JP2019189590A - Kinesin inhibitor, anticancer agent comprising that inhibitor, and method of screening kinesin inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel kinesin inhibitor, an anticancer agent containing the kinesin inhibitor, and a screening method for the novel kinesin inhibitor. Kinesin inhibitors of the present invention and anticancer agents containing the inhibitors include compounds having a structure represented by the following general formula 1: embedded image In the above general formula 1, R1 is H, or , Any organic group, and R2 is H or any organic group. [Selection diagram] None

Description

  The present invention relates to a kinesin inhibitor, an anticancer agent containing the inhibitor, and a screening method for a kinesin inhibitor.

  Cancer is the most common cause of death in Japan. Various treatment methods (for example, surgical treatment, radiation therapy, chemotherapy and immunotherapy) have been developed for cancer treatment, but there is still no perfect treatment, and each treatment depends on the condition of the cancer. The law is selected.

  Among them, chemotherapy is a general treatment method, but since there is not one gene that causes cancer, multi-drug combination therapy is used.

J. Wu., Et al., ACS Chem. Biol., 8, 2201-2208, 2013 B. Yang, et al., J. Med. Chem., 57, 9958-997- 2014 C. A. Watts, et al., Chem. Biol., 20, 1399-1410, 2013 W. Zhang, et al., Biochem J., 473, 1027-1035, 2016

  In the 2000s, various molecular targeted drugs that act on oncogenes and tumor suppressor genes have been put into practical use in the treatment of cancer, but the development of new molecular targeted drugs is still required.

  An object of one embodiment of the present invention is to provide a novel kinesin inhibitor, an anticancer agent containing the kinesin inhibitor, and a screening method for the kinesin inhibitor.

  In order to solve the above problems, the kinesin inhibitor according to one embodiment of the present invention has the following configuration.

  [1] A kinesin inhibitor containing a compound having a structure represented by the following general formula 1:

In the general formula 1, R ₁ is H or any organic group, and R ₂ is H or any organic group.

  [2] The kinesin inhibitor according to one embodiment of the present invention preferably contains a compound having a structure represented by the following general formula 2:

In the above general formula 2, R ₁ is H or any organic group, and R ₂ is H or any organic group.

[3] In the kinesin inhibitor according to one embodiment of the present invention, R ₁ and / or R ₂ may be an organic group containing an oxy group.

[4] The anticancer agent according to one embodiment of the present invention may be an anticancer agent containing the kinesin inhibitor according to any one of [1] to [3]:
[5] A screening method for a kinesin inhibitor according to one embodiment of the present invention includes a culturing step of culturing a transformant whose growth is inhibited by overexpression of a kinesin gene in a medium containing a candidate substance, In addition, the culture step preferably includes a selection step of selecting, as a kinesin inhibitor, a candidate substance contained in a medium in which the growth of the transformant is recognized to be recovered.

  According to one embodiment of the present invention, a novel kinesin inhibitor can be provided.

  According to one embodiment of the present invention, a novel anticancer agent containing a novel kinesin inhibitor can be provided.

  According to one embodiment of the present invention, an anticancer agent that is toxic to cancer cells at a low concentration can be provided.

  According to one embodiment of the present invention, a screening method for confirming that growth of a transformant whose growth is inhibited by overexpression of the kinesin gene is recovered. Thereby, a kinesin inhibitor and an anticancer agent can be easily screened.

It is a graph which shows each component refine | purified from the sample in an Example. It is the table | surface which shows the relationship between the image which shows the growth of TY-27 stock | strain in an Example, and the spot amount of each component, and a growth circle. It is a figure which shows the structure of the component A, the component B, and the component C in an Example. It is an image which shows the presence or absence of growth about TY-27 stock | strain, TY-94 stock | strain, and Host strain | stump | stock in an Example. It is a graph which shows the cell viability of HL60 in an Example. It is a graph which shows the cell viability of MCF-7 and MDA-MB-231 in an Example. FIG. 2 is a schematic diagram for explaining the structures of compounds included in General Formula 1, General Formula 2, and General Formula 3.

