binding site can be achieved by the skilled person according to known technical means, such as protein sequence analysis, X-ray and NMR structural determination. In particular, the "glycosaminoglycan binding site" may be RKQRRER or RKQRRQR.
According to a preferred embodiment, the ligands of the invention are capable of interacting with glycosaminoglycan (GAG) binding sites.
According to another preferred embodiment, the ligands of the invention are capable of mimicking glycosaminoglycan (GAG) binding.
According to a preferred embodiment, the ligand of the invention is capable of preventing the binding of at least one glycosaminoglycan (GAG) to the glycosaminoglycan binding site.
According to another preferred embodiment, the ligand of the invention is capable of reducing the binding of at least one glycosaminoglycan (GAG) to the glycosaminoglycan binding site.
According to a preferred embodiment, said glycosaminoglycan GAG is selected from the group consisting of heparan sulphate, heparin and chondroitin sulphate.
In a preferred embodiment, the effector molecule is selected from the group consisting of transcription factors, growth factors, signal transduction factors and coagulation cascades.
In a preferred embodiment, the effector molecule is selected from the axon-guiding factor and the family of homologous proteins, preferably Otx2 or axon-guiding factor 3A.
According to a preferred embodimentEmbodiments, ligands according to the invention comprise a polypeptide [ X ]]nOr from the polypeptide [ X ]]nComposition of said polypeptide [ X]nSelected from (ECAC)5、(ECAC)6、(ECCA)5And (ECCA)6The effector molecule is a family of homologous proteins, preferably Otx 2.
According to another preferred embodiment, the ligand according to the invention comprises a polypeptide [ X ]]nOr from the polypeptide [ X ]]nComposition of said polypeptide [ X]nSelected from (ECAC)5And (ECAC)6The effector molecule is an axon-homing factor, preferably axon-homing factor 3A.
The polypeptides [ X ] comprised by the ligands according to the invention can be prepared according to conventional and well-known techniques]n. Synthetic routes may use Fmoc strategy on Rink amide MBHA resin as known in the art. A 6-aminocaproic acid (Ahx) spacer may be introduced at the N-terminus, and the peptidyl resin may be acetylated (Ac) or acylated with biotinyl sulfone (Biot (SO 2)).
The peptide can be cleaved from the resin by TFA and precipitated in diethyl ether to obtain Ac-Ahx- (X) n-NH2, Ac-Ahx- (X) n-NH2, Biot (SO2) -Ahx- (X) n-NH2 or Biot (SO2) -Ahx- (X) n-NH 2. These precursors can be oxidized with performic acid to obtain the cysteic acid peptide, then neutralized with ammonia to obtain GAG mimetic peptides according to the present invention with acetyl or biotinyl sulfone at the N-terminus and amide at the C-terminus. The crude sulfopeptide can be desalted on Sephadex G25, purified (e.g., by reverse phase HPLC) and characterized by mass spectrometry. These GAG mimetic peptides can be stored as sodium salts at-20 ℃ for months.
It is also an object of the present invention to provide a ligand as described above according to any of the previous embodiments, for use as a medicament.
The invention further relates to a composition comprising at least one ligand as defined above and a pharmaceutically acceptable excipient.
As used herein, the term "pharmaceutically acceptable" refers to an excipient that does not produce an adverse, allergic, or other undesirable reaction when properly administered to an animal or human. As used herein, "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such excipients in pharmaceutically active substances is well known in the art.
