JP2023111649A - Ace2 inhibitory compound - Google Patents

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

The present invention relates to ACE2 inhibitory compounds. More specifically, the present invention relates to ACE2 inhibitors intended for prevention of coronavirus infection.

Recently, a new type of coronavirus infection called COVID-19 has spread all over the world and has become a problem.
There are multiple spike proteins on the surface of this novel coronavirus. It is known that this spike protein invades cells by binding to receptors present on the surface of human cells and causes human infection. This human receptor is called ACE2 (Angiotension II-Converting Enzyme), and when inoculated with vaccines developed by Pfizer, Moderna, AstraZeneca, etc., the vaccine blocks the spike protein and converts it to ACE2. It has been reported to inhibit binding and prevent infection.

In addition, there is a fact that new mutant strains are appearing one after another in the coronavirus. In particular, it has been reported that multiple mutations in the ACE2-binding region of the spike protein weaken the efficacy of vaccines and reduce the protection against infection. Therefore, as a countermeasure against coronaviruses that mutate one after another, additional administration of vaccines and new vaccine development by pharmaceutical companies are being attempted.
However, since coronavirus is an RNA virus in the first place, it is impossible to prevent the emergence of mutant strains. For this reason, as long as virus mutations appear, measures such as additional administration of vaccines and the development of new vaccines will be necessary.

JP 2019-112406 A

By the way, the inventors have so far invented an ACE inhibitory compound for suppressing the production of angiotensin II, which has a pressor action, and have filed a patent application (Patent Document 1). In the present application, based on this knowledge, we started developing an ACE2 inhibitory compound that can prevent coronavirus infection.
In other words, in order to prevent coronavirus infection, it is possible to prevent infection by blocking the spike protein present on the surface of the coronavirus with a vaccine, or by inhibiting ACE2 present on the surface of human cells. is considered useful. In the present application, ACE2 that can prevent coronavirus infection by inhibiting ACE2 itself on the partner side to which the spike protein, which is the entrance to infection, binds to the current coronavirus or a mutated coronavirus that may appear in the future. The aim is to develop inhibitory compounds.

As a result of extensive research, the inventors have found that elastin or elastin degradation products have an inhibitory effect on ACE2, and have completed the invention.
The present invention consists of the following configurations.
[1] An ACE2 inhibitory compound comprising elastin or elastin degradation products (including corresponding chemically synthesized compounds) as an active ingredient.

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

[6] Elastin degradation products are
The ACE2 inhibitory compound according to [3], which is a peptide having a molecular weight of less than 1000 and containing any one or more of SEQ ID NOS: 4 to 14.
[7] The ACE2 inhibitory compound according to [3], wherein the elastin degradation product is represented by any one of SEQ ID NOS: 4 to 14.
[8] The ACE2 inhibitory compound according to [3], wherein the elastin degradation product is represented by SEQ ID NO:7.

[9] A drug for coronavirus characterized by using the ACE2 inhibitory compound of [1] to [8] as an active ingredient and preventing or treating coronavirus infection by inhibiting ACE2 by the ACE2 inhibitory compound or supplement.
[10] Drugs or supplements for coronavirus characterized by using the ACE2 inhibitory compound of [1] to [8] as an active ingredient, and enhancing the effect of coronavirus vaccine by inhibiting ACE2 by the ACE2 inhibitory compound .

INDUSTRIAL APPLICABILITY The present invention has made it possible to provide useful ACE2 inhibitory compounds.
By using the ACE2 inhibitory compound of the present invention as a drug or supplement, not only the current novel coronavirus, but also future mutant strains can be prevented, treated, and enhanced vaccine effects. Useful effects can be expected.

The figure which showed the result of having investigated the ACE2 inhibitory activity of each compound. The figure which showed the result of having calculated _IC50 of each compound.

The ACE2 inhibitory compound of the present invention will be explained.

