ACE2 inhibitor compounds

Abstract

To develop an ACE2 inhibitory compound that can prevent coronavirus infection.SOLUTION: Provided is an ACE2 inhibitory compound containing elastin or elastin degradation products (including corresponding chemically synthesized compounds) as an active ingredient. By using the ACE2 inhibitory compound of the present invention as a drug or supplement, not only for the current new coronavirus, but also for future mutant strains, useful effects against new coronavirus infections, such as prevention, treatment, and enhancement of vaccine effects, can be expected.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an ACE2 inhibitory compound. More specifically, the present invention relates to an ACE2 inhibitor for the purpose of preventing coronavirus infection and the like.

Background Art

[0002] Recently, a novel coronavirus infection called COVID-19 has spread worldwide and has become a problem. On the surface of this novel coronavirus, there are a plurality of spike proteins. It is known that this spike protein binds to a receptor present on the surface of human cells and then invades the cells, causing infection in humans. This human receptor is called ACE2 (Angiotension II-Converting Enzyme), and it has been reported that when vaccinated with vaccines developed by companies such as Pfizer, Moderna, and AstraZeneca, the vaccine blocks the spike protein and inhibits binding to ACE2, preventing infection.

[0003] In addition, there is a fact that new mutant strains of the coronavirus keep emerging one after another. In particular, it has been reported that when a large number of mutations occur in the ACE2 binding region of the spike protein, the effect of the vaccine weakens and the prevention of infection decreases. Therefore, as a countermeasure against the continuously mutating coronavirus, attempts have been made to administer additional doses of the vaccine and for pharmaceutical companies to develop new vaccines. However, in the first place, the coronavirus is an RNA virus, and it can be said that it is impossible to prevent the emergence of mutant strains. From this, as long as virus mutant strains emerge, measures such as administering additional doses of the vaccine and developing new vaccines will be necessary.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

[0005] Incidentally, the inventors have previously invented an ACE inhibitor compound for suppressing the production of angiotensin II, which has a blood pressure-raising effect, and have filed a patent application (Patent Document 1). In this application, based on this knowledge, we have started developing an ACE2 inhibitor compound that may be able to prevent coronavirus infection, etc. In other words, to prevent coronavirus infection, it is considered useful to either block the spike protein present on the surface of the coronavirus with a vaccine, or to inhibit ACE2 present on the surface of human cells. This application aims to develop an ACE2 inhibitor compound that can prevent coronavirus infection by inhibiting ACE2 itself, which is the target of the spike protein that serves as the entry point for infection against current coronaviruses or mutated coronaviruses that may appear in the future. [Means for solving the problem]

[0006] As a result of diligent research, the inventors discovered that elastin or elastin degradation products have an inhibitory effect on ACE2, and thus completed the invention. The present invention consists of the following configuration. [1] An ACE2 inhibitory compound characterized by containing elastin or elastin degradation products (including chemically synthesized compounds equivalent thereto) as an active ingredient.

[0007] [2] The ACE2 inhibitor compound according to [1], wherein the molecular weight of elastin is less than 300,000, preferably less than 100,000, and particularly preferably the average molecular weight is between 20,000 and less than 30,000. [3] The ACE2 inhibitor compound according to [1], wherein the molecular weight of the elastin degradation product is 100 to less than 2,000, preferably 100 to less than 1,000. [4] Elastin degradation products are repeating sequences in elastin. An ACE2 inhibitor compound as described in [3], which is represented by any of the following in the sequence VGVAPGVGVAPGVGVAPGVGVAPG: dipeptide, tripeptide, tetrapeptide, pentapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide. [5] Elastin breakdown products are contained in elastin. An ACE2 inhibitory compound as described in [3], represented by one of the tripeptides X1X2P (X1 or X2 = an amino acid represented by A, G, S, V, L, T, I, C, M, D, N, E, Q, H, K, R, F, Y, W, P).

[0008] [6] Elastin breakdown products, An ACE2 inhibitor compound as described in [3], which is a peptide with a molecular weight of less than 1000, comprising one or more of Sequence IDs 4 to 14. [7] An ACE2 inhibitor compound as described in [3], wherein the elastin degradation product is represented by any of SEQ ID NOs: 4 to 14. [8] An ACE2 inhibitor compound described in [3], represented by the elastin degradation product of Sequence ID No. 7.