The compositions of the invention are suitably buffered for use in humans at physiological or slightly basic pH (e.g., about pH 7 to about pH 9). Suitable buffers include, but are not limited to, phosphate buffers (e.g., PBS), bicarbonate buffers, HEPES and PIPES buffers, and/or Tris buffers. The composition of the present invention may further comprise a diluent suitable for human or animal use. It is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength. Representative examples include sterile water, physiological saline (e.g., sodium chloride), ringer's solution, dextrose, trehalose or sucrose solution, hank's solution, and other physiologically balanced saline solutions (see, e.g., The latest version of Remington: The Science and Practice of Pharmacy, a. gennaro, Lippincott, Williams & Wilkins). The pharmaceutically acceptable carrier included in the compositions of the present invention must also be capable of maintaining its stability under the conditions of manufacture and under prolonged storage (i.e., at least one month, preferably at least one year) at refrigerated (e.g., -70 ℃, -20 ℃), chilled (e.g., 4 ℃) or ambient temperature. Other pharmaceutically acceptable excipients may be used to provide desired properties, including, for example, altering or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, dissolution rate of the formulation, altering or maintaining release or absorption into the human or animal body, facilitating transport across the blood barrier or permeation in a particular organ (e.g., the brain).
Compositions comprising any of the ligands described herein may be formulated for sustained or slow release (also referred to as timed or controlled release). Such compositions can generally be prepared using well known techniques and administered, for example, by oral, rectal, intradermal, intranasal or subcutaneous implantation or by implantation at the desired target site. Sustained release formulations may comprise a compound dispersed in a carrier matrix and/or contained in a reservoir surrounded by a controlled release membrane (rate controlling membrane).
The invention further relates to a ligand or composition according to any preceding embodiment, for use as a plasticity-modifying agent (plasticity-modifying agent).
As used herein, the term "plasticity-altering agent" refers to a substance or composition that, upon administration to a subject alone or in combination with one or more other substances or non-drug therapies, will result in a detectable change in the plasticity of at least a portion of the nervous system. Such changes may be evidenced by changes in nervous system function and/or structure, as compared to the function and/or structure observed in the absence of the agent.
According to a preferred embodiment, the plasticity altering agent is capable of reopening the critical period (e.g., the plasticity altering agent of the present invention is capable of initiating the critical period and/or controlling the timing and duration of the critical period). According to another preferred embodiment, the plasticity-altering agent is capable of inducing plasticity of the nervous system or a part thereof, and of altering (e.g. recombining) the structure and/or function of said nervous system or a part thereof. According to another preferred embodiment, the plasticity-altering agent is capable of stimulating neurogenesis.
The present invention further relates to a method for altering the plasticity of the nervous system or a part thereof in a subject in need thereof, comprising the step of administering a plasticity-altering agent in an amount effective to alter the plasticity of the nervous system, wherein the plasticity-altering agent is a ligand or composition according to the invention and interacts with the binding of an effector molecule and at least one glycosaminoglycan (GAG) in the nervous system or a part thereof of said subject in need thereof.
The present invention further relates to a method for altering the plasticity of the nervous system or a part thereof in a subject in need thereof, comprising the step of administering a plasticity altering agent in an amount effective to alter the plasticity of the nervous system, wherein the plasticity altering agent is a ligand or composition according to the invention, and prevents or reduces binding of one effector molecule to at least one glycosaminoglycan (GAG) in the nervous system or a part thereof of said subject in need thereof.
The present invention further provides a method for promoting reconstitution or restoration of the nervous system or a portion thereof in a subject, comprising the step of administering to a subject in need thereof a plasticity altering agent, wherein the plasticity altering agent is a ligand or composition according to the present invention, and interacts with the binding of at least one glycosaminoglycan (GAG) by an effector molecule in the nervous system or a portion thereof of the subject in need thereof, alone or in combination with one or more other agents, in an amount effective to promote reconstitution or restoration of the nervous system.
The present invention further provides a method for promoting reconstitution or restoration of the nervous system or a portion thereof in a subject, comprising the step of administering to a subject in need thereof a plasticity altering agent, wherein the plasticity altering agent is a ligand or composition according to the present invention, alone or in combination with one or more other agents, in an amount effective to promote reconstitution or restoration of the nervous system, and prevents or reduces binding of one effector molecule to at least one glycosaminoglycan (GAG) in the nervous system or a portion thereof of the subject in need thereof.
The plasticity altering agent can contribute to (e.g., enhance) recovery or reorganization of the subject's nervous system and/or promote functional normalization. In other words, the degree of reorganization or restoration or the degree of functional improvement of the nervous system is greater than if the agent was not administered to the subject.