The ACE2 inhibitory compound of the present invention is characterized by containing elastin or elastin degradation products (including corresponding chemically synthesized compounds) as an active ingredient. That is, the elastin shown in SEQ ID NO: 1 or its degradation product is used as an active ingredient to exert an ACE2 inhibitory effect.
The elastin may typically have a molecular weight of less than 300,000, preferably less than 100,000, particularly preferably having an average molecular weight of less than 20,000 to less than 30,000.
Further, the molecular weight of elastin degradation products can typically be from 100 to less than 2,000, preferably from 100 to less than 1,000.

When the ACE2 inhibitory compound of the present invention is produced by decomposing elastin, it can be produced using a known method (International Publication No. 2006/046626 pamphlet).
That is, although biological tissues such as aortic blood vessels obtained from mammals such as pigs, horses, cows, and sheep are used as raw materials, biological tissues such as avian arterial blood vessels and fish arterial bulbs may also be used as raw materials. First, these living tissues are degreased, homogenized, or the like to be finely divided. Subsequently, collagen or the like is removed from the subdivided sample using an alkaline solution or the like to obtain insoluble elastin. Further, the insoluble elastin is hydrolyzed with an acid or alkali or the like, and the treated solution is subjected to a separation operation to obtain water-soluble elastin. This water-soluble elastin is treated by enzyme treatment or the like, and the resulting treated solution is subjected to column purification such as membrane separation or gel filtration to obtain a fraction with a molecular weight of 100 to less than 2,000.

The ACE2 inhibitory compounds of the present invention can also be chemically synthesized. That is, since the ACE2 inhibitory compound of the present invention is composed of a peptide, it can be chemically synthesized using a peptide synthesizer or the like.

As a preferred embodiment of the elastin hydrolyzate, the elastin hydrolyzate is a dipeptide, tripeptide, tetrapeptide, pentapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, or undecapeptide contained in the sequence VGVAPGVGVAPGVGVAPGVGVAPG (SEQ ID NO: 2). It can be a compound with a molecular weight of less than 1000, represented by either a peptide or a dodecapeptide.
The compound represented by SEQ ID NO: 2 is represented by a one-letter amino acid code, with the N-terminus on the left and the C-terminus on the right (hereinafter the same).

Further, as a different preferred embodiment of the elastin degradation product, the elastin degradation product is X ₁ X ₂ P (X ₁ or X ₂ = A, G, S, V, L, T, I, C, M, D, N, E, Q, H, K, R, F, Y, W, or P)), and can be a compound having a molecular weight of less than 1000.

Furthermore, as a different preferred embodiment of the elastin hydrolyzate, the elastin hydrolyzate can be a peptide having a molecular weight of less than 1,000 containing any or more of SEQ ID NOS: 4 to 14, particularly preferably SEQ ID NOS: 4 to 14. It can be a peptide represented by either, most preferably a peptide represented by SEQ ID NO:7.
By using these elastin degradation products as preferred embodiments, the design and chemical synthesis of ACE2 inhibitory compounds are facilitated, and the ACE2 inhibitory compounds of the present invention can be obtained with high purity and high reproducibility.

Although the ACE2 inhibitory compound of the present invention has the chemical structure as described above, it is not intended to be limited to this.
That is, in view of the gist of the present invention that the compound having the chemical structure shown above exerts an ACE2 inhibitory effect, in addition to the case where the compound itself exhibits an ACE2 inhibitory effect, the present invention can be obtained by undergoing various metabolisms in the body. A so-called DDS-formed compound, which has a compound structure of and exerts an ACE2 inhibitory action, may also be included. In this sense, the ACE2 inhibitory compounds of the present invention may be partially chemically modified.

The ACE2 inhibitory compound of the present invention can be used in various applications without any particular limitation as long as it can exhibit ACE2 inhibitory action. Examples of such uses include pharmaceuticals, health foods (supplements), and cosmetics. As dosage forms, for example, tablets, powders/granules, capsules, injections, internal liquids/syrups, and the like can be used.

