An allosteric inhibitor of the USP15 protease HUBL region of compound EK143 and its application

Abstract

This invention discloses an allosteric inhibitor of the HUBL region of the USP15 protease, compound EK143, and its applications. Compound EK143, or a pharmaceutically acceptable salt, ester, optical isomer, stereoisomer, polymorph, solvate, isotope-labeled compound, metabolite, chelate, complex, inclusion compound, or prodrug thereof, acts as an allosteric inhibitor of the HUBL region (441aa-751aa) in the molecular structure of the deubiquitinating protease USP15. EK143 exhibits excellent biocompatibility and significantly enhanced antitumor activity and bioavailability.

Description

Technical Field

[0001] The present invention relates to a protease allosteric inhibitor of compound EK143 and its application, and particularly relates to an allosteric inhibitor of USP15 protease HUBL region of compound EK143 and its application. Background Art

[0002] Abnormalities in the ubiquitination system are closely linked to the development and progression of malignant tumors. The misplaced ubiquitination of some proteins involved in cell survival regulation is a key factor in their becoming oncogenic molecules. Therefore, targeting deubiquitinating enzymes with strong substrate selectivity can effectively address the highly variable molecular networks involved in tumor malignant transformation, addressing the narrow anticancer spectrum of classic targeted drugs and providing new insights into the design of broad-spectrum anticancer drugs based on pan-cancer activity.

[0003] The deubiquitinating enzyme USP15 is a potential target for tumor treatment, and the compound EK143 has been shown to target the deubiquitinating enzyme USP15, inhibiting its activity and blocking the ubiquitination modification function on downstream molecules. Compared with USP15-IN-1, the only known small molecule inhibitor of USP15, EK143 and its derivatives have better solubility, stability, and biomembrane permeability. In addition, EK143 and its derivatives have a lower killing effect on non-malignant proliferating cells. The preparation process of the existing inhibitor USP15-IN-1 is relatively complex and cannot be produced on a large scale. In addition, the physical and chemical properties of USP15-IN-1 are unstable, making it difficult to carry out clinical transformation. Summary of the Invention

[0004] The present invention provides the use of the compound EK143 represented by formula (1) or a pharmaceutically acceptable salt, ester, optical isomer, stereoisomer, polymorph, solvate, isotope-labeled compound, metabolite, chelate, complex, inclusion compound or prodrug thereof as an allosteric inhibitor of the HUBL region of USP15 protease.

[0005] More specifically, in the present invention, compound EK143 does not interact with the catalytic CD domain of USP15. Instead, it binds firmly to TYR607 of USP15 via a hydrogen bond via the sidearm of the 4-hydroxy-2-methyl-butenoate. The carbonyl group on the inner aliphatic ring of compound EK143 forms a hydrogen bond with cysteine 634 (CYS634) of USP15, and also binds to Ala596, Pro592, and other residues via π bonds or van der Waals forces. Compound EK143 binds firmly to the protein, acting as an allosteric inhibitor of the HUBL domain of the USP15 protease.

[0006] The present invention provides the use of the compound EK143 represented by formula (1) or a pharmaceutically acceptable salt, ester, optical isomer, stereoisomer, polymorph, solvate, isotope-labeled compound, metabolite, chelate, complex, inclusion compound or prodrug thereof for treating diseases or conditions related to USP15 regulation.

[0007]

[0008] In a preferred embodiment of the invention, the disease or disorder is cancer, neurodegenerative diseases, immune diseases, diabetes, bone and joint diseases, osteoporosis, arthritic inflammatory disorders, cardiovascular diseases, ischemic diseases, viral infections and diseases, viral infections and / or delays and bacterial infections.

[0009] In a preferred embodiment of the present invention, the disease or condition is cancer, and the cancer is selected from liposarcoma, neuroblastoma, glioblastoma, breast cancer, bladder cancer, glioma, adrenocortical carcinoma, multiple myeloma, colorectal cancer, colon cancer, prostate cancer, non-small cell lung cancer, human papillomavirus-associated cervical cancer, oropharyngeal cancer, penile cancer, ovarian cancer, anal cancer, thyroid cancer, vaginal cancer, Epstein-Barr virus-associated nasopharyngeal carcinoma, gastric cancer, rectal cancer, thyroid cancer, Hodgkin lymphoma, diffuse large B-cell lymphoma, and Ewing sarcoma.