The present invention further relates to a method for stimulating neurogenesis in the nervous system or a portion thereof in a subject in need thereof, comprising the step of administering a plasticity-altering agent in an amount effective to stimulate neurogenesis, wherein the plasticity-altering agent is a ligand or composition according to the invention and interacts with the binding of at least one glycosaminoglycan (GAG) by an effector molecule in the nervous system or a portion thereof of said subject in need thereof.
The present invention further relates to a method for stimulating neurogenesis in the nervous system or a portion thereof in a subject in need thereof, comprising the step of administering a plasticity altering agent in an amount effective to stimulate neurogenesis, wherein the plasticity altering agent is a ligand or composition according to the invention, and prevents or reduces binding of an effector molecule to at least one glycosaminoglycan (GAG) in the nervous system or a portion thereof of the subject in need thereof.
The plasticity altering agent can help stimulate neurogenesis in the nervous system of the subject. In other words, the degree of neurogenesis is greater than if the agent is not administered to the subject.
According to a preferred embodiment, in such a method, the effector molecule is a protein comprising a glycosaminoglycan binding site.
According to a preferred embodiment, in this method, the glycosaminoglycan GAG is selected from the group consisting of heparan sulfate, heparin and chondroitin sulfate.
In a preferred embodiment, in such a method, the effector molecule is selected from the group consisting of transcription factors, growth factors, signal transduction factors and coagulation cascades.
In a preferred embodiment, in this method, the effector molecule is selected from the axon-directing factor and the family of homologous proteins, preferably Otx2 or axon-directing factor 3A.
According to a particular embodiment, "nervous system or a part thereof" means the "central nervous system" (CNS), which includes the brain, spinal cord, visual, olfactory and auditory systems. The CNS includes neurons and glial cells (glia), which are supporting cells for accessory neuron function. Oligodendrocytes, astrocytes and microglia are glial cells within the CNS. A portion of a nervous system can be any functionally or structurally defined part, site, region, unit, or component of the nervous system (these terms are used interchangeably herein). Parts of the nervous system include the cortex, cerebellum, thalamus, hypothalamus, hippocampus, amygdala, basal ganglia (caudate nucleus, putamen and globus pallidus), midbrain, pons, medulla oblongata, nerve bundles, and the like, as well as sub-parts of any of the foregoing. For example, sub-regions of the cortex include the visual cortex, auditory cortex, somatosensory cortex, entorhinal cortex, olfactory cortex, and the like. It will be appreciated that these regions may themselves be made up of smaller sub-regions.
As used herein, the term "restore" refers to a process in which the nervous system, or a portion thereof, at least partially deprived of the ability to perform a previously performed function, at least partially restores the ability to perform that function.
The term "reorganization" as used with respect to a nervous system or a portion thereof refers to a process in which a portion of a nervous system fully or partially assumes (i.e., presents) a function (e.g., a sensory, motor, or cognitive function) that the portion of the nervous system did not previously perform. The functions or tasks may, but need not, have been previously performed by different parts of the nervous system. Functional recombination can, but need not, result in one or more aspects of structural recombination. Functional reorganization may also be referred to as functional rearrangement.
According to another embodiment, it is desirable to develop a non-invasive method of targeting a specific protein within the choroid plexus. By secreting CSF, the choroid plexus is important for brain homeostasis and transmits signals (including Otx2) that are associated with brain development, neurogenesis, and plasticity.
According to a particular embodiment, the invention relates to the use of a ligand according to the invention for treating a patient in need thereof by altering choroid plexus function.
In a preferred embodiment, the ligand of the invention is an anti-Otx 2 compound active in the intracellular space of choroid plexus cells.
In a preferred embodiment, the ligands of the invention are capable of sequestering Otx2 in the extracellular environment, thus altering ocular dominant plasticity (oculometric plasticity).