[0009] A drug or supplement for coronavirus, characterized in that it contains one of the ACE2 inhibitor compounds listed in [9][1] to [8] as an active ingredient, and the ACE2 inhibitor compound inhibits ACE2, thereby preventing or treating coronavirus infection. A drug or supplement for coronavirus, characterized in that it contains one of the ACE2 inhibitor compounds listed in

[10] [1] to [8] as an active ingredient, and the ACE2 inhibitor enhances the effect of the coronavirus vaccine by inhibiting ACE2. [Effects of the Invention]

[0010] This invention makes it possible to provide useful ACE2 inhibitory compounds. By using the ACE2 inhibitor compound of the present invention as a drug or supplement, useful effects against COVID-19, such as prevention, treatment, and enhancement of vaccine efficacy, can be expected not only against the current coronavirus but also against future variant strains. [Brief explanation of the drawing]

[0011] [Figure 1] This figure shows the results of investigating the ACE2 inhibitory activity of each compound. [Figure 2] This figure shows the results of calculating the IC50 for each compound. [Modes for carrying out the invention]

[0012] The ACE2 inhibitory compound of the present invention will now be described.

[0013] The ACE2 inhibitory compound of the present invention is characterized by containing elastin or elastin degradation products (including chemically synthesized compounds equivalent thereto) as an active ingredient. That is, it exerts ACE2 inhibitory activity using elastin or its degradation products as shown in Sequence ID No. 1 as an active ingredient. The molecular weight of elastin can typically be less than 300,000, preferably less than 100,000, and particularly preferably have an average molecular weight of 20,000 to less than 30,000. Furthermore, the molecular weight of the elastin degradation product can typically be between 100 and less than 2,000, preferably between 100 and less than 1,000.

[0014] When producing the ACE2 inhibitor compound of the present invention by decomposing elastin, it can be produced using known methods (International Publication No. 2006 / 046626). That is, biological tissues such as aortic blood vessels obtained from mammals such as pigs, horses, cows, and sheep are used as raw materials, but biological tissues such as avian arterial blood vessels and fish aortic bulbs may also be used as raw materials. First, these biological tissues are subdivided by degreasing, homogenizing, etc. Subsequently, the subdivided sample is treated with an alkaline solution or the like to remove collagen and the like to obtain insoluble elastin. Further, the insoluble elastin is hydrolyzed with an acid or an alkali or the like, and a separation operation is performed on this treatment solution to obtain water-soluble elastin. The water-soluble elastin is treated by enzymatic treatment or the like, and the obtained treatment solution is subjected to column purification by membrane separation or gel filtration to obtain a fraction having a molecular weight of 100 to less than 2,000.

[0015] The ACE2 inhibitory compound of the present invention can also be chemically synthesized. That is, since the ACE2 inhibitory compound of the present invention is composed as a peptide, it can be chemically synthesized by a peptide synthesizer or the like.

[0016] As a preferred embodiment of the elastin degradation product, the elastin degradation product can be a compound having a molecular weight of less than 1000, represented by any one of a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a heptapeptide, an octapeptide, a nonapeptide, a decapeptide, an undecapeptide, and a dodecapeptide, which is included in the sequence of VGVAPGVGVAPGVGVAPGVGVAPG (SEQ ID NO: 2). The compound represented by SEQ ID NO: 2 is represented by the single-letter notation of amino acids, with the left side being the N-terminus and the right side being the C-terminus (hereinafter, the same).

[0017] Also, as another preferred embodiment of the elastin degradation product, the elastin degradation product can be a compound having a molecular weight of less than 1000, represented by a tripeptide of X1X2P (where X1 or X2 is an amino acid represented by any one of A, G, S, V, L, T, I, C, M, D, N, E, Q, H, K, R, F, Y, W, P).

[0018] Furthermore, in other preferred embodiments of the elastin degradation product, the elastin degradation product may be a peptide with a molecular weight of less than 1,000, comprising any or more of SEQ ID NOs: 4 to 14, particularly preferably the peptide represented by any of SEQ ID NOs: 4 to 14, and most preferably the peptide represented by SEQ ID NO: 7. By adopting these preferred embodiments of elastin degradation products, the design and chemical synthesis of ACE2 inhibitory compounds become easier, and the ACE2 inhibitory compounds of the present invention can be obtained with high purity and reproducibility.

[0019] The ACE2 inhibitory compound of the present invention has the chemical structure described above, but is not limited thereto. In other words, in view of the present invention, which aims to exert an ACE2 inhibitory effect by a compound having the chemical structure shown above, the present invention may include not only compounds that exert an ACE2 inhibitory effect themselves, but also so-called DDS-modified compounds that undergo various metabolic processes in the body to form the compound structure of the present invention and exert an ACE2 inhibitory effect. In this sense, the ACE2 inhibitory compound of the present invention may be partially chemically modified.