[0010] The present invention provides a pharmaceutical composition for treating diseases or conditions associated with USP15 regulation, comprising a preventive or therapeutically effective amount of the compound EK143 represented by formula (1) or a pharmaceutically acceptable salt, ester, optical isomer, stereoisomer, polymorph, solvate, isotope-labeled compound, metabolite, chelate, complex, inclusion compound or prodrug thereof, and a pharmaceutically acceptable carrier.

[0011] In a preferred embodiment of the present invention, the pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle or a combination thereof.

[0012] In a preferred embodiment of the present invention, the pharmaceutical composition is in the form of tablets, capsules, injections, powder injections, powders, syrups, solutions, suspensions or aerosols.

[0013] The invented compound EK143 is a specific allosteric inhibitor of the HUBL region of the USP15 protease, potentially providing highly selective USP15 protease inhibition, thereby playing a role in the treatment of numerous diseases associated with the USP15 protease. The inventors further discovered that hexokinase HKDC1 is a substrate protein of the USP15 downstream signaling pathway targeted by compound EK143, and that HKDC1-mediated glycolipid metabolism pathways can be modulated by compound EK143.

[0014] Compared with the prior art, the advantages of the present invention are:

[0015] 1) EK143 of the present invention has good biocompatibility, and its anti-tumor activity and bioavailability are significantly improved;

[0016] 2) The synthetic route is feasible for large-scale production and has promising prospects for transformation and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The IC values of EK homologues for inhibiting proliferation of gastric cancer cell line AGS for 48 hours are shown. 50 ;

[0018] Figure 2 The inhibition rate of EK homologs against USP protease family members is shown;

[0019] Figure 3 It was shown that compound EK143 specifically inhibited USP15 activity;

[0020] Figure 4 Shown are the SPR results of compound EK143 binding to the USP15 HUBL domain;

[0021] Figure 5 The LC-MS / MS analysis results of the site where compound EK143 binds to USP15 are shown;

[0022] Figure 6 The figure shows the site where compound EK143 covalently binds to USP15; A represents the three-dimensional conformation of EK143 binding to USP15HUBL; B is a planar analysis diagram of the binding site;

[0023] Figure 7 The graph shows that compound EK143 has the effect of regulating the USP15 / HKDC1 signaling axis; AB represents protein molecules that interact with USP15; C represents that compound EK143 mediates the interaction between USP15 and HKDC1 in cells and regulates protein expression; D represents the effect of compound EK143 on USP15-mediated HKDC1 and its ubiquitination level; E represents that EK143 inhibits HKDC1-mediated glucose metabolism;

[0024] Figure 8 is the hydrogen spectrum of EK143;

[0025] Figure 9 This is the carbon spectrum of EK143.

[0026] It should be noted that, for the sake of simplicity in drawing, compound EK143 is abbreviated as EK in the accompanying drawings. DETAILED DESCRIPTION

[0027] The present invention is described below with reference to specific examples. Those skilled in the art will appreciate that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0028] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. The purchase information for some raw materials and reagents is as follows: ethanol, petroleum ether, ethyl acetate, and n-butanol were all analytically pure and purchased from Sigma, USA.

[0029] Example 1

[0030] Preparation method of Eupalinolide K and its derivative EK143:

[0031] Preparation of Eupalinolide K (abbreviated EK): The dried aerial part (7 kg) of Eupatorium lind leyanum DC. was crushed into coarse powder, soaked in 95% ethanol for 24 hours, and then extracted with 20 times 95% ethanol at room temperature. The crude extract was concentrated under vacuum to obtain a crude extract. After evaporation under reduced pressure, the ethyl acetate extract (194 g) was subjected to silica gel column chromatography and eluted with a polyethylene-ethyl acetate (20:1-0:1, v / v) gradient to obtain twelve fractions (Frs.AL). 5 g of Fr.K was separated using methanol / water as the mobile phase (50%, 70%, 100%, respectively, at a flow rate of 50 ml / min). A portion was subjected to preparative high performance liquid chromatography (55% methanol / water, at a flow rate of 15 ml / min) using 70% MeOH (215.2 mg) to obtain 66.7 mg of a colorless gelatinous substance. Scan its 1 H-NMR, 13 C-NMR, the compound Eupa l inol ide K was identified (Reference: Yang, B., Shen, JW, Zhou, DH, Zhao, YP, Wang, WQ, Zhu, Y., & Zhao, HJ (2019). ant i-tr ip le-negat ive breastcancer. Natura lproduct research,33(4),477-485.)