As used herein, the term "subject" refers to an individual to whom an agent is to be delivered. Preferred subjects are mammals, particularly primates or humans.
According to a particular embodiment, the subject in need thereof is suffering from a disease and/or a neurological condition and/or a neurological injury, more particularly a disease and/or a neurological condition and/or a neurological injury requiring stimulation of neuronal plasticity. According to a particular embodiment, the disease and/or neurological condition and/or neurological damage is modulated by Otx2 or Sema 3A.
According to a particular embodiment, the disease and/or neurological condition and/or neurological damage is selected from:
-nervous system injury, e.g. after a cerebrovascular accident, ischemic injury, hemorrhagic injury, neoplastic injury, degenerative injury, traumatic injury and/or neurodevelopmental injury;
post-event nervous system injury, such as after stroke or injury (e.g. due to accident or surgery);
-diseases and disorders, including but not limited to neurodegenerative diseases, such as multiple sclerosis, amyotrophic lateral sclerosis, subacute sclerosing panencephalitis, parkinson's disease, huntington's disease, muscular dystrophy, alzheimer's disease, idiopathic dystonia, spinal muscular atrophy or wilson's disease;
-disorders caused by nutritional deficiencies or toxins (e.g. neurotoxins, drugs of abuse);
-a neurologic developmental disease, such as autism or dyslexia, i.e. a disease in which at least a part of the nervous system fails to develop normal structure and/or function;
neuropsychiatric disorders, such as schizophrenia and bipolar disorder, i.e. disorders in which at least a part of the nervous system fails to reach its typical level of cognitive function;
-depression, epilepsy;
degenerative diseases affecting the eye or ear (i.e. visual or auditory), such as glaucoma or amblyopia.
As used herein, an "effective amount" of a plasticity-altering agent refers to an amount of the plasticity-altering agent sufficient to elicit a desired biological response. As will be appreciated by one of ordinary skill in the art, the absolute amount of a particular plasticity-altering agent that is effective may vary depending on factors such as the desired biological endpoint, the agent to be delivered, the target tissue, and the like. One of ordinary skill in the art will further appreciate that an "effective amount" may be administered in a single dose, or may be achieved by multiple dose administration. The desired biological response may be, for example, (i) a functional or structural reorganization of synaptic connections, dendrites, or axonal projections; (ii) maintaining the synaptic connections, dendrites, or axon projections under conditions that worsen the synaptic connections, dendrites, or axon projections; (iii) regeneration of a nerve or axon projection system, or maintenance of a nerve or axon projection system under conditions that worsen the nerve or axon projection system; (iv) improving performance of tasks requiring motor or sensory function; (v) improving performance of tasks requiring cognitive function, e.g., improving performance in tests that measure learning and/or memory; (vi) the rate of decline in motor, sensory and/or cognitive function is slowed.
As used herein, the term "function" with respect to the nervous system or a portion thereof is used broadly herein to refer to any function, action, task or activity performed by the nervous system or a component thereof. The term includes, but is not limited to, the ability to process and recall information, modulate behavior, stimulate endogenous chemical release, control motor function, receive and process sensory input, maintain consciousness, and the like.
The dosage of the plasticity altering agent administered according to the invention may depend on the condition of the subject, i.e., the stage of the disease and/or neurological disorder and/or neurological injury, the severity of symptoms caused by the disease and/or neurological disorder and/or neurological injury, the general health status, as well as age, sex, and weight and other factors apparent to those skilled in the medical arts. The plasticity altering agent may be administered in a manner appropriate to the disease and/or neurological condition and/or neurological injury to be treated, as determined by one of skill in the medical arts. In addition, the appropriate duration and frequency of administration of the plasticity-altering agent may also be determined or adjusted by factors such as the condition of the patient, the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. The optimal dosage of the plasticity-altering agent can generally be determined using experimental models and/or clinical trials. The optimal dosage may depend on the weight, weight or blood volume of the subject. It is generally preferred to use the minimum dose sufficient to provide effective treatment. The design and execution of preclinical and clinical studies of the plasticity altering agents described herein is well within the skill of those in the relevant art.
