Patent Application
20190145962
The present disclosure relates to a peptide having an ability to inhibit binding between amylospheroids (ASPD) and neurons as well as a method of evaluating the same and a method of screening the same.
Amylospheroids (ASPD; amylospheroids) each are a spherical Aß assembly that is formed of about 30 amyloid ß proteins (Aß) aggregated together and has a diameter of approximately 10 nm, and are a structure considered to play an important role in the irreversible stage at which Alzheimer's disease develops.
ASPD were initially isolated as an in vitro synthesized Aß assembly (i.e., synthetic ASPD) that exhibited strong neurotoxicity (Non-Patent Document 1). Antibodies specific to this synthetic ASPD have been produced (Patent Documents 1 and 2), and using these antibodies, ASPD formed in vivo (that is, native ASPD) were actually isolated from the brain of a human patient with Alzheimer's disease (Non-Patent Document 1).
Native ASPD and synthetic ASPD likewise selectively induce cell death of mature neurons. It was discovered that the target of ASPD in the neuronal cell death is the synaptic protein “alpha 3 subunit of Na+, K+-ATPase pump (hereinafter referred to as ‘NAKα3’)” that plays a very important role in neuronal survival and function, and it was revealed that the function of NAKα3 decreases due to the binding of ASPD and neurons are excited excessively, which results in death of neurons (Patent Document 3 and Non-Patent Document 2). Peptides capable of inhibiting the interaction between ASPD and NAKα3 have also been reported (Patent Document 3 and Non-Patent Document 2).
The most correlated with clinical symptoms in Alzheimer's disease is neuronal loss. It was revealed that the amount of native ASPD in the cerebral cortex of an Alzheimer's disease patient with neuronal loss increases relative to the severity of Alzheimer's disease and only a trace amount of native ASPD exists in the cerebellum of an Alzheimer's disease patient with little neuronal loss (Non-Patent Document 3). Therefore, ASPD are considered to play an important role in the irreversible stage at which Alzheimer's disease develops. Furthermore, native ASPD have also been detected from the brains of patients with Lewy body dementia (Non-Patent Document 3). Therefore, similarly in Lewy body dementia, ASPD are considered to play an important role in the development thereof.
Synthetic ASPD, which are considered to be equivalent to native ASPD, can be produced by slowly rotating a liquid containing Aß (Non-Patent Document 1 and Patent Document 4).
It is considered that if binding between ASPD and neurons can be inhibited, the interaction between ASPD and neurons can be inhibited, and thereby the neuronal cell death caused by ASPD can be avoided. Furthermore, it is considered that if a substance capable of inhibiting the binding between ASPD and neurons can be obtained, it can contribute to the development of treatment methods, for example, for Alzheimer's disease and Lewy body dementia.
Thus, in one aspect, the present disclosure provides a peptide capable of inhibiting binding between ASPD and neurons.
In one aspect, the present disclosure relates to a synthesized, isolated, or purified polypeptide, the polypeptide containing an amino acid sequence represented by Formula (I) below:
where X1 is R, H, or K,
X3 is any amino acid, and
In one aspect, the present disclosure relates to a synthesized, isolated, or purified polypeptide, the polypeptide containing an amino acid sequence represented by Formula (II) below:
where X1 is R, H, or K,
X2 is any amino acid,
In one aspect, the present disclosure relates to a synthesized, isolated, or purified polypeptide, the polypeptide having an amino acid sequence represented by HANW, HVNW, HFHW, or HFYW
In one aspect, the present disclosure relates to a peptide mimic having a structure mimicking the structure of a polypeptide according to the present disclosure, the peptide mimic having an ability to inhibit binding between ASPD and neurons.
In one aspect, the present disclosure relates to a vector for expressing a polypeptide according to the present disclosure, the vector containing a polynucleotide that encodes the polypeptide. In another aspect, the present disclosure relates to a composition containing a polypeptide according to the present disclosure or a peptide mimic according to the present disclosure.