  An embodiment of the present invention will be described as follows, but the present invention is not limited to this. The present invention is not limited to each configuration described below, and various modifications can be made within the scope shown in the claims, and technical means disclosed in different embodiments and examples respectively. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention. Moreover, all the literatures described in this specification are used as a reference in this specification. In the present specification, when “A to B” is described with respect to a numerical range, the description intends “A or more and B or less”.

[1. Outline of the present invention]
Kinesins (eg, meiotic kinesin HSET (kinesin-14 family)) are one of the microtubule motor proteins that cause centrosome clustering that results from excessive duplication of centrosomes.

  The present inventor thought that inhibiting the function of kinesin could inhibit centrosome clustering and consequently inhibit cancer cell growth. Therefore, the present inventor first developed a new screening method for kinesin inhibitors.

  The present inventor screened 328 kinds of known anticancer agents using a new kinesin inhibitor screening method. However, known anticancer agents have not been determined as kinesin inhibitors in the screening method for kinesin inhibitors.

  Furthermore, the present inventor screened 994 kinds of samples extracted from natural resources using the above kinesin inhibitor screening method. As a result, kinesin inhibitory activity was observed in nine types of samples (Seika Awadachiso, Karatachi, Avocado, Sawara, Hyamridhi, Sage, Licorice, Mitsuba and Yamaurushi). Among the samples, a particularly strong kinesin inhibitory activity was observed in a sample derived from the root of Solidago altissima (Rapid name: Solidago altissima). Therefore, a sample having strong kinesin inhibitory activity was further isolated and purified, and the structure of a group of compounds having kinesin inhibitory activity was identified.

  Each compound belonging to the above compound group was a novel kinesin inhibitor having a similar structure. Moreover, when each of the above-mentioned compounds was dropped onto the cancer cells, the survival rate of the cancer cells decreased. From this result, it was suggested that the novel structure which the obtained compound has contributed to anticancer property.

[2. Kinesin inhibitor)
The kinesin inhibitor in this embodiment contains a compound having a structure represented by the following general formula 1.

In the above general formula 1, R ₁ is H or any organic group, and R ₂ is H or any organic group. The compound having the structure represented by the general formula 1 can be any stereoisomer and / or any optical isomer.

  As used herein, “kinesin” intends any kinesin that can be classified into the kinesin superfamily. Examples of the kinesin include kinesin-14 family, (KIFC1 / HSET / KNSL2, KIFC2, KIFC3, KIF25 / KNSL3) and the like. In the present specification, the term “kinesin inhibitor” means an inhibitor having an activity that inhibits the function of kinesin (hereinafter referred to as kinesin inhibitory activity).

In the kinesin inhibitor according to one embodiment of the present invention, R ₁ and / or R ₂ may be an organic group containing an oxy group. According to the said structure, the kinesin inhibitor which shows higher kinesin inhibitory activity is realizable.

  Examples of the organic group containing the oxy group include a tigloyloxy group, an acetyloxy group, a benzyloxy group, and an acyloxy group.

  Examples of the compound having the structure represented by the general formula 1 include collabic acid, soridagonic acid, 6β-tigloyloxycolabenic acid, corabic acid analog (6β-tigloyloxycolabenic acid analog), and the like.

  The compound having the structure represented by the general formula 1 may be a synthesized compound or a compound extracted from a natural resource.

  The natural resource containing the above compound can be selected from, for example, (Apiaceae, Karachi, Avocado, Sawara, Hyamridhi, Sage, Licorice, Mitsuba, Porcupine) and the like.

  Moreover, it is preferable that the kinesin inhibitor in this embodiment contains the compound which has a structure shown by the following general formula 2.

In the above general formula 2, R ₁ is H or any organic group, and R ₂ is H or any organic group. According to the said structure, a kinesin inhibitor with remarkably high kinesin inhibitory activity is realizable (refer (a) of FIG. 7).

In the general formula 2, R ₁ and / or R ₂ may be an organic group containing an oxy group. According to the said structure, the kinesin inhibitor which shows remarkably high kinesin inhibitory activity is realizable.

  Examples of the organic group containing the oxy group include a tigloyloxy group and an acyloxy group.

  The compound having the structure represented by the above general formula 2 is preferably a corabenic acid analog (6β-tigloyloxycolabenic acid analog).

  Moreover, it is preferable that the kinesin inhibitor in this embodiment contains the compound which has a structure shown by the following general formula 3.

