Application of high capsaicin II in preparation of targeted driver protein KIF11 antitumor drug

Abstract

The invention discloses an application of high capsaicin II in preparation of a targeted kinin KIF11 anti-tumor drug, the high capsaicin II is directly combined with a Tyr-104 / Thr-300 / Arg-355 site of KIF11 and stabilizes kinin KIF11, the kinin KIF11 stabilizes cholesterol biosynthesis and induces centrosome amplification, and the high capsaicin II and kinin KIF11 mediated centrosome separation synergistically drive multipolar splitting, so that the high capsaicin II can be used for preparing the targeted kinin KIF11 anti-tumor drug. And finally, ferroptosis is triggered. According to the invention, the toxicity of the traditional KIF11 inhibitor is overcome, the tumor specific induction of ferroptosis is realized, and the indications of the targeted therapy of the kinin KIF11 are expanded.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to an application of capsaicin II in the preparation of an anti-tumor drug targeting the kinesin KIF11. Background Art

[0002] Kinesin KIF11 (also known as Eg5 or Kinesin-5) is an important microtubule-associated motor protein belonging to the kinesin superfamily. It plays a key role in cell division and mitosis and is a potential target for the development of anti-cancer drugs. The conventional function of kinesin KIF11 is to maintain the bipolarity of the mitotic spindle, regulating microtubule dynamics and centrosome separation through ATPase activity. It is highly expressed in solid tumors such as colorectal cancer and is associated with chemotherapy resistance and poor prognosis. Traditional KIF11 inhibitors (such as ispinesib and filanesib) induce mitotic arrest and apoptosis by competitively inhibiting ATPase activity. However, because normal cells (such as hematopoietic stem cells) rely on KIF11 to maintain division, it leads to severe toxicity such as bone marrow suppression, hindering clinical development.

[0003] Capsaicinoids are primarily extracted from chili peppers. Homocapsaicin II (Hp II) is a subtype of capsaicinoids. Homocapsaicin II has been reported to inhibit tumors. Therefore, Hp II is expected to become a lead compound for regulating the kinesin KIF11 and KIF11-related diseases, potentially enabling the development of drugs targeting KIF11. Summary of the Invention

[0004] The purpose of the present invention is to provide the use of capsaicin II in the preparation of anti-tumor drugs targeting the kinesin KIF11. On the one hand, the present invention can overcome the toxicity of traditional KIF11 inhibitors: by stabilizing rather than inhibiting KIF11, it avoids the systemic toxicity caused by normal cell mitosis arrest; on the other hand, it can achieve tumor-specific induction of ferroptosis: by utilizing the metabolic-mitotic dual dependence of KIF11-high-expressing tumors, ferroptosis is accurately triggered through the synergistic effect of cholesterol synthesis and multipolar fission; on the other hand, it can expand the indications of KIF11 targeted therapy: for tumors that overexpress KIF11 but are ineffective against traditional inhibitors (such as chemotherapy-resistant colorectal cancer), a new treatment method is provided.

[0005] To achieve the above-mentioned purpose of the invention, the present invention provides the use of capsaicin II in the preparation of anti-tumor drugs targeting the kinesin KIF11.

[0006] Furthermore, capsaicin II specifically targets the kinesin KIF11, regulating the dynamic balance between cholesterol metabolism and mitosis, and inducing ferroptosis in colorectal cancer cells.

[0007] Furthermore, capsaicin II directly binds to the Tyr-104 / Thr-300 / Arg-355 site of KIF11 and stabilizes the kinesin KIF11. Stabilization of the kinesin KIF11 activates cholesterol biosynthesis, induces centrosome amplification, and synergistically drives multipolar fission with the centrosome separation mediated by the kinesin KIF11, ultimately triggering ferroptosis.

[0008] To achieve the above-mentioned purpose of the invention, the present invention also provides a pharmaceutical composition targeting the kinesin KIF11, wherein the pharmaceutical composition contains capsaicin II as an active ingredient and contains pharmaceutically acceptable excipients.

[0009] Preferably, the tumor is colorectal cancer or pancreatic cancer with high expression of the kinesin KIF11 (IHC score ≥2+).

[0010] Preferably, the dosage form of the pharmaceutical composition is an external dosage form or an internal dosage form.

[0011] Preferably, the dosage forms of the pharmaceutical composition include oral liquid, mixture, tablet, tincture, powder, capsule, pill, paper mold, injection solvent, oil, suspension, crystal, ointment, tincture, liniment, lotion, drops, suppository, film coating, adhesive patch, and implantable slow-dissolving tablet.

[0012] To achieve the above-mentioned object of the invention, the present invention also provides a pharmaceutical composition targeting the kinesin KIF11, which contains a plant extract containing capsaicin II and a pharmaceutically acceptable excipient.

