Composition for diagnosing diseases associated with COX2 overexpression and screening method therefor

Abstract

A composition for diagnosing diseases associated with COX2 overexpression, containing a compound with a structural feature of exhibiting strong binding activity to a COX2 protein; and a screening method therefor.

Claims

1. An analysis method comprising: measuring binding between a compound and a cyclooxygenase 2 (COX2) protein defined by SEQ ID NO: 1 in a sample obtained from a subject, wherein the compound is a compound defined by Chemical Formula 1: ##STR00011## wherein, at least one atom of the compound defined by Chemical Formula 1 is a radioactive isotope, R.sub.1 is hydrogen or C.sub.1-C.sub.7 alkylcarbonyl, R.sub.2 and R.sub.3 are each independently hydrogen, C.sub.1-C.sub.7 alkylcarbonyl, or ##STR00012## and n is an integer of 5 to 15, and wherein at least one of R.sub.1, R.sub.2, and R.sub.3 is a C.sub.1-C.sub.7 alkylcarbonyl.

2. The method of claim 1, wherein the compound contains a functional group that interacts with the COX2 protein at one or more amino acids selected from the group consisting of N181, T564, S567, and S565.

3. The method of claim 1, wherein the binding is a hydrogen bond with at least one amino acid selected from the group consisting of N181, T564 and S567 or a nucleophilic acyl substitution reaction with S565.

4. The method of claim 3, wherein at least one atom of the C.sub.1-C.sub.7 alkylcarbonyl of at least one of R.sub.1, R.sub.2, and R.sub.3 is a radioactive isotope.

5. The method of claim 3, wherein the compound defined by Chemical Formula 1 is labeled with one or more radioactive isotopes selected from the group consisting of .sup.2H, .sup.3H, .sup.11C, .sup.13C, .sup.14C, .sup.13N, .sup.15N, .sup.15O, and .sup.17O.

6. The method of claim 3, wherein the compound defined by Chemical Formula 1 above is selected from compounds defined by the following Chemical Formulas 2 to 11: ##STR00013## ##STR00014## and, wherein in all compounds defined by Chemical Formulas 2 to 11, an atom of an acetyl group is a radioactive isotope.

7. The method of claim 1, wherein the measuring is done by photographic film or scintillation counters.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a diagram illustrating confirming and quantifying binding energy at which compounds according to the present invention directly bind to COX2 through a docking simulation.

(2) FIG. 2 is a schematic diagram illustrating that N-acetyl sphingosine (N-AS) directly binds to COX2 through hydrogen bonds with N181, T564, and S567 of a COX2 protein and a nucleophilic interaction with S565.

(3) FIG. 3 illustrates a result of evaluating the degree of binding between N-acetyl sphingosine and wild type COX2 or mutant COX2 (M181A, T564A, S565A or S567A).

(4) FIGS. 4A to 4C illustrate a result of showing a concentration remaining in the brain for each time period after oral (p.o. 10 mg/kg) or intravenous administration (i.v. 1 mg/kg) of N-acetyl sphingosine to normal mice (FIG. 4A), a result of showing a concentration remaining in the brain after 24 hours (FIG. 4B), and a pharmacokinetic test analysis result for a brain distribution (FIG. 4C) (n=3/group).

(5) FIG. 5 illustrates a result of confirming expression levels of COX2 protein in microglia (CX3CR1.sup.+) derived from wild-type or APP/PS1 mice at 3 months, 5 months and 9 months (WT: wild type, AD: Alzheimer's animal model).

(6) FIG. 6 illustrates a result of confirming the degree of binding of the COX2 protein and [.sup.14C] in microglia isolated from the brain after administration of [.sup.14C]N-acetyl sphingosine to 5-month-old wild-type or APP/PS1 mice (WT: wild type, AD: Alzheimer's animal model).

(7) FIG. 7 illustrates results of confirming and quantifying through Western blot that the expression of the COX2 protein is increased in a neuroblastoma cell line (neuroblastoma SH-SY5Y).

(8) FIG. 8 illustrates a result showing a result of detecting an amount of [.sup.14C] binding to the COX2 protein by collecting cells after treatment with [.sup.14C]N-acetyl sphingosine in a neuroblastoma cell line (neuroblastoma SH-SY5Y) for 1 hour.

MODE FOR CARRYING OUT INVENTION

(9) Hereinafter, the present invention will be described in detail.

(10) However, the following Examples are just illustrative of the present invention, and the contents of the present invention are not limited to the following Examples.