In the above general formula 3, R ₁ is H or any organic group, and R ₂ is H or any organic group. According to the said structure, the kinesin inhibitor which shows high kinesin inhibitory activity is realizable (refer (b) of FIG. 7).

In the kinesin inhibitor according to one embodiment of the present invention, R ₁ and / or R ₂ may be an organic group containing an oxy group. According to the said structure, the kinesin inhibitor which shows high kinesin inhibitory activity is realizable.

  Examples of the organic group containing an oxy group include an acyloxy group and an acetyloxy group.

  Examples of the compound having the structure represented by the above general formula 3 include collabic acid, soridagonic acid, 6β-tigroyloxycolabenic acid, and the like. The compound having the structure represented by the above general formula 3 is preferably soridagonic acid or cholavenic acid.

  The kinesin inhibitor may contain one or more of the compounds represented by the general formula 1, the general formula 2 and the general formula 3. Furthermore, the kinesin inhibitor may contain a derivative of the above compound.

  In the present specification, the “derivative” means a group of compounds generated by substituting a part of the compound in the molecule with another functional group or another atom for the specific compound.

  Examples of the other functional groups include alkyl groups, alkoxy groups, alkylthio groups, aryl groups, aryloxy groups, arylthio groups, arylalkyl groups, arylalkoxy groups, arylalkylthio groups, arylalkenyl groups, arylalkynyl groups, allyls. Group, amino group, substituted amino group, silyl group, substituted silyl group, silyloxy group, substituted silyloxy group, arylsulfonyloxy group, alkylsulfonyloxy group, nitro group and the like. Examples of the other atoms include a carbon atom, a hydrogen atom, an oxygen atom, a nitrogen atom, a sulfur atom, a phosphorus atom, and a halogen atom.

  Content of active ingredient (specifically, compound represented by general formula 1, compound represented by general formula 2, compound represented by general formula 3 or derivative of said compound) contained in the kinesin inhibitor Although it is not particularly limited, when the kinesin inhibitor is 100 wt%, it may be 0.001 wt% to 100 wt%, may be 0.01 wt% to 100 wt%, 0.1% to 100% by weight, 0.1% to 95% by weight, 0.1% to 90% by weight, 0.1% by weight ~ 80 wt%, 0.1 wt% to 70 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 50 wt% It may be 0.1 wt% to 40 wt%, 0.1 wt% to 3 wt% May be weight percent, it may be 0.1 wt% to 20 wt% may be 0.1 wt% to 10 wt%.

  In addition, as long as the kinesin inhibitor is a component that does not inhibit the kinesin inhibitory activity of the kinesin inhibitor, the compound represented by general formula 1, general formula 2 and general formula 3, and components different from these derivatives May be included.

  The above different components include buffers, pH adjusters, isotonic agents, preservatives, antioxidants, high molecular weight polymers, excipients, carriers, diluents, solvents, solubilizers, stabilizers, fillers. , Binders, surfactants, stabilizers, and the like.

  Examples of the buffer include phosphoric acid, phosphate, boric acid, borate, citric acid, citrate, acetic acid, acetate, carbonic acid, carbonate, tartaric acid, tartrate, ε-aminocaproic acid, and And trometamol. Examples of the phosphate include sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate. Examples of the borate include borax, sodium borate, and potassium borate. Examples of the citrate include sodium citrate, disodium citrate, and trisodium citrate. Examples of the acetate include sodium acetate and potassium acetate. Examples of the carbonate include sodium carbonate and sodium bicarbonate. Examples of the tartrate salt include sodium tartrate and potassium tartrate.

  Examples of pH adjusters include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, potassium hydroxide, and the like.

  Examples of the tonicity agents include ionic tonicity agents (such as sodium chloride, potassium chloride, calcium chloride, and magnesium chloride) and nonionic tonicity agents (glycerin, propylene glycol, sorbitol, And mannitol).

  Examples of the preservative include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl paraoxybenzoate, propyl paraoxybenzoate, and chlorobutanol.

  Examples of the antioxidant include ascorbic acid, tocophenol, dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate, and sodium sulfite.