For the treatment of neurodegenerative diseases, the plasticity altering agent may be administered locally, in particular by injection or infusion, to the target brain site. The plasticity altering agent may also be administered using a controlled release device, such as an osmotic mini-pump connected to a catheter implanted in the brain.
The disclosures of all patents, publications, and database entries cited above are hereby expressly incorporated by reference in their entirety to the same extent as if each such individual patent, publication, or entry was specifically and individually indicated to be incorporated by reference.
Drawings
In addition to the above, the present invention also encompasses other aspects which will be disclosed in the specification with reference to exemplary embodiments and with reference to the accompanying drawings, wherein:
figure 1 the ligand interacts with Otx 2. (a) Dot Blot (DB) examples of biotinylated sulfonyl peptides incubated with Otx2 protein or not with Otx2 protein. (b) Quantification of DB chemiluminescence for at least 3 replicates per data point.
Figure 2. the ligand interacts with the GAG binding site of Otx 2. (a) When compared with the HexaCSE sum (EC 'AC')5After incubation, electrophoretic mobility analysis (EMSA) of biotinylated IRBP1 DNA probe and Otx2 protein showed a loss of mobility. (b) Incubated with GAG motif peptide (RKpep) or control peptide (AApep, SCpep) (EC 'AC')6Dot Blot (DB). (c) Quantification of DB. All values are: n is 3; average + -SEM; single factor analysis of variance with Bonferonni post hoc tests; p<0.01。
Figure 3 ligand pull-down experiments using adult mouse visual cortex lysate. (a) Comparison of GAG binding motifs. (b) Western Blot (WB) of Sema-3A after pull-down with ligand retained on streptavidin beads (control beads alone). (c) Quantification of WB. (d) With incubation with GAG motif peptide (RKpep) or control peptide (AApep, SCpep) (EC 'AC')6Pull down the WB of post Sema-3A. (e) Quantification of WB. All values are: n is a radical of3; average + -SEM; single factor analysis of variance with Bonferonni post hoc tests; p<0.05，**P<0.01，***P<0.001。
Figure 4. ligand has in vivo activity. (a) With or without injection of 40fmol (EC 'AC')6In the adult mouse primary visual cortex IV, representative images of Wisteria Floribunda Agglutinin (WFA), which is labeled PNN, staining and small albumin (PV) staining. (b) Quantification of WFA + cell number. (c) Quantification of PV cell number. Scale bar 100 μm. All values are: n is 3-6; average + -SEM; t, checking; p<0.05，**P<0.01，***P<0.001。
Figure 5 GAG mimics the peptide scaffold of the invention: EC ' C ' a and EC ' AC ', wherein C ' represents cysteic acid.
Detailed Description
Examples
Materials and methods:
compound (I)
Sequences (ECCA) n and (ECAC) n (n ═ 3-6) were synthesized by Fmoc strategy on Rink amide MBHA resin. A 6-aminocaproic acid (Ahx) spacer was introduced at the N-terminus and the peptidyl resin was acetylated (Ac) or acylated with biotinyl sulfone (Biot (SO 2)). The peptide was cleaved from the resin by TFA and precipitated in diethyl ether to yield Ac-Ahx- (ECAC) n-NH2, Ac-Ahx- (ECCA) n-NH2, Biot (SO2) -Ahx- (ECAC) n-NH2 or Biot (SO2) -Ahx- (ECCA) n-NH 2. Oxidation of these precursors with performic acid to obtain cysteic acid (C ') peptides, followed by neutralization with ammonia to obtain GAG mimetics (EC ' AC ') N and (EC ' C ' a) N with an acetyl or biotin sulfone at the N-terminus and an amide at the C-terminus. The crude sulfopeptide was desalted on Sephadex G25, purified by reverse phase HPLC and characterized by mass spectrometry. These GAG mimetic peptides can be stored as sodium salts at-20 ℃ for months.