Experimental Materials and Experimental Methods

(11) 1. Compounds

(12) The structures and names of compounds used in experiments in the present invention were shown below:

(13) ##STR00009## ##STR00010##
2. Docking Simulation

(14) The three-dimensional structure of a COX2 protein (PDB code: 3HS5) and the molecular docking of each compound according to the present invention were analyzed using Discovery Studio 2018 implemented based on a DS-CDOCKER protocol. A docking position of the compound was simulated by designating a substrate binding site region including R106, Y341, Y371 and S516 of an amino acid sequence of a COX2 protein (GeneBank accession No.AAR23927.1). Arachidonic acid (AA), a substrate for COX2, was used as a positive control. A specific experimental method was performed with reference to Nat Commun. 2018 Jan. 9; 9(1):128, etc.

(15) 3. Enzymatic Analysis of COX2

(16) The binding activity of wild type COX2 or mutant COX2 (N181A, T564A, S565A, S567A) and N-acetyl sphingosine (N-AS) was analyzed by filter binding assay (Nat Commun. 2018 Apr. 16; 9(1):1479).

(17) The binding rate (Vbinding) of [.sup.14C]N-acetyl sphingosine (American Radiolabeled Chemicals, ARC1024) to wild-type COX2 and mutant COX2 (N181A, T564A, S565A, S567A) was expressed as the concentration of N-acetyl sphingosine. The nonlinear regression analysis of a saturation plot showed the binding activity of N-acetyl sphingosine and wild-type COX2 or mutant COX2 (N181A, T564A, S565A, S567A) using K.sub.cat (catalyst constant) and K.sub.M(Michaelis-Menten constant). By using the calculated K.sub.cat and K.sub.M, K.sub.cat/K.sub.M (catalytic efficiency), which has been widely used as a measure of enzyme performance, was calculated from wild-type COX2 and mutant-type COX2 (N181A, T564A, S565A, S567A).

(18) 4. Mouse

(19) Mouse experiments have been approved by the Kyungpook National University Institutional Animal Care and Use Committee (IACUC). A transgenic mouse line overexpressing APPswe (hAPP695swe) or PS1 (presenilin-1M146V) based on C.sub.57BL/6 mice (Charles River, UK) was used [Hereinafter, APP mouse: refers to a mouse overexpressing APPswe, PS1 mouse: refers to a mouse overexpressing presenilin-1M146V; GlaxoSmithKline]

(20) 5. Immunofluorescence

(21) Microglia were isolated from the cerebrum of a 3-month-old, 5-month-old or 9-month-old wild type or APP/PS1 mouse, treated with anti-COX2 (rabbit, 1:10, Abcam) and anti-CX3CR1 (mouse, 1:100, Biolegend) antibodies, and then cultured. The microglia were quantified and analyzed for the percentage of cells stained with anti-COX2 among all microglia using an Operetta CLS High-Content Analysis System (PerkinElmer, USA).

(22) 6. Method of Measuring Binding Degree Between COX2 and [.sup.14C]

(23) After oral administration of 10 Ci of [.sup.14C]N-acetyl sphingosine (American Radiolabeled Chemicals, ARC1024) to 5-month-old wild-type and APP/PS1 mice, microglia were isolated from the cerebra of the mice after 1 hour.

(24) In addition, neuroblastoma SH-SY5Y was treated with 2 Ci of [.sup.14C]N-acetyl sphingosine (American Radiolabeled Chemicals, ARC1024) for 1 hour, and then cells were collected.

(25) The COX2 protein of isolated microglia and neuroblastoma SH-SY5Y were isolated by immunoprecipitation, and then liquid scintillation counting was performed on [.sup.14C].

(26) 7. Western Blot

(27) The expression of the proteins was analyzed using Western blotting. First, antibodies against COX2 (abcam) and -actin (Santa Cruz) were used, and the densitometric quantification was performed using ImageJ software (US National Institutes of Health).

(28) 8. Statistical Analysis

(29) A T-test of students was performed to compare two groups, while for comparison of multiple groups, repeated measurement analysis of a Tukey's HSD test and a variance test was performed according to an SAS statistical package (release 9.1; SAS Institute Inc., Cary, NC). *p<0.05, **p<0.01, and ***p<0.001 were considered significant.

Experimental Results

(30) 1. Confirming that Compounds of the Present Invention Bind Directly to COX2

(31) The docking simulation was performed to confirm whether the compounds of the present invention listed in the experimental methods directly bind to COX2, and the binding energy of these compounds to COX2 was compared with the binding energy of arachidonic acid, a substrate of COX2, to COX2.

(32) The result thereof was illustrated in FIG. 1.

(33) As illustrated in FIG. 1, it was confirmed that the binding energy of COX2 and arachidonic acid (AA) and the binding energy of the compounds according to the present invention to COX2 had similar values.