[0032] Table 1: 1 H-(600MHz)and 13 C-NMR(150MHz)chemica l sh ifts of Eupa l ino lide K in CD3OD

[0033]

[0034]

[0035] Preparation of EK143: Eupalinolide K (compound 1) was dissolved in N,N-dimethylformamide (DMF), and an equal amount of imidazole and a catalytic amount of tert-butyldimethylsilyl chloride (TBDMS-Cl) were added. The mixture was stirred at room temperature under nitrogen for 24 hours. Extraction and column purification were performed to obtain compound 2 in an 82% yield. Compound 2 was dissolved in dichloromethane (DCM), and an excess of pyridinium chlorochromate (PCC) was added. The mixture was reacted at room temperature for 1 hour, and compound 3 was extracted with a 60% yield. Compound 2 was dissolved in methanol (MeOH), mixed with sodium borohydride (NaBH4), and stirred in an ice bath (0°C) for 2 hours. Compound 4 was extracted after extraction. Compound 4 was esterified with acetic anhydride (Ac2O) and stirred in pyridine at room temperature for 12 hours to obtain compound 5. Excess hydrogen fluoride (HF) was added, and the mixture was reacted in an ice bath (0°C) for 1 hour to remove the TBDMS protecting group. Compound 6 was extracted with a 61% yield. Finally, Noyori's catalyst was selected for asymmetric hydrogenation. Compound 6 was dissolved in MeOH and reacted in the presence of a catalytic amount of (S)-BINAP-Ru(II) at a temperature gradient of 0°C to 40°C under hydrogen protection for 4 hours to obtain the final product EK143 (compound 7) with a yield of 45%.

[0036] The synthetic route is as follows:

[0037]

[0038] The hydrogen and carbon spectra of EK143 synthesized in this example are shown in Figure 8 and Figure 9 .

[0039] Example 2

[0040] EK143 and its homologues inhibit gastric cancer cell proliferation test:

[0041] To verify the activity of EK143 and its homologues in inhibiting gastric cancer cell proliferation, this example used the CCK8 assay to test the inhibitory activity of the compounds of the present invention against the proliferation of various tumor cells. Gastric cancer cell line HGC27 was seeded into 96-well plates at a density of 5000 cells / well. After 24 hours of culture under conventional conditions, 5.0 μM or different concentrations of the compounds of the present invention were added sequentially to final concentrations of 50.0, 10.0, 5.00, 1.00, 0.50, 0.10, 0.05, and 0.01 μM, respectively. An equal amount of DMSO was added to the control group (three replicates per group). After 48 hours of culture, 100 μL of CCK8 solution was added to each well, and the cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. The culture plate was then shaken to mix the color, and the absorbance of each well was measured at a wavelength of 450 nM using an enzyme-linked immunosorbent assay (ELISA). Finally, the inhibitory effect of the target compounds on tumor cell proliferation was further statistically analyzed.

[0042] The test results are as follows Figure 1 As shown in Table 1, Figure 1 It was found that the vast majority of the compounds of the present invention, such as EK143, EK001, and EK024, exhibited significant inhibitory activity against the proliferation of gastric cancer cells HGC27 at a concentration of 1.0 μM. Among them, EK143 exhibited the strongest inhibitory activity against gastric cancer cell proliferation, with an IC50 of approximately 3.82 μM, demonstrating that the compounds of the present invention effectively inhibited the proliferation of gastric cancer cells.