Dot blot
For competition analysis, 400pmol of biotinylated ligand or hexaCSE was incubated for 30 min at 37 ℃ in 100mM ammonium acetate containing 1. mu.g of the Otx2 protein (internal) and 3. mu.g of the RK-, AA-or SC-peptide. Then, each solution was spotted on a nitrocellulose membrane and biotin was detected by incubation with streptavidin-hrp (thermo fisher Scientific) for 30 minutes followed by chemiluminescence (#34580, thermo fisher Scientific). The membrane was digitized with LAS-4000(Fujifilm) and quantified densitometrically with ImageJ.
Gel migration
The Otx2 protein (0.1. mu.g) was reacted in 50 ng/. mu.l of dIdC, PBS with 40fmol of biotinylated IRBP1 oligonucleotide and 4pmol (EC 'AC')5Or hexaCSE, at room temperature for 30 minutes. Samples were separated on 6% native polyacrylamide gels at 100V in TBE, then transferred to nylon membranes at 380mA for 45 min and UV (120,000. mu.J/cm)2Amersham). For detection using the LightShift chemiluminescent EMSA kit (#89880, ThermoFisher Scientific), the membranes were digitized with LAS-4000(Fujifilm) and quantified densitometrically with ImageJ.
Immunoprecipitation
Adult mice were dissected for visual cortex and placed in homogeneous buffer (0.32M sucrose, 5mM HEPES, 10mM MgCl)2And protease inhibitors). The samples were centrifuged (8 min, 1700g) at 4 ℃ and the supernatant incubated with 5nmol of GAG mimic for 2 hours at 37 ℃. For competition assays, the ligands were preincubated with 50nmol of RK-, AA-or SC-peptide for 30 min at 37 ℃ followed by overnight incubation with streptavidin-coupled Dynabeads (Life technologies) at 4 ℃. The loaded beads were washed with homogenization buffer and heated in Laemmli buffer (containing DTT) at 95 ℃ for 10 min to isolate the protein for western blot analysis.
Western blot
Immunoprecipitated proteins were separated at 200V for 1 hour on a NuPAGE 4-12% Bis-Tris precast gel (Invitrogen) and transferred to a methanol activated PVDF membrane at 400mA for 1 hour. The membranes were blocked with 5% nonfat dry milk for 1 hour and then incubated with primary anti-Sema 3A antibody (rabbit, 1/1000, Millipore) overnight at 4 ℃. The membrane was washed and incubated with anti-rabbit HRP-linked secondary antibody (Cell Signaling) for 1 hour. The membrane was digitized with LAS-4000(Fujifilm) and quantified densitometrically with ImageJ.
Brain infusion and immunohistochemistry
Three month old C57BL/6J mice (Janvier) were infused with different concentrations of ligand (4pM, 400pM or 4 μ M) for 7 days using an Alzet mini-osmotic pump (0.5 μ L/h) V1(λ: x ═ 1.7mm, y ═ 0mm, z ═ 0.5 mm). Animals were then perfused with PBS and 4% paraformaldehyde. Frozen sections (20 μm) were incubated overnight with anti-small albumin primary antibody (rabbit, 1/500, Swant) and WFA-FITC (1/100, Vector) followed by anti-rabbit Alexa Fluor-546 secondary antibody (1/2000, Molecular Probes) for 1 hour. Images were collected with a Leica SP5 confocal microscope and quantified analytically with ImageJ.
Statistical analysis
Analysis was performed using Prism 6 (GraphPad). Single term comparisons were performed by t-test and multiple sets of analyses were performed by analysis of variance and subsequent Bonferonni post-hoc tests.
As a result:
to evaluate biotinylated (EC 'C' A)nAnd (EC 'AC')nAffinity of the library for Otx2 protein, we performed dot blots with nitrocellulose membranes, where retention of the sulfopeptide requires interaction with the protein (fig. 1 a). Although the binding of Otx2 was not more biased towards a motif than other motifs, the affinity increased significantly with increasing n repeats (fig. 1 b). For comparison, dot blots were performed with a hexaCSE, which previously demonstrated to bind to Otx2 and interfere with its in vivo activity in mouse brain. Sequences with n-4 or 5 repeats were found to bind equally to hexaCSE with Otx2, while those with n-6 were found to bind 2 to 3 times better than hexaCSE.