  Examples of the high molecular weight polymer include methylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, carboxymethylcellulose sodium, hydroxypropylmethylcellulose acetate succinate, hydroxypropylmethylcellulose phthalate Carboxymethyl ethyl cellulose, cellulose acetate phthalate, polyvinyl pyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyethylene glycol, and atelocollagen.

  The content of the different components contained in the kinesin inhibitor is not particularly limited, but may be 0 wt% to 99.999 wt% when the kinesin inhibitor is 100 wt%, or 0 wt% % To 99.99% by weight, 0% to 99.9% by weight, 5% to 99.9% by weight, or 10% to 99.9%. % By weight, 20% by weight to 99.9% by weight, 30% by weight to 99.9% by weight, 40% by weight to 99.9% by weight, 50 wt% to 99.9 wt%, 60 wt% to 99.9 wt%, 70 wt% to 99.9 wt%, 80 wt% % To 99.9% by weight, or 90% to 99.9% by weight.

[3. Anticancer agents including kinesin inhibitors]
The anticancer agent in this embodiment is an anticancer agent containing a kinesin inhibitor. Here, since the kinesin inhibitor contained in the anticancer agent in the present embodiment is the same as described above, the description thereof is omitted here.

  The cancer to which the anticancer agent can be applied is not particularly limited, for example, breast cancer, bladder cancer, colon cancer, lung cancer, hepatocellular carcinoma, gastric cancer, melanoma, thyroid cancer, endometrial cancer, kidney cancer, cervical cancer, Examples include pancreatic cancer, esophageal cancer, prostate cancer, brain cancer, ovarian cancer, small cell lung cancer, non-small cell lung cancer, head and neck cancer, and leukemia.

  The use of the anticancer agent of this embodiment is appropriately determined according to the pathological condition of cancer. For example, when the anticancer agent of this embodiment is used, it is preferably used for breast cancer.

  The cancer cell which can use the anticancer agent of this embodiment is not specifically limited. In the present invention, for example, chondrocytes, osteoblasts, fibroblasts, epidermal cells, epithelial cells, adipocytes, hepatocytes, pancreatic cells, muscle cells and the like can be mentioned. The animal species from which the cells are derived is not particularly limited, and the animal species may be a non-human mammal or a human. Examples of non-human mammals include primates (monkeys, apes, etc.), cloven-hoofers (cattle, wild boar, pigs, sheep, goats, etc.), odd-hoofed animals (such as horses), rodents (mouse, rats, hamsters). , Squirrel, etc.), rabbit eyes (rabbit, etc.), and meat (dogs, cats, ferrets, etc.). The non-human mammal described above may be a domestic animal, a companion animal, or a wild animal.

  The content of the kinesin inhibitor contained in the anticancer agent is not particularly limited, but may be 0.001 wt% to 100 wt% when the anticancer agent is 100 wt%, or 0.01 wt% % To 100% by weight, 0.1% to 100% by weight, 0.1% to 95% by weight, 0.1% to 90% by weight It may be 0.1 wt% to 80 wt%, 0.1 wt% to 70 wt%, or 0.1 wt% to 60 wt%. 0.1 wt% to 50 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt% % To 20% by weight, or 0.1% to 10% by weight.

  In addition, the anticancer agent is a component that does not inhibit the kinesin inhibitory activity, and other components different from the kinesin inhibitor (for example, the buffer, pH adjuster, tonicity agent, preservative, and antioxidant described above) , High molecular weight polymers, excipients, carriers, diluents, solvents, solubilizers, stabilizers, fillers, binders, surfactants, stabilizers, and the like.

  The content of the component different from the kinesin inhibitor contained in the anticancer agent is not particularly limited, but may be 0 wt% to 99.999 wt% when the anticancer agent is 100 wt%, and 0 wt% % To 99.99% by weight, 0% to 99.9% by weight, 5% to 99.9% by weight, or 10% to 99.9%. % By weight, 20% by weight to 99.9% by weight, 30% by weight to 99.9% by weight, 40% by weight to 99.9% by weight, 50 wt% to 99.9 wt%, 60 wt% to 99.9 wt%, 70 wt% to 99.9 wt%, 80 wt% % To 99.9% by weight, or 90% to 99.9% by weight.