[0020] The ACE2 inhibitory compound of the present invention is not particularly limited as long as it can exert an ACE2 inhibitory effect and can be used in a variety of applications. For example, it can be used in the form of pharmaceuticals, health foods (supplements), cosmetics, etc. In terms of dosage form, it can be used in the form of tablets, powders / granules, capsules, injections, oral solutions / syrups, etc.

[0021] Preferred applications for the ACE2 inhibitor compound of the present invention include the prevention of coronavirus infection. In other words, when a person ingests or inoculates with the ACE2 inhibitor compound of the present invention, the binding of the coronavirus spike protein to ACE2 is inhibited, resulting in prevention of coronavirus infection or enhancement of the effectiveness of coronavirus vaccines. In this embodiment, the present invention can be configured as a drug or supplement for coronavirus, characterized in that the ACE2 inhibitor compound is an active ingredient, and the ACE2 inhibitor compound prevents or treats coronavirus infection by inhibiting ACE2. In a different embodiment, the present invention can be configured as a drug or supplement for coronavirus, characterized in that the ACE2 inhibitor compound is an active ingredient, and the ACE2 inhibitor enhances the effect of the coronavirus vaccine by inhibiting ACE2.

[0022] The ACE2 inhibitory compound of the present invention may contain various additives. That is, when the ACE2 inhibitory compound of the present invention is used as an active ingredient in a composition such as a drug or supplement, appropriate additives can be selected depending on the dosage form. Examples of such additives include excipients, stabilizers, antioxidants, pH adjusters, emulsifiers, solubilizers, and isotonic agents.

[0023] Examples of excipients include sugar alcohols such as D-sorbitol, mannitol, xylitol, and propylene glycol; sugars such as glucose, sucrose, lactose, and fructose; crystalline cellulose; calcium hydrogen phosphate; wheat starch; rice starch; corn starch; potato starch; dextrin; β-cyclodextrin; light anhydrous silicic acid; titanium dioxide; magnesium aluminometasilicate; talc; kaolin; olive oil; gelatin; casein; and pectin.

[0024] Examples of stabilizers include pectin, dextran, glycerin, polysaccharides (guar gum, gum arabic, xanthan gum, gellan gum, sclero gum, tamarind seed gum), starches (hydroxypropyl starch, phosphate monoesterified phosphate cross-linked starch), agar, gelatin, and methylcellulose.

[0025] Examples of antioxidants include L-ascorbic acids (L-ascorbic acid, calcium L-ascorbate, stearate L-ascorbic acid, palmitate L-ascorbic acid), erythorbic acid, butylhydroxyanisole, γ-oryzanol, catechin, licorice oil extract, quercetin, citric acid, glyceryl monocitrate, tocotrienol, tocopherols (d-α-tocopherol, d-γ-tocopherol, d-δ-tocopherol), lecithin, ferulic acid, Melaloika essential oil, sunflower seed extract, grape seed extract, propolis extract, tree fern / ginkgo extract, bayberry extract, and rosemary extract.

[0026] Examples of pH adjusters that can be used include citric acid, gluconic acid, succinic acid, malic acid, phosphoric acid, maleic acid, tartaric acid, lactic acid, calcium lactate, ammonium acetate, meglumine, and glucono-δ-lactone.

[0027] Examples of emulsifiers include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; and shellac wax, beeswax, carnauba wax, whalebone, lanolin, liquid lanolin, reduced lanolin, hard lanolin, and cyclic lanolin. Examples include waxes such as norin, lanolin wax, candelilla wax, Japanese wax, montan wax, shellac wax, and rice wax; sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, glycerin fatty acid esters, calcium stearoyl lactylate, calcium citrate, calcium phosphate salts; pagoda tree saponin, soybean saponin, tea seed saponin, beet saponin; sphingolipids, tomato glycolipids, plant sterols, plant lecithin, enzyme-hydrolyzed lecithin, egg yolk lecithin, yucca foam extract, barley husk extract, and quillaja extract.

[0028] Examples of solubilizing agents include macrogol 1500, macrogol 4000, macrogol 6000, macrogol 20000, lauromacrogol, polyoxyl stearate 40, polysorbate 80, polyoxyethylene hydrogenated castor oil 60, D-mannitol, sodium citrate, anhydrous citric acid, tartaric acid, sodium bisulfite, and arginine.

[0029] Examples of isotonic agents include glycerin, propylene glycol, sodium chloride, and salt. Examples include potassium hydroxide, sodium dihydrogen phosphate, potassium dihydrogen phosphate, citric acid, sodium citrate, sorbitol, mannitol, glucose, and boric acid. [Examples]

[0030] The ACE2 inhibitory compound of the present invention will be further explained using experimental examples.