Preferred uses of the ACE2 inhibitory compounds of the present invention include uses such as prevention of coronavirus infection.
That is, when humans ingest or inoculate the ACE2 inhibitory compound of the present invention, it inhibits the binding between the spike protein of coronavirus and ACE2, and as a result, prevents coronavirus infection and enhances the effect of coronavirus vaccines. and so on.
As such an embodiment, the ACE2 inhibitory compound of the present invention is used as an active ingredient, and the ACE2 inhibitory compound inhibits ACE2 to prevent or treat coronavirus infection. Can be configured.
As a different embodiment, the ACE2 inhibitory compound of the present invention is used as an active ingredient, and the ACE2 inhibitory compound inhibits ACE2 to constitute a drug or supplement for coronaviruses, which is characterized by enhancing the effect of a coronavirus vaccine. can be done.

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

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, and corn starch. , potato starch, dextrin, β-cyclodextrin, light anhydrous silicic acid, titanium oxide, magnesium aluminometasilicate, talc, kaolin, olive oil, gelatin, casein, pectin and the like.

Stabilizers include, for example, pectin, dextran, glycerin, polysaccharides (guar gum, arabic gum, xanthan gum, gellan gum, sclero gum, tamarind seed gum), starches (hydroxypropyl starch, phosphate monoesterified phosphate cross-linked starch). , agar, gelatin, and methyl cellulose.

Examples of antioxidants include L-ascorbic acids (L-ascorbic acid, L-calcium ascorbate, L-ascorbyl stearate, L-ascorbate palmitate), 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, melaleuca essential oil, Sunflower seed extract, grape seed extract, propolis extract, hego ginkgo biloba extract, bayberry extract, rosemary extract and the like.

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.

Examples of emulsifiers include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate; polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose. , methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose; hydrophilic polymers such as shellac wax, beeswax, carnauba wax, whale wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, cyclic lanolin, lanolin wax, candelilla wax , Japanese wax, montan wax, shellac wax, rice wax and other waxes, sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, glycerin fatty acid ester, calcium stearoyl lactylate, calcium citrate, calcium phosphate salts, pagoda saponin, soybean saponin, tea seeds saponins, beet saponins, sphingolipids, tomato glycolipids, plant sterols, plant lecithins, enzymatically degraded lecithins, egg yolk lecithins, yucca foam extracts, barley shell extracts, quillaya extracts and the like.

Examples of solubilizers include macrogol 1500, macrogol 4000, macrogol 6000, macrogol 20000, lauromacrogol, polyoxyl stearate 40, polysorbate 80, polyoxyethylene hydrogenated castor oil 60, D-mannitol, citric acid sodium, anhydrous citric acid, tartaric acid, sodium hydrogen sulfite, arginine, and the like.

Examples of tonicity agents include glycerin, propylene glycol, sodium chloride, potassium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate, citric acid, sodium citrate, sorbitol, mannitol, glucose, boric acid, and the like. be done.

The ACE2 inhibitory compound of the present invention will be further explained using Experimental Examples.

<<ACE2 inhibition test with elastin-derived oligopeptide>>
<Outline of experiment>
1. The experiment was performed according to the protocol of ACE2 Inhibitor Screening Kit (K310, Bio Vision, USA). The ACE2 Enzyme used is a human recombinant protein produced in CHO cells from a human-derived sequence.
2. The ACE2 inhibitory ability was examined using compounds Nos. 1 to 11 shown in FIG. 1 and swine-derived water-soluble elastin, a total of 12 compounds.

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

4. FIG. 1 shows the results of examining the inhibitory activity.
(1) Compound 04 (SEQ ID NO: 7) had the best ACE2 inhibitory activity, 92% at a peptide concentration of 320 µM and 91% at a peptide concentration of 80 µM.
(2) Compound 03 (SEQ ID NO: 6) showed 51% at a peptide concentration of 320 µM as a compound that showed an inhibitory activity of 50% or more.