[4. Screening method for kinesin inhibitor]
In the screening method of this embodiment, the transformant in which growth is inhibited by overexpression of the kinesin gene is cultured in a medium containing a candidate substance, and in the above-described culture step, A screening method for a kinesin inhibitor, comprising a screening step of selecting a candidate substance contained in a medium whose growth has been recovered as a kinesin inhibitor.

  In the culturing step in the screening method of the present embodiment, the growth of the transformant is inhibited by overexpressing the kinesin gene. Yeast etc. are mentioned as a host of the above-mentioned transformant. From the viewpoint that a gene disruption strain or an overexpression strain can be easily prepared, yeast is preferably used as the host. One reason for this is that when screening is performed, it is preferable to delete the multidrug excretion transporters (for example, Bfr1 and Pmd1) of the host in order to increase the permeability to various drugs.

  Examples of yeast used as the host include fission yeast (Shizosaccharomyces pombe), budding yeast (Saccharomyces cerevisiae), and the like. From the viewpoint of superiority in research on genes related to human diseases, it is preferable to use fission yeast as a host.

Examples of the fission yeast used as a host in the culture step include, for example, the YA8 strain, the SAK931 strain (h ^- caf5 :: bsdR pap1Δpmd1Δmfs1Δbfr1Δdnf2Δerg5 :: ura4 ⁺ leu1 ura4 ade6-M210: A Takemoto et al., J. Cell Sci. 129, 1250-1259, 2016, provided by Dr. Shigehiro Kawashima (University of Tokyo).

  As a kinesin gene to be overexpressed, a gene encoding a kinesin protein involved in centrosome clustering is intended. Examples of the kinesin gene include Kinesin-14 family (specifically, KIFC1 / HSET / KNSL2, KIFC2, KIFC3, KIF25 / KNSL3) and the like.

  More specifically, as a kinesin gene, Accession No. NC — 000006.12 (KIFC1), Accession No. NC — 000008.11 (KIFC2), Accession No. NC — 000016.10 (KIFC3), or Accession No. NC — 018917.2 (KIF25) ), But of course the present invention is not limited to these.

  The kinesin gene to be overexpressed may be a HSET yeast homolog (eg, pkl1 or klp2) or a human homolog (eg, KIFC2, KIFC3, or KIF25 described above). The present inventor has newly discovered that cell growth inhibition is caused by overexpression of human-derived HSET (M. Yukawa et al., J. Cell Sci. 131, 1-11, 2018). Therefore, the screening method for kinesin inhibitors in the present invention may be a screening method using an overexpression system of not only yeast homologs but also human homologs (for example, KIFC2, KIFC3, and KIF25 described above).

  The technique for overexpressing the above kinesin gene is not particularly limited, and a known technique is used. If the kinesin gene introduced into the host is constantly expressed, the host may be killed. Therefore, it is preferable that the kinesin gene introduced into the host is controlled so that expression can be induced. The control can be performed using a known promoter.

  The components and form of the medium used in the culture process are not particularly limited. For example, the form of the medium to be detected may be liquid or solid. In addition, the culture conditions used in the culture step can be appropriately selected according to the host.

  In addition, the screening method of the present embodiment includes a selection step of selecting, as a kinesin inhibitor, a candidate substance contained in a medium in which the growth of the transformant is recognized to have been recovered in the culture step.

  As a method for confirming that the growth of the transformant has been recovered in the culturing step, the following methods may be mentioned.

  For example, when a solid medium is used as a medium, a kinesin inhibitor candidate substance is dropped on a solid medium coated with a transformant overexpressing the kinesin gene, and the dripped substantially circular region (hereinafter, When the growth of the transformant is observed in the vicinity of the growth circle), and in the vicinity thereof, it can be determined that the growth of the transformant has been recovered. Further, in the screening step, a candidate substance contained in the medium when the growth of the transformant is recovered can be selected as a kinesin inhibitor.

  The candidate substance used for screening in the present embodiment may be an artificially synthesized compound or a compound extracted from a natural resource.

  One embodiment of the present invention will be described below.