[0031] <<ACE2 Inhibition Test Using Elastin-Derived Oligopeptides>> <Experiment Overview> 1. The experiment was conducted according to the protocol of the ACE2 Inhibitor Screening Kit (K310, Bio Vision, USA). The ACE2 enzyme used was a human recombinant protein produced in CHO cells from a human-derived sequence. 2. Regarding ACE2 inhibitory activity, we investigated using compounds numbered 1 to 11 shown in Figure 1, as well as porcine-derived water-soluble elastin, for a total of 12 compounds.

[0032] 3. Compounds 1-11 have the following origins: Specifically, all compounds are derived from tropoelastin, a porcine elastin precursor shown in Sequence ID No. 1. (1) Compounds 1 to 5 and 11 are derived from the VGVAPG repeat sequence (VGVAPGVGVAPGVGVAPGVGVAPG, SEQ ID NO: 2) in the amino acid residues from the 496th Val residue to the 519th Gly residue of tropoelastin, an elastin precursor derived from pigs. (2) Compound No. 6 is a PRP sequence derived from the 692nd to 694th Pro amino acid residues of tropoelastin, an elastin precursor derived from pigs. (3) Compound No. 7 is a VSP sequence derived from the 370th Val residue to the 372nd Pro residue and the 659th Val residue to the 661st Pro residue of tropoelastin, an elastin precursor derived from pigs. (4) Compound number 8 is a TRP sequence derived from amino acid residues 682 to 684 of tropoelastin, an elastin precursor derived from pigs. (5) Compound No. 9 is a GAP sequence derived from the amino acid residues 249 to 251, 283 to 285, 306 to 308, 468 to 470, and 531 to 533 of tropoelastin, an elastin precursor derived from pigs. (6) Compound No. 10 is a sequence included in the amino acid sequences of tropoelastin, a pig-derived elastin precursor, between Ala residues 73 and 81, between Ala residues 235 and 246, between Ala residues 286 and 292, between Ala residues 373 and 380, between Ala residues 427 and 431, between Ala residues 471 and 479, between Ala residues 534 and 544, between Ala residues 589 and 591, between Ala residues 623 and 629, and between Ala residues 662 and 665.

[0033] 4. The results of the investigation into inhibitory activity are shown in Figure 1. (1) Compound 04 (SEQ ID NO: 7) exhibited the best ACE2 inhibitory activity, with 92% at a peptide concentration of 320 μM and 91% at 80 μM. (2) Among the compounds that showed inhibitory activity of 50% or more, compound 03 (SEQ ID NO: 6) showed 51% at a peptide concentration of 320 μM.

[0034] 5. IC 50 The results of the calculation are shown in Figure 2. (1) Compound 04 (SEQ ID NO: 7) showed a relatively low value of 4.6 μM. From this, it was considered that compound 04 (SEQ ID NO: 7) has excellent inhibitory activity against ACE2. (2) Furthermore, water-soluble porcine elastin typically has an average molecular weight of 20,000 Da to 30,000 Da. Therefore, assuming that the average molecular weight of water-soluble porcine elastin is 25,000 Da, IC 50 This is 18 μM. This is the IC of compound 04. 50 Although larger, it was thought to possess superior inhibitory activity.

[0035] 6. These results indicate that elastin, or elastin degradation products, inhibit ACE2. In particular, compound 04 showed excellent results, with an IC50 of 4.6 μM and inhibitory activity of over 90%, indicating its usefulness as an ACE2 inhibitor. 7. Furthermore, the sequence of compound 04 (SEQ ID NO: 7), which exhibits the strongest inhibitory activity, exists as four consecutive repeats between positions 496 and 519 in porcine elastin (corresponding to SEQ ID NO: 2). The sequence of compound 03 (SEQ ID NO: 6), which exhibits the next strongest inhibitory activity, exists in two discontinuous locations between positions 496 and 519 in porcine elastin (corresponding to SEQ ID NO: 2). From this, it was inferred that porcine elastin degradation products are useful for ACE2 inhibition.

[0036] 8. Based on these experimental results, it can be assumed that ingesting or inoculating porcine elastin or porcine elastin-derived peptides will have a preventive effect against COVID-19 infection in clinical trials. Currently, in response to the global spread of infection, taking porcine elastin or porcine elastin-derived peptides as a supplement, or taking them in combination with vaccination, can be expected to have a significant preventive effect against infection.

Claims

1. An ACE2 inhibitory composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, or both, as an active ingredient.

2. The ACE2 inhibitory composition according to Claim 1, A drug or supplement used to prevent or treat coronavirus infection.

3. The ACE2 inhibitory composition according to Claim 1, A drug or supplement to enhance the effectiveness of the COVID-19 vaccine.

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