In another aspect, the present disclosure relates to a method of evaluating a substance that inhibits binding between ASPD and neurons, the method including: contacting a liquid mixture containing a substance to be evaluated and ASPD with cultured neurons or contacting the ASPD with the cultured neurons in the presence of the substance to be evaluated, thereafter providing the ASPD and the neurons with different labels from each other to perform imaging, and obtaining an indicator of binding of the ASPD to the neurons from data obtained by the imaging to evaluate the substance to be evaluated using the indicator.
In another aspect, the present disclosure relates to a method of screening a substance that inhibits neurotoxicity caused by ASPD, the method including:
contacting a liquid mixture containing a candidate substance and ASPD with cultured neurons or contacting the ASPD with the cultured neurons in the presence of the candidate substance,
thereafter providing the ASPD and the neurons with different labels from each other to perform imaging, and
obtaining an indicator of binding of the ASPD to the neurons from data obtained by the imaging to select the candidate substance using the indicator.
In one aspect, the present disclosure relates to first and second peptides described below. That is, in one aspect, the present disclosure relates to a peptide having an amino acid sequence represented by Formula (I) or (II) below.
[First Peptide]
In one aspect, the present disclosure relates to a synthesized, isolated, or purified polypeptide thereinafter also referred to as a “first peptide”) containing an amino acid sequence represented by Formula (I) below:
where X1 is arginine (R), histidine (H), or lysine (K),
X2 is arginine (R), aspartic acid (D), glutamine (Q), histidine (H), lysine (K), threonine (T), tryptophan (W), or tyrosine (Y),
X3 is any amino acid, and
X4 is tryptophan (W), phenylalanine (F), or tyrosine (Y).
In Formula (I), in one or more embodiments, X1 is preferably R or H, more preferably H, from the viewpoint of inhibiting binding between ASPD and neurons.
In Formula (I), in one or more embodiments, from the same viewpoint, X2 is: preferably H, W, Q, Y, or T, and more preferably H or T.
In Formula (I), in one or more embodiments, from the same viewpoint, X3 is: preferably alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine leucine (L), lysine (K), methionine (M), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), or valine (V),
more preferably N, L, G, I, W, R, M, D, E, A, V, K, H, or Y,
further preferably N, L, I, W, R, D, K, H, or Y, and
further more preferably N, R, K, H, or Y.
In Formula (I), in one or more embodiments, X1 is preferably W from the same viewpoint.
One or more embodiments of the amino acid sequence represented by Formula (I) include the amino acid sequences of the peptides 02 to 11 indicated in Table 1 below, include HHNW, HWNW, HQNW, HYNW, and HTNW, or include HHNW and HTNW.
[Second Peptide]
In one aspect, the present disclosure relates to a synthesized, isolated, or purified polypeptide (hereinafter also referred to as a “second peptide”) containing an amino acid sequence represented by Formula (II) below:
where X1 is arginine (R), histidine (H), or lysine (K),
X2 is any amino acid,
X3 is leucine (L), glycine (G), isoleucine tryptophan (W), arginine (R), methionine (ND, aspartic acid (D), glutamic acid (E), alanine (A), valine (V), or lysine (K), and
X4 is phenylalanine (F), tryptophan (W), or tyrosine (Y).
In Formula (II), in one or more embodiments, X1 is preferably R or H, more preferably H, from the viewpoint of inhibiting binding between ASPD and neurons.
In Formula (II), in one or more embodiments, from the same viewpoint, X2 is: preferably alanine (A), arginine (R), aspartic acid (D), glutamine (Q), glycine (G), histidine (H), isoleucine leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), threonine (T), tryptophan (W), tyrosine (Y), or valine (V),
more preferably F, R, K, D, H, W, Q, Y, T, A, or V,
further preferably F, H, W, Q, Y, T, A, or V, and
further more preferably F, H, T, A, or V.
In formula (II), in one or more embodiments, from the same viewpoint, X3 is:
more preferably L, I, W, R, D, or K, and
further preferably R or K.