<1. Construction of HSET overexpressing fission yeast strain TY-27>
At the time of screening, in order to further increase drug permeability, the fission yeast strain YA8 (h ⁻ bfr1 :: ura4 ⁺ pmd1 :: hisG) previously lacking two major multidrug efflux transporters (Bfr1 and Pmd1) leu1-32 ura4-D18: Y. Arita, Mol Biosyst 7, 1463-1472 (2011), Dr. Yokoshiro Yatsuta and Dr. Satoshi Yoshida (RIKEN) were used as hosts. A HSET overexpression plasmid (pREP41-GFP-HSET) was introduced into this strain to construct a TY-27 strain.

  A method for preparing a HSET overexpression plasmid (pREP41-GFP-HSET) is described below. Using HSET cDNA (DNAForm, Clone ID: 40117894, Yokohama, Japan) as a template, a DNA fragment of HSET cDNA was amplified by a PCR method using the following oligo DNA primers into which restriction enzyme sites of SalI and BamHI had been introduced in advance. The obtained DNA fragment and the vector plasmid pREP41-GFP (RA Craven, Gene, 221, 59-68 (1998)) are cleaved with restriction enzymes (SalI and BamHI) to express a fusion protein of HSET and GFP. The DNA fragment was inserted into the vector plasmid pREP41-GFP as possible.

MY280: 5'-CCCAGGGGGTCGACTATGGACCCGCAGAGGTCCCCCCTATT-3 '(SEQ ID NO: 1)
MY281: 5′-CTTTCCCCGGATCCTCACTTCCTGTTGGCCTGAGCAGTAC-3 ′ (SEQ ID NO: 2).

<2. Setting of screening conditions and measurement of growth recovery activity in TY-27 strain>
The TY-27 strain whose growth was suppressed by overexpression of HSET was used. As a culture condition in which the growth recovery of the TY-27 strain can be detected with the naked eye when the activity of HSET is inhibited, 4.6 × 10 ⁴ cells are planted per 1 mL of the medium, and the exposure condition for the sample is as follows. 28 ° C. and 3 days.

The PY medium (Pombe Minimal Glutamate medium) containing thiamine was used for preculture and main culture of the TY-27 strain. When OD ₅₉₀ = 0.8 during the main culture, the cells were washed twice with PMG medium from which thiamine was removed, and the washed cells were 400 μl, and PMG agar from which thiamine was kept at 50 ° C. was removed. Four plates containing 12.5 ml of PMG agar were prepared by mixing in 49.6 ml of medium. A natural resource sample (described later) used for screening was extracted with MeOH and adjusted to 10 mg / ml, spotted on a plate at 2 μl or 5 μl, and growth recovery activity was evaluated under the above conditions.

Below, the composition of PMG medium (Pombe Minimal Glutamate medium) (per 1 L) is shown.
Potassium hydrogen phthalate: 3g
Disodium hydrogen phosphate: 2.2 g
L-glutamic acid-sodium salt: 3.75 g
Glucose: 20g
Adenine: 225mg
Histidine: 225mg
Uracil: 225mg
Lysine hydrochloride: 225mg
Thiamine: 5mg
* ₁ Salts stock: 20ml
* ₂ Vitamins Stock: 1ml
* ₃ Minerals Stock: 100μl
(In the case of agar medium) Agar: 20g
[* ₁ Salts stock]
MgCl · 6H ₂ O: 52.5 g / L
CaCl ₂ · 2H ₂ O: 0.735 g / L
KCl: 50g / L
Na ₂ SO ₄ : 2 g / L
[* ₂ Vitamins stock]
Pantothenic acid: 1 g / L
Nicotinic acid: 10g / L
Inositol: 10g / L
Biotin: 10mg / L
[* ₃ Minerals stock]
Boric acid: 5g / L
MnSO ₄ : 4g / L
ZnSO ₄ · 7H ₂ O: 4 g / L
_{FeCl 2 · 6H 2 O: 2g} / L
Molybdic acid: 0.4 g / L
KI: 1 g / L
_{CuSO 4 · 5H 2 O: 0.4g} / L
Citric acid: 10 g / L
In addition, as a natural resource sample, a 994 plant / food-derived methanol extract library, which is a natural resource independently collected, purchased or externally provided by the present inventor, was used. Among the 994 plant / food-derived methanol extract libraries, growth recovery activity was observed in nine libraries of Sequoia serrata, Karatachi, Avocado, Sawara, Hyamridhi, Sage, Licorice, Honeybee, and Porcupine.