(34) That is, it was found that the compounds of the present invention can bind to COX2 well in the same level as that arachidonic acid (AA), a substrate of COX2, binds to COX2.

(35) 2. Confirmation of New Binding Site of N-Acetyl Sphingosine (N-AS) to COX2

(36) Referring to Experimental Result 1, it was confirmed that among 10 kinds of compounds applied in the experiment, the N-acetyl sphingosine (N-AS) compound exhibited the highest binding energy with COX2.

(37) Accordingly, the present inventors selected the N-AS compound and analyzed the docking simulation result to confirm the structural characteristics of these compounds binding to COX2.

(38) The result thereof was illustrated in FIG. 2.

(39) As illustrated in FIG. 2, it was confirmed that the N-AS bound to COX2 through hydrogen bonds (FIG. 2, dotted lines) with N181, T564, and S567 of the COX2 protein, and through these hydrogen bonds, S565 had a nucleophilic acyl substitution reaction (arrow). In addition, it was confirmed that a site at which the N-AS bound to COX2 included a binding site (R106, Y341, Y371, S516) of arachidonic acid (AA), a substrate of COX2 known in the related art (FIG. 2).

(40) 3. Confirmation of Binding Degree of N-Acetyl Sphingosine (N-AS) to COX2

(41) In Experimental Result 2, it was confirmed that the positions of N181, T564, S567 and S565 of the COX2 protein played a very important role in binding to N-AS through a docking simulation.

(42) Accordingly, the present inventors attempted to confirm once again through an enzymatic analysis method that a hydrogen bond at each amino acid position of the COX2 protein played an important role in binding to N-AS.

(43) That is, after preparing a mutant COX2 protein in which asparagine (N181), threonine (T564), or serine (S567, S565) capable of forming a hydrogen bond as a polar amino acid was substituted with alanine (A), a non-polar amino acid, which level of the binding activity to the N-AS was exhibited was compared by comparing each mutant COX2 with wild-type COX2.

(44) The result thereof was illustrated in FIG. 3.

(45) As illustrated in FIG. 3, the binding of the N-AS to the wild-type COX2 was saturated as the concentration of N-AS increased, and the K.sub.M and K.sub.cat values were 46.01 m and 0.48 min-1, respectively. Through this, it was confirmed once again that the N-AS bound well to the COX2.

(46) On the other hand, in the case of mutant COX2 (N181A, T564A, S565A and S567A) in which the amino acid at the N-AS binding site of COX2 confirmed in Experiment 2 was substituted with alanine, it was confirmed that when the catalytic efficiency (K.sub.cat/K.sub.M), which was widely used as a measure of enzyme performance, was compared with the catalytic efficiency of the wild-type COX2, the catalytic efficiency of the mutant COX2 decreased compared to the wild-type COX2. Among them, it was confirmed that when S565 was mutated, the catalytic efficiency decreased the most, and these results showed that the N-AS directly bound to the COX2 protein through the hydrogen bonds with N181, T564, S565, and S567 of COX2 and the nucleophilic acyl substitution reaction.

(47) Therefore, it was found that a compound capable of having the hydrogen bonds with N181, T564, S565, and S567 of COX2 and the nucleophilic acyl substitution reaction may strongly bind to COX2.

(48) 4. Confirmation of Brain Distribution of N-Acetyl Sphingosine

(49) The present inventors have confirmed that the compounds of the present invention are very excellent in binding activity to COX2 through Experimental Result 1. Therefore, it could be determined that these compounds could be used for diagnosis of various diseases caused by overexpression of the COX2 protein. In particular, it was confirmed whether the compounds of the present invention could be used for degenerative brain diseases in which the COX2 protein is overexpressed.

(50) In order to apply the compounds of the present invention to the diagnosis of degenerative brain diseases, it is important to distribute the compounds well to the brain after administration. To confirm this, the N-AS was administered orally (10 mg/kg) or through tail vein (1 mg/kg), and then the brain was extracted by time to measure the concentration of N-AS, and after 24 hours, the brain was extracted to measure the concentration of the remaining N-AS.

(51) The result thereof was illustrated in FIG. 4.

(52) As a result, it was confirmed that the concentration of N-AS was high in the brain (FIGS. 4A and 4B). On the other hand, as a result of confirming pharmacokinetic parameters in the brain, it was confirmed that the brain distribution value was 3.18 for oral administration and 2.16 for tail vein administration (FIG. 4C).

(53) From these results, it can be seen that the N-AS exhibits a high brain distribution in terms of pharmacokinetics to be very usefully used in the development of diagnostic substances for brain diseases such as neurodegenerative diseases.