[0013] The natural compound Homocapsaicin II (Hp II) specifically targets the kinesin family member 11 (KIF11) protein, regulating the homeostasis between cholesterol metabolism and mitosis, thereby inducing ferroptosis in colorectal cancer cells. This study reveals for the first time that KIF11 is a direct target of Hp II and elucidates its novel mechanism of stabilizing the KIF11 protein to trigger a metabolic-mitotic synergistic lethality. This provides a novel strategy for the precision treatment of tumors with high KIF11 expression.

[0014] This study, published in Nature Communications, demonstrates that Hp II disrupts traditional KIF11 targeting strategies by directly binding to and stabilizing the KIF11 protein, rather than inhibiting its ATPase activity. KIF11 stabilization activates cholesterol biosynthesis, inducing centrosome amplification (CA), and synergistically drives multipolar fission with KIF11-mediated centrosome separation, ultimately triggering ferroptosis. This mechanism is only activated in tumor cells with high KIF11 expression. Normal cells, due to low basal KIF11 expression and intact cholesterol homeostasis, exhibit significant tumor selectivity for Hp II.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) Breakthrough targeting mechanism

[0017] Precise targeting of KIF11: For the first time, it was revealed that Hp II stabilizes KIF11 protein by binding to its Tyr-104 / Thr-300 / Arg-355 sites (without inhibiting ATPase activity), subverting the mode of action of traditional KIF11 inhibitors and avoiding toxicities such as bone marrow suppression caused by normal cell mitosis arrest.

[0018] Tumor selectivity: Ferroptosis is activated only in tumors such as colorectal cancer with high KIF11 expression (IC50≈80-100μM). Normal intestinal epithelial cells (IC50>200μM) have significant tolerance (survival rate>80%) due to low KIF11 expression and intact cholesterol metabolism homeostasis.

[0019] (2) Metabolic-mitotic synergistic lethal effect

[0020] Cholesterol metabolic reprogramming: KIF11 upregulates enzymes such as FDFT1 / DHCR7, inducing cholesterol overload and driving centrosome amplification (CA), providing a metabolic basis for ferroptosis.

[0021] Multipolar division induction: KIF11 overexpression promotes centrosome separation, cooperates with CA to form a multipolar spindle, and directly triggers Fe 2+ Accumulation (increased 2.5 times) and lipid peroxidation (LPO increased 3 times), achieving multi-pathway synergistic killing.

[0022] (2) Therapeutic advantages of high efficiency and low toxicity

[0023] The in vivo efficacy was significant: Hp II (25 mg / kg / d) inhibited the growth of colorectal cancer xenografts by 60.7% (vs. 5-FU: 44.5%) without causing toxicity such as weight loss (p=0.07).

[0024] Overcoming drug resistance: By stabilizing KIF11, we can bypass traditional inhibitor resistance mechanisms (such as ATP-binding domain mutations) and be effective against chemotherapy-resistant tumors.

[0025] (3) Clinical application potential

[0026] Integrated diagnosis and treatment: Screening of sensitive patients based on KIF11 expression (IHC score ≥2+) and centrosome abnormalities (CA ≥3) improves treatment response rate (AUC = 0.87).

[0027] Combination drug expansion: Combined use with ferroptosis enhancers (such as erastin) or PLK4 inhibitors can synergistically enhance the anti-tumor effect (synergistic index CI < 0.8).

[0028] (4) Technical barriers and patent barriers

[0029] Mechanistic Uniqueness: The metabolic-mitotic synergistic mechanism of KIF11 stabilization and ferroptosis has not been reported in the literature, circumventing existing KIF11 inhibitor patents (such as ispinesib).

[0030] Wide application: The indications cover various solid tumors such as colorectal cancer and pancreatic cancer with high KIF11 expression, and the market potential is significant. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The molecular formula of capsaicin II (A) and its HPLC detection chart (B) are shown;

[0032] Figure 2 Figure 2 is the nuclear magnetic resonance (NMR) test result of capsaicin II; (A) is 1 H-NMR, (B) 1 C-NMR;

[0033] Figure 3 The mass spectrometry results of capsaicin II are shown in Figure 1; (A) The mass-to-nuclear ratio of the mass spectrometry result is 8.27×10 4 , (B) is the mass spectrometry result with a mass-to-nuclear ratio of 4.28×10 4 ;

[0034] Figure 4 Molecular docking diagram (A), epoxy agarose fishing diagram (B), microcalorimetric binding energy detection diagram (C);

[0035] Figure 5 KIF11 half-life detection diagram (A), KIF11 ubiquitination modification detection diagram (B);

[0036] Figure 6Figure 3 shows the heat map of transcriptome cholesterol synthesis and metabolism gene expression differences (A), fluorescence quantitative PCR detection (B), cholesterol level detection (C), centrosome amplification detection (D), and centrosome amplification detection of the cholesterol synthesis pathway (E).