In the formula (II), in one or more embodiments, from the same viewpoint, X1 is preferably W.
One or more embodiments of the amino acid sequence represented by Formula (II) include the amino acid sequences of peptides 12 to 24 indicated in Table 2 below, include HFLW, HFIW, HFWW, HFRW, HFDW, and HFDW, or include HFRW and HFDW.
[Third Peptide]
In another aspect, the present disclosure relates to a synthesized, isolated, or purified polypeptide (hereinafter also referred to as a “third peptide”) containing an amino acid sequence represented by HANW, HVNW, HFHW, or HFYW.
It is preferable that the first to third peptides according to the present disclosure can inhibit binding between ASPD and neurons in one or more embodiments.
It is preferable that the first to third peptides according to the present disclosure have an ability to inhibit the interaction between ASPD and NAKα3 in one or more embodiments.
It is preferable that the first to third peptides according to the present disclosure have an ability to bind to ASPD in one or more embodiments.
It is preferable that the first to third peptides according to the present disclosure can suppress ASPD-induced cell death of neurons in one or more embodiments.
In the present disclosure, when simply a term “amylospheroids (ASPD)” is used, it may include native ASPD and synthetic ASPD.
In the one or more embodiments, from the viewpoint of inhibiting binding between ASPD and neurons, it is preferable that the first to third peptides according to the present disclosure have, at its N terminus, an amino acid sequence represented by the Formula (I) or (II) or HANW, HVNW, HFHW, or HFYW, that is, the first amino acid at the N terminus is X1 or H.
In one or more embodiments, the lengths of the first to third peptides according to the present disclosure can be, for example, 4 or more but 50 or less, 25 or less, 20 or less, 15 or less, 10 or less, or 5 or less. Therefore, examples of the lengths of the peptides include 4 to 50, 4 to 25, 4 to 20, 4 to 15, 4 to 10, or 4 to 5.
In one or more embodiments, the first to third peptides according to the present disclosure can be in the form in which an amino acid sequence represented by the Formula (I) or (II) or HANW, HVNW, HFHW, or HFYW and a brain-migrating peptide are bound to each other directly or through a linker peptide. Binding a brain-migrating peptide can increase the efficiency of migration of the polypeptide of the present disclosure across the blood brain barrier into the brain. For the brain-migrating peptide, conventionally known ones can be used.
The first to third peptides according to the present disclosure can also include those in the forms in which said peptides have been modified. In one or more embodiments, the forms in which the terminals of said peptides and/or peptide bonds have been modified can also be included. Examples of the terminal modification include removal or amidation of the C-terminal carboxyl group and removal or acetylation of the N-terminal amino group. Examples of the modification of the peptide bonds include N-alkylation.
In one or more embodiments, from the viewpoint of inhibiting binding between ASPD and neurons, in the first to third peptides according to the present disclosure, the C-terminal carboxyl group preferably has been removed or amidated and more preferably has been removed. Examples of the amidation of the C-terminal carboxyl group include —CONH2, —CONHR, and —CONRR′ (R and R′ are hydrocarbon groups), and preferably it is —CONH2.
The N-terminal amino group may be left as it is or may be acetylated or removed, but preferably it is left as it is from the viewpoint of inhibiting binding between ASPD and neurons.
In one or more embodiments, with respect to the first to third peptides according to the present disclosure, from the viewpoint of promoting the binding of said peptides to ASPD, at least one of the peptide bonds may have been N-alkylated. Examples of N-alkylation include N-methylation, N-ethylation, and N-cyclopropylation.
The first to third peptides according to the present disclosure can be prepared by appropriate synthesis according to an ordinary synthesis method, and purification.
[Peptide Mimic]
In another aspect, the present disclosure relates to peptide mimics having structures mimicking the structures of the first to third peptides according to the present disclosure.
One or more embodiments of the “peptide mimic” in the present disclosure include peptide-like compounds or synthetic compounds that do not have the structure of a peptide bond and the structure of a modified peptide bond.