To confirm that the biotinylated ligand interacted with Otx2 through its previously identified GAG binding site, we performed DNA tracking and peptide binding experiments (figure 2). In Otx2, the GAG binding motif is located in the first helix of its DNA binding domain, so specific binding of GAG molecules may interfere with DNA binding. Analysis showed that (EC 'AC')5The mimic was able to follow IRBP1 DNA probe from Otx2 to the same extent as the biotinylated hexaCSE positive control (fig. 2 a). GAG binding sites in Otx2(RKQRRER) contain arginine-lysine pairs (RK) that no longer interact with the ECM when mutated to alanine pairs (AA)Combined and at Otx2+/AACausing critical phase defects in the mouse model. Dot blot analysis using peptides based on this motif (15-mer) to evaluate (EC 'AC')6Whether it binds specifically (FIG. 2 b). Although the wild type peptide (RKpep: RKQRRERTTFTRAQL) retained the ligand, the mutated peptide (AApep: AAQRRERTTFTRAQL) did not, nor did the scrambled peptide (SCpep: RTQTRFRTRARLEQK) which contained the same residues as RKpep but was randomly ordered (FIG. 2 c). These analyses demonstrate that the interaction is not only electrostatic, but also requires a specific residue sequence.
To measure the in vivo activity and specificity of biotinylated GAG mimetics, we performed biochemical and immunohistochemical analyses. Both Otx2 and axon-guiding factor 3A (Sema-3A) are key roles in visual cortical plasticity and have similar motifs for binding CS-E (fig. 3A). We first focused on Sema-3A because the level of cortex Otx2 was too low to be reliably detected biochemically. In a pull-down experiment of adult mouse visual cortex lysate by using ligand, we found (EC 'AC')5And (EC 'AC')6Interact with Sema-3A, but (EC 'C' A)6Does not interact with Sema-3A (fig. 3 b-fig. 3 c). RKpep specifically disrupts Sema-3A vs (EC 'AC') in these lysates6The interaction of (FIGS. 3 d-3 e) suggests the involvement of the Sema-3A GAG binding motif.
Infusion in the visual cortex of adult mice (EC 'AC')5Or (EC 'AC')6After 7 days duration, 40fmol resulted in reduced PNN assembly (fig. 4 a-4 b). Infusion is up to 105Multiple more of each mimetic provided the same reduction. There is a feedback loop between Otx2 accumulation in the PV cell and the assembly of the surrounding PNN; PNN attracts extracellular Otx2, while Otx2 activity in PV cells increases PNN expression. These results suggest that these ligands interfere with Otx2 signaling sufficiently to break the feedback loop and reduce PNN assembly. Furthermore, it has been shown that interfering with Otx2 signaling in the adult visual cortex will reverse the PV cell maturation state and induce plasticity. Here, only infusion (EC 'AC')6(>40fmol) resulted in a significant reduction in PV expression (fig. 4 c). This modest reduction has previously been demonstrated (>25%) was sufficient to re-turn on the brain's plasticity.
These pull-down and infusion experiments confirmed the in vitro findings that longer ligands have higher affinity for GAG binding proteins; only when (EC 'AC')nWe observed a sufficient effect on PV cell maturation when n-6 of the mimetic (fig. 4 c). They also suggest, (EC 'AC')nAnd (EC 'C' A)nMimetics can provide specificity and selectivity. Although Otx2 showed no preference for either motif in vitro (fig. 1b), Sema-3A was neutralized in brain lysate (EC 'AC')nMotifs interact with each other, but not with (EC 'C' A)nMotifs interact (FIG. 3 c). Thus, these GAG mimetics contain an effective electrostatic pattern to replicate the specific sulfation (sulfation) pattern present in natural GAGs.
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