[0037] Figure 7 Fluorescence detection of cell multipolar division (A), statistical analysis of cell multipolar division (B), detection of cell multipolar division after KIF11 expression inhibition (C), statistical analysis of cell multipolar division in Figure C (D);

[0038] Figure 8 Iron ion level detection chart (A), lipid peroxide LPO level analysis chart (B), GSH / GSSG ratio analysis chart (C);

[0039] Figure 9 Detection of iron ion levels in multipolar dividing cells (A), detection of iron ion levels in cells that promote centrosome amplification and multipolar dividing cells (B), detection of lipid peroxides in cells that promote centrosome amplification and multipolar dividing cells (C).

[0040] Figure 10 KIF11 expression levels in colorectal cancer tissues and adjacent adjacent tissues (A); screening of cell clones with different centrosome amplification levels in a clonal formation experiment (B); and normalized cell survival rate (C). DETAILED DESCRIPTION

[0041] The present invention is further described in detail below with reference to specific embodiments.

[0042] Unless otherwise specified, the raw materials and reagents used in the following examples were obtained from commercial sources.

[0043] Capsaicin II was purchased from Sichuan Dester Biological Company with CAS number: 71240-51-2 and molecular formula: C 19 H 29 NO3, molecular weight: 319.44. Structural formula: Figure 1 As shown in A, the purification method is HPLC, and the purity is 99.64% ( Figure 1 B). The compounds were identified using NPM and Mass. The identification results are shown in Figure 2 and Figure 3 .

[0044] Example 1

[0045] Molecular validation of Hp II targeting KIF11

[0046] Molecular docking: AutoDock Vina was used to simulate the binding mode of Hp II and KIF11 (PDB: 6L9K) and screen the key interacting residues (Tyr-104 / Thr-300 / Arg-355) ( Figure 4 A).

[0047] Epoxy agarose pull-down assay: Hp II was coupled to epoxy-activated agarose gel (Sepharose 6B) and incubated with HCT116 cell lysate. Western blot was used to detect the bound KIF11 ( Figure 4 B).

[0048] MST analysis: To verify whether Hp II can bind to KIF11 protein, MST was used to analyze the interaction between Hp II and fluorescently labeled KIF11 protein: labeled KIF11 was replaced by desalting column into MST buffer (20mM HEPES, 150mM NaCl, 5mM MgCl2, 1mM DTT, pH 7.5), mixed with equal volumes of serially diluted Hp II (500nM to 15.25μM, DMSO ≤ 1%), incubated at 25°C in the dark for 15 minutes, and then detected by Monolith NT.115 instrument (20% LED, 40% MST power, 30 seconds of thermophoresis, 5 seconds of delay). The ΔFnorm data of three independent experiments (three technical replicates each) were fitted with the Hill equation to calculate the Kd value, and unlabeled KIF11 and buffer were used as negative controls to verify binding specificity. ( Figure 4 C).

[0049] Example 2

[0050] KIF11 stabilization and ubiquitination regulation

[0051] CHX tracking experiment: HCT116 cells were pretreated with Hp II (100 μM), and cycloheximide (CHX) was added to block protein synthesis. Western blot analysis showed that the half-life of KIF11 was prolonged ( Figure 5 A).

[0052] Ubiquitination detection: After immunoprecipitation of KIF11, Western blot was used to detect the dose-dependent inhibition of its ubiquitination by Hp II ( Figure 5 B).

[0053] Example 3

[0054] KIF11-dependent cholesterol metabolism and CA induction

[0055] Cholesterol synthase detection: transcriptome and qPCR verification of Hp II upregulating the expression of key genes in the cholesterol synthesis pathway ( Figure 6AB). And upregulate intracellular cholesterol levels ( Figure 6 C).

[0056] Increased cholesterol induces CA: Exogenous addition of cholesterol can induce CA ( Figure 6 D); Inhibition of cholesterol synthesis pathway can inhibit CA ( Figure 6 E).

[0057] CA quantification: Immunofluorescence staining (γ-tubulin / DAPI) was used to count the proportion of CA cells. Knockout of KIF11 showed a decrease in the CA rate of >50%.

[0058] Example 4

[0059] Co-validation of multipolar division and ferroptosis

[0060] Multipolar spindle detection: After HCT116 cells were treated with Hp II, the frequency of multipolar divisions was counted by α-tubulin / γ-tubulin staining ( Figure 7 AB). KIF11 mediates Hp II-induced multi-stage division ( Figure 7 CD).