One or more embodiments of the peptide-like compounds include, for example, peptoid (S. M. Miller, R. J. Simon, S. Ng, R. N. Zuckermann, J. S. Ken; W. H. Moos, Bioorg. Med. Chem Lett., 4, 2657 (1994)) and nonpeptide compounds mimicking the ß/γ turn structure of proteins (M. Kahn Tetrahedron, 49, 3433 (1993), “Combinatorial Chemistry” published by Kagaku Dojin, p 44-64, 1997).
In one or more embodiments, it is preferable that the peptide mimic according to the present disclosure can inhibit binding between ASPD and neurons. In one or more embodiments, the peptide mimic according to the present disclosure preferably has an ability to inhibit the interaction between ASPD and NAKα3. In one or more embodiments, the peptide mimic according to the present disclosure preferably has an ability to bind to ASPD. In one or more embodiments, it is preferable that the peptide mimic according to the present disclosure can suppress ASPD-induced cell death of neurons.
[Polynucleotide and Vector]
In another aspect, the present disclosure relates to a polynucleotide that encodes the polypeptides of the present disclosure, and to a vector that contains a polynucleotide that encodes the polypeptides of the present disclosure and is used for expressing the above-mentioned polypeptides. Preferably, the vector has an expression cassette containing, for example, a regulatory sequence that enables the expression of the polynucleotide of the present disclosure. The vector is not particularly limited as long as it allows gene transfer of the polynucleotide, but an AVV (adeno-associated virus) vector is preferred from the viewpoint of safety. Preferred embodiments of the vector of the present disclosure include those having a form in which the vector is bound to a brain-migrating peptide. Binding of a brain-migrating peptide to the vector can increase the efficiency of migration of the vector of the present disclosure across the blood brain barrier into the brain. For the brain-migrating peptide, conventionally known ones can be used.
[Composition]
As another aspect, the present disclosure relates to a composition containing a peptide according to the present disclosure, a peptide mimic according to the present disclosure, or a vector according to the present disclosure. In one or more embodiments, the above-mentioned composition is a pharmaceutical composition.
[Pharmaceutical Composition]
When the present disclosure is a pharmaceutical composition, the dosage form thereof can be suitably selected according to the administration method, and examples thereof include injections, liquid formulations, capsules, chews, tablets, suspensions, creams, and ointments. Furthermore, the method of administration is also not particularly limited and examples thereof include oral administration and parenteral administration. The pharmaceutical composition of the present disclosure may contain conventionally known additives (for example, excipients or diluents) depending on the administration form and dosage form.
Examples of the dosage form suitable for oral administration include; solid formulations such as tablets, particles, liquid- or powder-containing capsules, troches, chews, multiparticles and nanoparticles, gels, and films; and liquid formulations such as suspensions, solutions, syrups, and elixirs. Examples of the excipient include carriers such as cellulose, calcium carbonate, dicalcium phosphate, mannitol, and sodium citrate; granulating binders such as polyvinylpyrrolidine, hydroxypropyl cellulose, hydroxypropyl methylcellulose, and gelatin; disintegrators such as sodium starch glycolate and silicate; lubricants such as magnesium stearate and stearic acid; humectants such as sodium lauryl sulfate; preservatives, antioxidants, flavoring agents, and colorants.
Examples of parenteral administration of the pharmaceutical composition of the present disclosure include direct administration into a bloodstream, a muscle, or an internal organ. Examples of parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, and subcutaneous administrations. Parenteral administration can be performed, for example, with a needle syringe, a needleless syringe, and other injection techniques. Furthermore, examples of a dosage form suitable for parenteral administration include an aqueous solution containing an excipient and/or a buffer agent.
[Prevention, Amelioration, and/or Treatment Method]
In one or more embodiments, the pharmaceutical composition according to the present disclosure can be used for the prevention, amelioration, and/or treatment of Alzheimer's disease and/or Lewy body dementia.