[0043] Table 1 Inhibitory effects of some compounds on the proliferation of gastric cancer cell lines HGC27, MKN28, and AGS

[0044] Compound number <![CDATA[HGC27IC 50 (μM)]]> <![CDATA[MKN28IC 50 (μM)]]> <![CDATA[AGS IC 50 (μM)]]> EK012 10.92 12.97 18.50 EK006 8.24 9.54 10.67 EK061 7.68 8.76 11.51 EK032 7.73 8.89 18.87 EK033 7.56 9.65 12.76 EK045 7.08 9.97 13.54 EK023 6.99 8.76 10.26 EK107 6.65 8.98 9.26 EK151 6.34 8.45 9.65 EK080 5.56 8.96 10.76 EK081 5.44 6.27 10.62 EK027 5.07 6.25 10.67 EK024 4.57 5.33 11.87 EK001 4.26 5.16 9.21 EK143 3.82 4.97 10.24

[0045] Note: Cell survival rate (%) = OD value of drug-treated group / OD value of DMSO group × 100%

[0046] Example 2

[0047] To verify the selectivity of EK143 and its partial homologs against other USP subtypes, we selected three subtypes with high homology to USP15, USP11, USP4, and USP7, as representative representatives for testing. Different His-Ubs were used in the Ub-Nluc reporter gene system. We first incubated the compound with His-USP protein in 50 μL of 1× reaction buffer at 37°C for 30 minutes to allow for full binding and interaction. The compound was then added to 100 μL of 1× reaction buffer containing pre-coated His-Ub-Nluc Ni-NTA beads and allowed to react at 37°C for 45 minutes. After centrifugation, 50 μL of the supernatant was transferred to a non-adsorbing 96-well white plate. The substrate was then added and read in the dark.

[0048] The test results are as follows Figure 2 As shown in the results, EK143 and its homologues have low selectivity for other isoforms such as USP11, USP4 and USP7. Among the compounds tested, EK143 and its homologues have excellent selective inhibition of USP15, indicating that these compounds are selective USP15 inhibitors.

[0049] Example 3

[0050] USP15 activity inhibition test of EK143:

[0051] The detection method is as follows:

[0052] USP15 activity was measured by the fluorescence intensity of the ubiquitin-rhodamine substrate (Ub-Rh110). The activity of USP15 was measured at a final concentration of 5 nM in a black polystyrene 96-well plate. The substrate concentration was set to 25 nM, and EK143 was diluted in a DMSO gradient from a maximum concentration of 64 μM. Buffer (20 mM Tris-HCl, pH 8.0, 2 mM CaCl2, 1 mM reduced glutathione, 0.01% v / v Triton X-100, 0.01% v / v Pr ionex) was used as the reaction solution. After adding the substrate, the fluorescence intensity (485 nm excitation / 520 nm emission) was measured every 60 seconds using a fluorescence spectrophotometer, and the detection was continued for 50 minutes. The kinetic curve was drawn, and the results were shown in FIG. Figure 3 As shown, the enzyme activity constant k is calculated inact and K I value.

[0053] Test results:

[0054] Compound EK143 has a carbonyl moiety in the inner ring and can covalently modify protein cysteine residues. Enzyme activity assay results showed that EK143 inhibited the hydrolysis of Ub-Rh110 by USP15 in a dose-dependent manner. The inactivation rate of USP15 was k inact and inhibition constant K I 0.0065s respectively -1 and 5.28 μM.

[0055] Example 4

[0056] EK143 binding to USP15 domain detection:

[0057] Detection method:

[0058] SPR determines the binding domain: USP15 is a ubiquitination-specific protease composed of more than a thousand amino acids. It is roughly divided into three domains according to its specific function: the N-terminal TRAF domain and the catalytic domain (CD domain).

[0059] and a five-repeat UBL domain (HUBL domain) at the C-terminus. To preliminarily determine the approximate location of EK143 binding to USP15, we constructed plasmids covering different domains and expressed recombinant proteins of each truncation in an E. coli protein expression system. These recombinant proteins were then validated by SPR binding to EK143 to identify the specific USP15 domains that bind to EK143. Full-length USP15 and its truncated proteins were diluted with 10 mM sodium acetate (pH 4.5) to a 1 mg / mL sample solution and injected into the sample channel at a flow rate of 5 μL / min; the reference channel was not injected. After coupling was completed, uncoupled active groups on the chip were blocked with ethanolamine solution. A gradient of EK143 concentrations was prepared in TBST buffer containing 5% DMSO, with the following concentrations: 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.562 μM, 0.781 μM, and 0.39 μM. Samples were injected sequentially at a flow rate of 30 μL / min, with an association time of 60 s and a dissociation time of 120 s. Data were collected and analyzed using Biacore Evolution software (T200 Version 1.0).