5. Figure 2 shows the results of calculating the _IC50 .
(1) Compound 04 (SEQ ID NO: 7) showed a relatively low value of 4.6 µM. From this, compound 04 (SEQ ID NO: 7) was considered to have excellent inhibitory activity against ACE2.
(2) Water-soluble porcine elastin usually has an average molecular weight of 20,000 Da to 30,000 Da. Assuming that the average molecular weight of water-soluble porcine elastin is 25,000 Da, the IC ₅₀ is 18 μM. Although this was greater than the _IC50 of compound 04, it was considered to have excellent inhibitory activity.

6. These results indicate that elastin or elastin degradation products inhibit ACE2. In particular, compound 04 had an IC50 of 4.6 µM and an inhibitory activity of 90% or more, which was excellent, and was found to be useful as an ACE2 inhibitory compound.
7. In addition, the sequence of compound 04 (SEQ ID NO: 7), which has the strongest inhibitory activity, exists as a continuous four repeat sequence between positions 496 to 519 (corresponding to SEQ ID NO: 2) in porcine elastin. The sequence of compound 03 (SEQ ID NO: 6), which has the next strongest inhibitory activity, exists in two discontinuous positions between positions 496 and 519 (corresponding to SEQ ID NO: 2) in porcine elastin. From this, it was presumed that porcine elastin degradation products are useful for ACE2 inhibition.

8. Based on these experimental results, it can be assumed that porcine elastin or porcine elastin-derived peptide intake or inoculation is effective in preventing infection with the novel coronavirus in clinical trials. Against the spread of infection, which is currently spreading all over the world, taking porcine elastin or porcine elastin-derived peptides as a supplement or in combination with vaccination is expected to have a significant effect in preventing infection.

Claims (10)

An ACE2 inhibitory compound comprising elastin or elastin degradation products (including corresponding chemically synthesized compounds) as an active ingredient.
2. ACE2 inhibitory compounds according to claim 1, wherein elastin has a molecular weight of less than 300,000, preferably less than 100,000, particularly preferably an average molecular weight of from 20,000 to less than 30,000.
2. The ACE2 inhibitor compound according to claim 1, wherein the elastin degradation product has a molecular weight of 100 to less than 2,000, preferably 100 to less than 1,000.
Elastin degradation products are repeated sequences in elastin
ACE2 according to claim 3, represented by any one of dipeptide, tripeptide, tetrapeptide, pentapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, and dodecapeptide contained in the sequence of VGVAPGVGVAPGVGVAPGVGVAPG Inhibitory compounds.
Elastin degradation products are contained in elastin
X ₁ X ₂ P (X ₁ or X ₂ = A, G, S, V, L, T, I, C, M, D, N, E, Q, H, K, R, F, Y, W, 4. The ACE2 inhibitory compound according to claim 3, which is represented by any tripeptide of amino acids represented by any of P).
Elastin degradation products are
4. The ACE2 inhibitory compound according to claim 3, which is a peptide having a molecular weight of less than 1000 and containing any one or more of SEQ ID NOS: 4 to 14.
4. The ACE2 inhibitory compound according to claim 3, wherein the elastin degradation product is represented by any one of SEQ ID NOS: 4 to 14.
4. The ACE2 inhibitory compound according to claim 3, wherein the elastin degradation product is represented by SEQ ID NO:7.
A drug or supplement for coronavirus, comprising the ACE2 inhibitory compound according to any one of claims 1 to 8 as an active ingredient, wherein the ACE2 inhibitory compound inhibits ACE2 to prevent or treat coronavirus infection.
9. A drug or supplement for coronavirus, comprising the ACE2 inhibitory compound according to any one of claims 1 to 8 as an active ingredient, wherein the ACE2 inhibitory compound inhibits ACE2, thereby enhancing the effect of a coronavirus vaccine.

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