In the present disclosure, the prevention of Alzheimer's disease and/or Lewy body dementia refers to the inhibition of the development of Alzheimer's disease and/or Lewy body dementia or the prevention of the progression of the pathological condition of reversible mild cognitive impairment. Furthermore, in the present disclosure, the amelioration of Alzheimer's disease and/or Lewy body dementia includes stopping the progression of the pathological condition or improving the pathological condition of reversible mild cognitive impairment of Alzheimer's disease and/or Lewy body dementia. Furthermore, in the present disclosure, the treatment of Alzheimer's disease and/or Lewy body dementia includes delaying or almost stopping the progression of the pathological condition.
[Evaluation Method]
In another aspect, the present disclosure relates to a method of evaluating a substance that inhibits binding between ASPD and neurons.
A first evaluation method may include contacting a liquid mixture containing a substance to be evaluated and ASPD with cultured neurons, thereafter providing the ASPD and the neurons with different labels from each other to perform imaging, and obtaining an indicator of the amount of the ASPD bound to the neurons from data obtained by the imaging to evaluate the substance to be evaluated using the indicator.
A second evaluation method may include contacting ASPD with cultured neurons in the presence of a substance to be evaluated, thereafter providing the ASPD and the neurons with different labels from each other to perform imaging, and obtaining an indicator of the amount of the ASPD bound to the neurons from data obtained by the imaging to evaluate the substance to be evaluated using the indicator.
According to the first evaluation method, it is possible to evaluate the binding inhibitory ability, with respect to the binding between ASPD and neurons, of a substance that binds to or interacts with ASPD.
According to the second evaluation method, it is possible to evaluate the binding inhibitory ability, with respect to the binding between ASPD and neurons, of a substance that binds to or interacts with neurons.
Preferably, the neurons are mature neurons. In one or more embodiments, mature neurons can be those having at least one axon and at least one dendrite.
In one or more embodiments, examples of the imaging method include immunofluorescence staining and fluorescence microscopic observation. In one or more embodiments, the neurons and the ASPD may be immobilized after the contacting but before the labeling.
In this aspect, when calculating the amount of the ASPD bound or the indicator thereof, it is easy to eliminate, from image data, information on large aggregates of APSD and/or information on nonspecific ASPD not bound to neurons. This makes it possible to improve the quantitativeness and/or the sensitivity of the evaluation in one or more embodiments.
In one or more embodiments, the indicator of the amount of ASPD bound may be the amount itself of the ASPD bound to neurons or may be ED50 or the ratio thereof as described in the Examples. For example, it can be evaluated that the higher the ED50 in the case where the substance to be evaluated and ASPD coexist, the higher the ability of inhibiting ASPD from binding to neurons.
[Screening Method]
In another aspect, the present disclosure relates to a method of screening a substance that inhibits neurotoxicity caused by ASPD.
A first screening method may include contacting a liquid mixture containing a candidate substance and ASPD with cultured neurons, thereafter providing the ASPD and the neurons with different labels from each other to perform imaging, and obtaining an indicator of binding of the ASPD to the neurons from data obtained by the imaging to select a candidate substance using the indicator.
A second screening method may include contacting ASPD with cultured neurons in the presence of a candidate substance, thereafter providing the ASPD and the neurons with different labels from each other to perform imaging, and obtaining an indicator of binding of the ASPD to the neurons from data obtained by the imaging to select a candidate substance using the indicator.
According to the first screening method, a substance that binds to or interacts with ASPD and has an ability to inhibit binding between ASPD and neurons can be selected as a candidate substance for a substance that inhibits neurotoxicity caused by ASPD.
According to the second screening method, a substance that binds to or interacts with neurons and has an ability to inhibit binding between ASPD and neurons can be selected as a candidate substance for a substance that inhibits neurotoxicity caused by ASPD.