[0060] Test results:

[0061] The results showed that neither the TRAF domain protein alone nor the TRAF+CD domain fusion protein could bind to EK143. The response signal of the TRAF+CD domain was negative because the signal value was lower than the background noise. Therefore, it was believed that this binding was non-specific adsorption of the molecule-protein, so the TRAF+CD domain did not bind to EK143. However, for the chip coupled with the HUBL domain protein, the signal intensity of the binding between the two also increased significantly with the increase of the concentration of the added compound, and the KD reached 2.70μM, showing a strong binding ability. Figure 4 Thus, we confirmed that the direct binding site between EK143 and USP15 protein is located in the HUBL allosteric regulatory domain.

[0062] Example 5

[0063] Detection of EK143 and USP15 binding sites:

[0064] Detection method:

[0065] LC-MS / MS identification of the EK143-USP15 binding site: Recombinant USP15 protein was diluted to 20 μM in PBS buffer and divided equally into a blank control group and a drug-treated group. EK143 solution or an equal volume of DMSO was added to each USP15 protein solution at a final concentration of 1 mM, mixed, and incubated overnight. After completion of the reaction, an appropriate amount of loading buffer was added and the mixture was denatured by heating in a metal bath at 98°C or higher for 10 min. The protein bands were separated by SDS-PAGE, digested in-gel with trypsin, and filtered to obtain peptide sample solutions. 10 μL of the peptide sample solution was injected onto an EASY-nLC-II liquid chromatograph for analysis. Chromatographic separation was performed using an RP-C18AQ capillary liquid chromatography column with a mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, with a gradient elution rate of 300 nL / min. The mass spectrometer was an LTQ-Orbitrap Velos Pro MS / MS system with an electrospray ionization (ESI) voltage of 1.8 kV. Data were acquired in positive ion mode with a scan range of m / z 350-2000. The Thermo Proteomics Insights platform was used to search the acquired secondary mass spectrometry peptide data and ultimately identify the protein components and their response values.

[0066] Test results:

[0067] In this example, mass spectrometry was used to identify the amino acid sites where EK143 binds to the HUBL domain. First, the protein sample was enzymatically hydrolyzed into peptide fragments. Due to the binding of EK143, the mass of the peptide fragments increased. Therefore, the changes in the peptide mass fingerprint (PMF) after binding to the small molecule were analyzed by Q-Trap 4500MS, thereby identifying the exact site where E4072B binds to the HUBL domain protein. Figure 5 As shown, after comparison of the results, it was found that EK143 covalently bound to cysteine 634 of the HUBL domain of USP15.

[0068] Example 6

[0069] HUBL-EK143 crystal structure determination

[0070] Detection method:

[0071] Crystals of HUBL were screened using Mosquito in a sitting-drop vapor diffusion experiment at 4°C. Crystals were obtained in a solution of 2% v / v Tacsimate pH 7.0, 0.1 M imidazole pH 7.0, 8% w / v polyethylene glycol 3350, and 5% v / v 2-propanol. To determine the structure of the HUBL-EK143 complex, HUBL crystals were soaked in a solution containing 10 mM EEK143, 2% v / v Tacsimate pH 7.0, 0.05 M imidazole pH 7.0, 14% w / v polyethylene glycol 3350, 5% v / v 2-propanol, 5% PEG400, and 5% DMSO for 40 hours. Stored crystals were cryoprotected with drops of 20% ethylene glycol and then flash-frozen in liquid nitrogen. Diffraction data for the apo-HUBL and HUBL-EK143 complex structures were collected on beamlines BL17U1 and BL19U1 at the National Center for Protein Sciences (NCPSS). Data were processed using the XDS package. The structures were solved using molecular replacement with Phaser, using the previously reported HUBL structure (PDB ID 2YLM) as a search model. The final models were obtained through iterative cycles of manual reconstruction using COOT and refinement using phoenix. All structural figures were generated using PyMOL.