<3. Isolation and Purification of Active Substances from Solidago serrata>
862.4 g of Rootweed Root was extracted with methanol at room temperature for 3 days, concentrated to dryness to obtain 23.0 g of extract. After the extract was dissolved in a small amount of methanol, the dissolved extract was dispersed in 10 times the amount of water, and the mixture was extracted twice with an equal amount of ethyl acetate and concentrated to dryness. .4 g of extract was obtained.

  4.2 g of the extract was subjected to silica gel column chromatography, and the active substance was eluted using an eluent (chloroform: methanol = 9: 1).

  352.6 mg of the active substance obtained after elution was purified by HPLC (elution condition: 85% methanol-0.1% acetic acid). Specifically, components corresponding to the three peaks detected during purification (see (a) and (b) of FIG. 1) were isolated and designated as component A, component B, and component C, respectively. . Component A and component B were isolated as a white powder, and component C was isolated as a yellow oil.

<4. Identification of the structure of the active substance>
Aforementioned components A, component B, and the structure of the components C, EIMS, HREIMS, 1-dimensional ^{NMR (1 H, 13 C)} , were identified DEPT, 2-dimensional NMR (COSY, HMQC, HMBC, NOESY) and the like. The result is shown in FIG. FIG. 3 is a diagram showing the structures of component A, component B, and component C. Component A is solidagonic acid (see (a) of FIG. 3), component B is kolavenic acid analog (6β-tigloyloxycolabenic acid analog) (see (b) of FIG. 3), and component C is kolavenic It was found to be acid (see (c) of FIG. 3). In addition, the structure of component B kolavenic acid analog is the structure of 6β-tigloyloxycorabenic acid that has been reported so far (Hiroyuki Koshino, Naoki Kurisawa, Kenichi Kimura, Toshigo Koichi, Hiroko Sato, CAST / evaluation of attribution and the structure of the ¹³ C NMR of Clerodane type diterpene using CNMR system, 56th NMR debate, November 14 to 16 days, Tokyo metropolitan University, different from the Abstracts p38), a new structure Met.

<Evaluation of growth recovery activity against TY-27 strain using three kinds of components>
<2. Setting of screening conditions and measurement of growth recovery activity in TY-27 strain> Evaluation of growth recovery activity for TY-27 strain when component A, component B and component C were used in the same manner as described above. went.

  The result is shown in FIG. FIG. 2 shows an image showing the growth recovery activity of the TY-27 strain, and the relationship between the spot amount of each component and the growth circle. Here, the image of (a) in FIG. 2 shows the result using the component A, the image of (b) using the component B, and the image of (c) showing the result using the component C. Further, the “growth circle” refers to a substantially circular region in which the growth of the transformant was observed by spotting component A, component B and component C onto the medium. Further, the “inhibition circle” refers to a substantially circular region where the growth of the transformant was not observed when component A, component B and component C were spotted on the medium. The numbers shown in the image correspond to the growth circle numbers (No.) described in the respective tables of FIG. 2, and the dotted circle seen in the image is the minimum of each component. The growth circle of the spot where the amount of spot was dropped is shown.

  As shown in FIG. 2, a growth circle was observed in any of (a) to (c). Further, in the images of (a) to (c), the minimum spot amount in which a growing circle was recognized was 310 μg for (a), 0.2 ng for (b), and 10 ng for (c). From this, it was found that Component B showed growth recovery activity at the lowest concentration. Furthermore, comparing the concentration of the minimum spot amount in which a growth circle was recognized, the growth recovery activity of component B was the strongest, the growth recovery activity of component C was the second strongest, and the growth recovery activity of component A was the third. I found it strong.

<Evaluation of growth recovery activity against TY-94 strain using 6.3 types of components>
Evaluation of growth recovery activity for strains overexpressing pkll1 which is a yeast homologue of HSET by the same methods and components as those described in 2 and 5 above (hereinafter referred to as TY-94 strain) went. The result is shown in FIG.