The neurons and the imaging method can be the same as those used for the evaluation method according to the present disclosure. Furthermore, in one or more embodiments, the indicator of the amount of ASPD bound may be the amount itself of the ASPD bound to neurons or may be ED50 or the ratio thereof. For example, it can be evaluated that the higher the ED50 in the case where the candidate substance and ASPD coexist, the higher the ability of inhibiting neurotoxicity of ASPD.
In this aspect, when calculating the amount of the ASPD bound or the indicator thereof, it is easy to eliminate, from image data, information on large aggregates of APSD and/or information on nonspecific ASPD not bound to neurons. This makes it possible to improve the quantitativeness and/or the screening sensitivity in one or more embodiments.
In one or more embodiments, the present disclosure may relate to the following:
[1] A synthesized, isolated, or purified polypeptide,
the polypeptide containing an amino acid sequence represented by Formula (I) below:
where X1 is arginine (Arg), histidine (His), or lysine (Lys),
X2 is arginine (Arg), lysine (Lys), aspartic acid (Asp), histidine (His), tryptophan (Trp), glutamine (Gln), tyrosine (Tyr), or threonine (Thr),
X3 is any amino acid, and
X4 is tryptophan (Trp), phenylalanine (Phe), or tyrosine (Tyr).
[2] The polypeptide according to [1], wherein X3 is alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), or valine (Val).
[3] The polypeptide according to [1] or [2], wherein X2 is histidine (His), tryptophan (Trp), glutamine (Gin), tyrosine (Tyr), or threonine (Thr).
[4] A synthesized, isolated, or purified polypeptide,
the polypeptide containing an amino acid sequence represented by Formula (II) below:
where X1 is arginine (Arg), histidine (His), or lysine (Lys),
X2 is any amino acid,
X3 is leucine (Leu), glycine (Gly), isoleucine (Ile), tryptophan (Trp), arginine (Arg), methionine (Met), aspartic acid (Asp), glutamic acid (Glu), alanine (Ala), valine (Val), or lysine (Lys), and
X4 is tryptophan (Trp), phenylalanine (Phe), or tyrosine (Tyr).
[5] The polypeptide according to [4], wherein X2 is alanine (Ala), arginine (Arg), aspartic acid (Asp), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), or valine (Val).
[6] The polypeptide according to [4] or [5], wherein X3 is leucine (Leu), isoleucine (Ile), tryptophan (Trp), arginine (Arg), aspartic acid (Asp), or lysine (Lys).
[7] A synthesized, isolated, or purified polypeptide, the polypeptide having an amino acid sequence represented by HANW, HVNW, HFHW, or HFYW.
[8] The polypeptide according to any one of [1] to [7], the polypeptide having, at its N terminus, an amino acid sequence represented by the Formula (I) or (II), or HANW, HVNW, HFHW, or HFYW.
[9] The polypeptide according to any one of [1] to [8], wherein the amino acid sequence has a length of 4 to 50 amino acids.
[10] The polypeptide according to any one of [1] to [9], wherein its C-terminal carboxyl group has been removed or amidated.
[11] The polypeptide according to any one of [1] to [10], wherein at least one of peptide bonds has been N-alkylated.
[12] The polypeptide according to any one of [1] to [11], wherein the polypeptide has an ability to inhibit binding between ASPD and neurons.
[13] A peptide mimic having a structure mimicking the structure of a polypeptide according to any one of [1] to [12], the peptide mimic having an ability to inhibit binding between ASPD and neurons.
[14] A vector for expressing a polypeptide according to any one of [1] to [12], the vector containing a polynucleotide that encodes the polypeptide.
[15] A composition containing a polypeptide according to any one of [1] to [12] or a peptide mimic according to [13].