[0072] Test results:

[0073] USP7 HUBLThe crystal structure of the USP15-EK143 complex reveals that EK143 is anchored in the negatively charged cavity between Ub l 1 and Ub l 2. The carbonyl group of the inner lipoyl ring of EK143 acts as a reactive Michael acceptor, forming a covalent bond with cysteine 634 (CYS634) of USP15. PRO592, ALA596, and TYR607 stabilize the binding to EK143 through hydrogen bonds and van der Waals interactions.

[0074] Example 7

[0075] Testing the allosteric mechanism of EK143-induced USP15:

[0076] Detection method:

[0077] Conformational changes of USP15 were determined by tryptophan fluorescence spectroscopy: EK143 at corresponding concentration gradients was added to 20 μL of recombinant USP15 solution and incubated at 25°C for 150 min. Fluorescence emission spectra were scanned between 300 and 500 nm under excitation at 280 nm in a black polystyrene 96-well plate.

[0078] Test results:

[0079] The conformational changes of USP15 induced by EK143 were verified by tryptophan fluorescence spectroscopy analysis. Figure 7 As shown, the fluorescence intensity of USP15 was significantly reduced after allosteric regulation in cells. This is consistent with our previous hypothesis that EK143 induces an overly open conformation of USP15. Our results suggest that the allosteric inhibition mechanism of USP15 is achieved by EK143 targeting the non-catalytic HUBL domain.

[0080] Example 8

[0081] Downstream signaling pathway testing of EK143-induced USP15 inhibition:

[0082] Detection method:

[0083] Pull-down assay: Recombinant USP15 protein was diluted to 20 μg / mL in PBS buffer and divided equally into blank control, binding control, and different drug competition concentrations (10 μM, 50 μM, and 100 μM). An equal volume of EK143-bonded agarose gel was added to each USP7 protein solution (the blank control group added an equal volume of blank gel). Subsequently, the corresponding concentration of EK143 solution or an equal volume of DMSO was added, mixed, and incubated at 4°C. After completion of the reaction, each sample was centrifuged at 10,000 rpm for 3 minutes at 4°C. The supernatant was discarded, and the sample was eluted six times. The appropriate amount of loading buffer was added and the sample was denatured by heating in a metal bath at 98°C or above for 10 minutes. The target protein levels in each sample were detected by silver staining mass spectrometry and Western blot.

[0084] Test results:

[0085] It is known that the deubiquitinase USP15 can protect substrate proteins from degradation by removing ubiquitination tags, while EK143, as a USP15 inhibitor, can induce the degradation of certain proteins. Figure 8 As shown, Co-IP experiments demonstrated that hexokinase HKDC1 is the substrate protein of EK143 acting on the USP15 downstream signaling pathway; Western blot detection showed that the ubiquitination level of HKDC1 increased in HGC27, AGS, and MKN28 cells treated with EK143, as well as the changes in glycolytic activity mediated by HKDC1.

Claims

1. Use of an allosteric inhibitor of the HUBL region of USP15 protease in the preparation of a pharmaceutical composition for treating diseases related to USP15 regulation, characterized in that: The inhibitor is compound EK143, which acts as an allosteric inhibitor of the HUBL 441aa-751aa region in the molecular structure of the deubiquitinating protease USP15. Compound EK143 firmly binds to TYR607 of USP15 via a hydrogen bond through the side arm of 4-hydroxy-2-methyl-butenoate. The carbonyl group on the internal aliphatic ring of compound EK143 forms a hydrogen bond with cysteine CYS634 at position 634 of USP15, and binds to Ala596 and Pro592 via π bonds or van der Waals forces. Compound EK143 firmly binds to the protein, thereby acting as an allosteric inhibitor of the HUBL region of the USP15 protease. The structure of the compound EK143 is as follows: ； The disease associated with USP15 regulation is gastric cancer.

2. The application according to claim 1, characterized in that The pharmaceutical composition is in the form of tablets, capsules, injections, powder injections, powders, syrups, solutions, suspensions or aerosols.

3. The application according to claim 1, characterized in that The compound EK143 acts on the substrate protein hexokinase HKDC1 of the USP15 downstream signaling pathway.

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