[0061] Ferroptosis marker analysis: FerroOrange staining for free iron ions; HPLC determination of GSH / GSSG ratio; C11-BODIPY staining for LPO levels ( Figure 7 AC).

[0062] CA+ multipolar division induces ferroptosis: the iron ion level in multipolar dividing cells increases ( Figure 8 A); PLK4 overexpression induces CA+KIFC knockdown induces multipolar division and jointly induces ferroptosis ( Figure 8 BC).

[0063] Fe 2+ Level detection: Ferro Orange staining kit was used to assess intracellular Fe 2+ The cells were cultured at 1×10 5 Cells were seeded at a density of 100 μM in 12-well plates and treated after culture. Ferro Orange probe (1 μM) was added and incubated for 30 minutes. The cells were then rinsed twice with PBS. Fluorescence images were captured under green laser (532 nm) excitation.

[0064] LPO detection: To verify whether the cells undergo ferroptosis, the lipid peroxide (LPO) content assay kit was used to measure LPO. First, the LPO sample was extracted into chloroform. Subsequently, each sample was mixed with 500 μl of chloroform extract, 450 μl of chloroform-methanol solvent, and 50 μl of developer solution (composed of equal volumes of FTS reagent 1 and FTS reagent 2). The reaction was incubated at room temperature for 5 minutes and analyzed using a spectrophotometer set at 500 nm (Epoch microplate reader, BioTek, USA). Optical density readings indicate LPO levels.

[0065] Quantification of glutathione (GSH) and oxidized glutathione (GSSG): To verify whether cells underwent ferroptosis, glutathione (GSH) and oxidized glutathione (GSSG) levels were measured. Quantification was performed using GSH and GSSG detection kits according to the manufacturer's instructions. Cells were frozen twice in liquid nitrogen and thawed in a 37°C water bath, followed by centrifugation at 10,000 g for 10 minutes at 4°C. The resulting supernatant was used for GSH and GSSG analysis, and absorbance was read at 450 nm using a BIO-DL microplate reader. Optical density values ​​represent GSH and GSSG concentrations.

[0066] Example 5

[0067] Patient stratification and diagnostic kit development

[0068] KIF11 IHC score: Colorectal cancer tissue sections were stained with KIF11 antibodies, and patients with a score ≥2+ were included in the population suitable for Hp II treatment.

[0069] Centrosome abnormality sensitivity test to Hp II: Cell clone formation experiment was used to obtain cell clone sub-cell lines with different centrosome amplification rates, and MTT experiment was used to detect the activity inhibition level of cell clones with different CA levels under Hp II treatment conditions. Figure 9 As shown in Figure 3, the results showed that the higher the CA rate, the more sensitive it was to Hp II.

[0070] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0071] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. Application of capsaicin II in the preparation of anti-tumor drugs targeting the kinesin KIF11.

2. The use of capsaicin II according to claim 1 in the preparation of anti-tumor drugs targeting the kinesin KIF11, characterized in that: Capsaicin II specifically targets the kinesin KIF11, regulates the dynamic balance of cholesterol metabolism and mitosis, and induces ferroptosis in colorectal cancer cells.

3. The use of high capsaicin II according to claim 2 in the preparation of anti-tumor drugs targeting the kinesin KIF11, characterized in that: Capsaicin II directly binds to the Tyr-104 / Thr-300 / Arg-355 site of KIF11 and stabilizes the kinesin KIF11. Stabilization of the kinesin KIF11 activates cholesterol biosynthesis, induces centrosome amplification, and synergistically drives multipolar fission with the centrosome separation mediated by the kinesin KIF11, ultimately triggering ferroptosis.

4. The use of high capsaicin II according to claim 1 or 2 in the preparation of anti-tumor drugs targeting the kinesin KIF11, characterized in that: The tumor is colorectal cancer or pancreatic cancer with high expression of the kinesin KIF11.

5. A pharmaceutical composition targeting the kinesin KIF11, characterized in that: The pharmaceutical composition uses capsaicin II as an active ingredient and contains pharmaceutically acceptable excipients.

6. The pharmaceutical composition according to claim 5, characterized in that The dosage form of the pharmaceutical composition is an external dosage form or an internal dosage form.

7. The pharmaceutical composition according to claim 6, characterized in that The dosage forms of the pharmaceutical composition include oral liquid, mixture, tablet, tincture, powder, capsule, pill powder, paper mold, injection solvent, oil, suspension, crystal, ointment, tincture, liniment, lotion, drops, suppository, film coating, adhesive patch, and implantable slow-dissolving tablet.

8. A pharmaceutical composition targeting the kinesin KIF11, comprising a plant extract containing capsaicin II and a pharmaceutically acceptable excipient.

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