  FIG. 4 is an image showing the presence or absence of growth recovery activity for the TY-27 strain, the TY-94 strain, and the Host strain (control into which an empty vector was introduced). Here, the image of (a) shows the result of using component A, the image of (b) shows the result of using component B, and the image of (c) shows the result of using component C. As shown in FIG. 4, no growth recovery activity was observed in the host strain into which the empty vector was introduced. On the other hand, the growth recovery activity similar to that of the TY-27 strain was observed in the TY-94 strain.

<Evaluation of toxicity to HL60 using three components>
The toxicity of the above three components on HL60, a human acute promyelocytic leukemia cell, was evaluated. As the medium, RPMI-1640 medium (containing L-glutamine, Phenol red, 10% inactivated FBS, 1% Penicillin (50 units / ml) Streptomycin (50 μg / ml)) was used.

HL60 was diluted to 1 × 10 ⁵ cells / ml and the diluted cells were seeded in 98 μl each in a 96-well plate. 2 μl of each sample of the above-mentioned component A, component B, and component C diluted to an arbitrary concentration with MeOH was added to the medium and cultured at 37 ° C. for 2 days.

  After culture, MTT assay was performed to determine cell viability. The cell viability was calculated as [absorbance of drug-treated cells (560 nm) / absorbance of control (methanol-treated) cells (560 nm)] × 100 (%).

The result is shown in FIG. FIG. 5 is a graph showing the cell viability of HL60. Here, (a) in FIG. 5 shows the result using component A, (b) using component B, and (c) using component C. The IC ₅₀ of HL60 to which each component was dropped was 100 μM or more in (a), 10.8 μM in (b), and 78.7 μM in (C). Therefore, it was confirmed that the component B was the strongest, the component C was the second strongest, and the component A was the third strongest in toxicity to human acute promyelocytic leukemia cells. That is, the ranking of the strength of toxicity to cancer cells of each component was the same as the ranking of the strength of recovery activity verified in 5 above.

<8. Evaluation of toxicity against MCF-7 and MDA-MB-231 using component B>
The toxicity of component B described above on MCF-7, which is a human breast cancer cell that does not overexpress HSET, and MDA-MB-231, which is a human breast cancer cell that overexpresses HSET, was evaluated. Specifically, RPMI-1640 medium (containing L-glutamine, Phenol red, 10% inactivated FBS, 1% Penicillin (50 units / ml) Streptomycin (50 μg / ml)) was used as the medium.

MDA-MB-231 was diluted to 2 × 10 ⁵ cells / ml and MCF-7 to 5 × 10 ⁴ cells / ml, and 98 μl each was seeded on 96 well plate. 2 μl of the above-mentioned component B diluted to an arbitrary concentration with MeOH was added to the medium and cultured at 37 ° C. for 2 days. After the culture, the cell viability was calculated according to the method described above.

The result is shown in FIG. FIG. 6 is a graph showing cell viability in MCF-7 and MDA-MB-231. As shown in FIG. 6, the IC _{50 of} MCF-7 to which each component was dropped was 126.6 μM. On the other hand, the IC ₅₀ of MDA-MB-231 to which each component was dropped was 85.4 μM. From this result, about 1.5 times higher toxicity was observed in MDA-MB-231 overexpressing HSET than MCF-7 not overexpressing HSET.

  From the above results, using the growth recovery activity of HSET overexpressing fission yeast as an index, a component that acts on human cancer cells can be isolated from natural resources, and the component is highly resistant to human breast cancer cells overexpressing HSET. It was confirmed to work.

  The present invention can be widely used in the medical field.

Claims (5)

A kinesin inhibitor containing a compound having a structure represented by the following general formula 1:
In the above general formula 1,
R ₁ is H or any organic group,
R ₂ is H or any organic group. The kinesin inhibitor according to claim 1, comprising a compound having a structure represented by the following general formula 2:
In the above general formula 2,
R ₁ is H or any organic group,
R ₂ is H or any organic group. The kinesin inhibitor according to claim 1 or 2, wherein R ₁ and / or R ₂ is an organic group containing an oxy group.   The anticancer agent containing the kinesin inhibitor of any one of Claims 1-3. It was observed that the transformant in which growth was inhibited by overexpression of the kinesin gene was cultured in a medium containing a candidate substance, and that the growth of the transformant was recovered in the above culture step. A screening method for a kinesin inhibitor, comprising a selection step of selecting a candidate substance contained in the medium as a kinesin inhibitor.