[16] A method of evaluating a substance that inhibits binding between amylospheroids (ASPD) and neurons, the method including:
contacting a liquid mixture containing a substance to be evaluated and ASPD with cultured neurons, or contacting ASPD with cultured neurons in the presence of the substance to be evaluated,
thereafter providing the ASPD and the neurons with different labels from each other to perform imaging, and
obtaining an indicator of binding of the ASPD to the neurons from data obtained by the imaging to evaluate the substance to be evaluated using the indicator
[17] A method of screening a substance that inhibits neurotoxicity caused by amylospheroids (ASPD), the method including:
contacting a liquid mixture containing a candidate substance and ASPD with cultured neurons or contacting the ASPD with the cultured neurons in the presence of the candidate substance,
thereafter providing the ASPD and the neurons with different labels from each other to perform imaging, and
obtaining an indicator of binding of the ASPD to the neurons from the data obtained by the imaging to select a candidate substance using the indicator.
Hereinafter, one or more embodiments of the present invention will be further described using examples.
1. Evaluation of ASPD-Inhibitory Peptides
Evaluation of peptides that inhibits binding of ASPD to neurons was performed by the following method.
Step 1: Adding a liquid mixture containing peptide and ASPD, which have been mixed together and then incubated, to cultured mature neurons.
Step 2: Degenerating (immobilizing) the neurons.
Step 3: Immunostaining the neurons and the ASPD, respectively, to perform imaging.
Step 4: Analyzing the image to quantify the ASPD bound to the neurons.
Step 5: Evaluating the peptides using the amount of the ASPD bound and/or an analytical value obtained therefrom.
Steps 1 to 3 were performed as follows:
A liquid mixture of peptide and synthetic ASPD was incubated at 4° C. for 15 minutes to pretreat the ASPD and this liquid mixture was added to primary cultured rat hippocampal neurons cultured in a 96-well plate.
After incubation at 37° C. for 15 minutes in a CO2 incubator, the wells were washed with PBS. Then, 2% paraformaldehyde (50 μl/well) was added thereto for immobilization performed for 20 minutes at 37° C. and further 4% paraformaldehyde (50 μl/well) was added thereto for immobilization performed for 20 minutes at 37° C. After washing and blocking, a primary antibody solution containing anti-ASPD antibody (mASD3) and anti-MAP2 antibody (trade name: sc-20172, manufactured by SANTACRUZ) was added, which then was left overnight. After washing, a secondary antibody solution containing Alexa Fluoro (trademark) 568 anti-mouse antibody (trade name: A11031, manufactured by life technologies), Alexa Fluoro (trademark) 488 anti-rabbit antibody (trade name: A11034, manufactured by life technologies), and DAPI (trade name: D523, manufactured by Dojindo) was added and then fluorescence microscopic observation was performed.
Examples of the results of the images obtained by microscopic observation are shown in
Subsequently, steps 4 to 5 were performed as follows: From the microscopic observation data, a calibration curve as shown in
Titer=ED50[ASPD+Peptide]/ED50[ASPD]
As shown in
In addition, as shown in
2. Surface Plasmon Resonance (SPR)
Synthetic ASPD were solid-phased on a chip and each peptide shown in Table 3 below was allowed to flow as an analyte. Then the dissociation constant (KD) was determined by surface plasmon resonance (SPR).
The peptides used herein are the peptides 01, 05, 08, 09, 15, and 18 shown in
The results are shown in Table 3 below.
As shown in Table 3 above, it was shown that peptides having an effect of inhibiting the binding of ASPD to neurons in the evaluation performed in section 1 above had an ability to bind to ASPD.
3. Treatment of Peptide Terminus
The ASPD-binding inhibitory ability (titer) was calculated in the same manner as described above using peptides obtained by amidating (—CONH2) or removing the COOH at the C-terminus of the peptide 22 (HFKW) and a peptide from which the amino group at the N-terminus was removed. The results are shown in
4. N-Methylation of Peptide Bond
One or two of the peptide bonds of the peptide 22 (HFKW) were N-methylated, and the dissociation constant (KD) was determined by surface plasmon resonance (SPR) in the same manner as in section 2 described above. As a result, each of the N-methylated peptides 22 was found to have an ability to bind to ASPD.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2016-081031 | Apr 2016 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2017/015090 | 4/13/2017 | WO | 00 |