(54) 5. Confirming that COX2 Expression in Microglia was Increased from the Early Stage of Alzheimer's

(55) According to previous studies, it has been reported that the expression level of COX2 protein was increased in brain microglia of patients with degenerative neuroinflammatory diseases including Alzheimer's (Curr Neuropharmacol. 2010 March; 8(1): 62-68).

(56) The present inventors confirmed from when the COX2 expression in microglia increased in an Alzheimer's environment.

(57) As a result, it was confirmed that the COX2 expression in microglia was significantly increased from 5 months, which was an early stage of the occurrence of Alzheimer's, compared with a control as illustrated in FIG. 5.

(58) Through the results, it was confirmed that the COX2 expression in microglia was increased from the early stage of the occurrence of Alzheimer's, and it could be determined that the compounds of the present invention, which have excellent brain distribution and direct binding activity with the COX2 protein, may be used for diagnosis or prognosis prediction from the early stage of Alzheimer's.

(59) 6. Confirming that COX2 Expression in Microglia was Increased from the Early Stage of Occurrence of Alzheimer's by Using the Compound of the Present Invention

(60) The present inventors confirmed that the expression of the COX2 protein in brain microglia was increased from the early stage of the occurrence of Alzheimer's in Experimental Result 5, and then attempted to determine whether the increased expression of the COX2 protein may be directly detected in the Alzheimer's brain using the compounds of the present invention.

(61) In other words, [.sup.14C]N-acetyl sphingosine ([.sup.14C]N-AS), in which carbon of N-AS which strongly bound to COX2 and had a brain distribution was substituted with an isotope, was administered orally (10 Ci) to a 5-month-old Alzheimer's animal model. After 1 hour, the COX2 in microglia was isolated from the control and the Alzheimer's animal model by immunoprecipitation, and the amount of COX2 labeled with [.sup.14C] was confirmed.

(62) The result thereof was illustrated in FIG. 6.

(63) As can be seen in FIG. 6, it was confirmed that the [.sup.14C]-labeled COX2 was increased in the Alzheimer's animal model compared to a control (WT).

(64) Therefore, it can be determined that the compounds of the present invention can be used for diagnosis or prognosis prediction of various diseases caused by overexpression of the COX2 protein, including Alzheimer's.

(65) 7. Confirming that COX2 Expression of Cancer Cells, Neuroblastoma SH-SY5Y, was Increased.

(66) According to previous studies, it has been reported that the expression level of the COX2 protein was increased in cancer cells (J Cell Physiol. 2019 May; 234(5):5683-5699).

(67) Accordingly, the present inventors confirmed whether the COX2 expression was increased in cancer cells.

(68) As a result, it was confirmed that the COX2 expression of the cancer cells, neuroblastoma SH-SY5Y was significantly increased compared to a control (normal nerve cells) as illustrated in FIG. 7.

(69) Through the results, it was confirmed that the expression of COX2 was increased in cancer cells, and it was determined that the compounds of the present invention having very excellent direct binding activity with the COX2 protein may be useful for diagnosis or prognosis prediction of cancer.

(70) 8. Confirming that COX2 Expression of Cancer Cells, Neuroblastoma SH-SY5Y, was Increased by Using the Compound of the Present Invention

(71) The present inventors confirmed that the expression of the COX2 protein in cancer cells, neuroblastoma SH-SY5Y was increased in Experimental Result 7, and then attempted to confirm whether the increased expression of the COX2 protein may be directly detected in the cancer cells using the compounds of the present invention.

(72) In other words, [.sup.14C]N-acetyl sphingosine ([.sup.14C]N-AS), in which carbon of N-AS strongly binding to COX2 was substituted with an isotope, was treated to the cancer cells (2 Ci). After 1 hour, the COX2 was isolated from the control and the cancer cells, neuroblastoma SH-SY5Y by immunoprecipitation, and the amount of COX2 labeled with [.sup.14C] was confirmed.

(73) The result thereof was illustrated in FIG. 8.

(74) As can be seen in FIG. 8, it was confirmed that the [.sup.14C]-labeled COX2 was increased in the cancer cells, neuroblastoma SH-SY5Y compared to a control.

(75) Therefore, it can be determined that the compounds of the present invention can be used for diagnosis or prognosis prediction of various diseases caused by overexpression of the COX2 protein, including Alzheimer's and cancer.

INDUSTRIAL APPLICABILITY

(76) The compounds provided in the present invention not only have very excellent binding force with COX2, but also have very high blood-brain barrier (BBB) permeability, and can be very useful in diagnosing and predicting prognosis of diseases associated with COX2 overexpression including neurodegenerative diseases. Therefore, the compounds of the present invention have very excellent industrial applicability.