Application of calycosin in preparation of medicine for treating palmoplantar wart

Abstract

The invention relates to the technical field of medicines, in particular to application of calycosin in preparation of a medicine for resisting human papilloma virus infection. The invention also provides application of calycosin in preparation of drugs for treating palmoplantar warts. According to the present invention, the calycosin is determined to be adopted as the key active component of the Weijing wart eliminating formula, and the anti-virus effect of the calycosin is further evaluated in the organ-like model; by integrating clinical tests, proteomics, component analysis, functional verification and targeted protein degradation, the invention provides comprehensive scientific evidence for the curative effect of calycosin on palmoplantar warts.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and specifically, to the application of calycosin in the preparation of a medicament for treating verruca palmaris et plantaris. Background Art

[0002] Verruca palmaris et plantaris (PWs), also known as common warts, are common diseases on the palms, soles, fingers and toes, caused by human papillomavirus (HPV) infection, and the incidence rate in the general population is 7 - 12%. They account for about 20% of all warts. Compared with adults, children and adolescents have a higher incidence rate, but there is no obvious gender difference. Current treatment methods include cryotherapy, surgical resection and electrocautery laser treatment, but these methods have problems such as long treatment time, easy recurrence, discomfort, and possible complications such as scars and pigmentation.

[0003] The pathogenesis of verruca plantaris is closely related to the ability of human papillomavirus (HPV) to evade the host immune response. Studies have shown that the replication cycle of HPV is independent of keratinocyte proliferation, and the virus can survive in the host by inhibiting the release of cytokines that induce immune responses. The HPV oncogenes E6 and E7 disrupt the cell cycle by promoting the degradation of the tumor suppressor proteins p53 and Rb, creating a favorable environment for viral replication and epithelial hyperplasia. HPV not only utilizes its own proteins but also cellular small nucleolar RNAs (snoRNAs) and long non-coding RNAs (lncRNAs) to assist the E6 and E7 proteins in inhibiting apoptosis. In addition, suppressing the local immune microenvironment is also an important feature after HPV infection. For example, Carmen et al. confirmed that HPV infection inhibits CXCR4 signaling, which is a key checkpoint for the migration of Langerhans cells and dendritic cells from the skin to the lymph nodes. In addition, the HPV8 E7 protein inhibits C / EBPβ-induced constitutive CCL20 gene expression, thereby impairing Langerhans cell migration. These findings highlight the complexity of HPV infection and the necessity of treatment strategies targeting viral replication and immune regulation. A number of studies have shown that traditional Chinese medicines have antiviral effects, and there is a large amount of literature documenting that traditional Chinese medicines can effectively treat verruca plantaris (Chen, H.Y., et al., Painless and non-invasive treatment of plantar warts utilizing traditional Chinese medicine soaking: A case report. Explore (NY), 2023. Zhao, Y., et al., Successful treatment of plantar warts using topical Zijinding, a traditional Chinese medicine preparation: A case series. J Cosmet Dermatol, 2020. 19(4): p. 946-950.). However, the specific mechanism of traditional Chinese medicine in treating verruca plantaris is still unclear. For many years, the research group of the inventor has widely used Weiren Xiaoyou Formula (WRXYF) (Patent No.: ZL202210023461.5) in Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Shanghai University of Traditional Chinese Medicine, achieving remarkable clinical efficacy and few adverse reactions.

[0004] Organoid models have become a powerful tool for studying diseases in a physiologically relevant context. By mimicking the three-dimensional structure and microenvironment of tissues, organoids provide an ideal platform for studying pathogen-host interactions and evaluating therapeutic interventions. For HPV-related diseases, organoid models derived from patient samples can study virus replication, immune evasion, and the effects of antiviral compounds. Using organoids to simulate plantar warts provides a unique opportunity to explore the efficacy and mechanisms of treatments such as WRXYF in a controlled but clinically relevant system.

[0005] There is currently no report on the application of the key active ingredients of WRXYF in the preparation of drugs for treating verruca plantaris. Summary of the Invention

[0006] The purpose of the present invention is to explore the mechanism of WRXYF in treating verruca plantaris, identify the effective active ingredients of WRXYF, so as to explore its potential molecular mechanism, and promote its wider application and acceptance in evidence-based medicine.

[0007] Through a randomized controlled trial, the present invention obtained preliminary clinical evidence and elucidated the molecular mechanism of WRXYF in the treatment of plantar warts. High-performance liquid chromatography-mass spectrometry (HPLC-MS) combined with molecular docking revealed calycosin (hereinafter referred to as CAL) as the key active ingredient of WRXYF, and further evaluated its antiviral effect in an organoid model. By integrating clinical trials, proteomics, component analysis, functional verification, and targeted protein degradation, the present invention provides comprehensive scientific evidence for the efficacy of WRXYF and calycosin in treating plantar warts.

[0008] Based on the above technical solution, in the first aspect of the present invention, there is provided the application of calycosin in the preparation of a drug for anti-human papillomavirus (HPV) infection.

[0009] Further, the molecular formula of the calycosin is C 16 H 12 O5, and its chemical structural formula is shown as Formula I below:

[0010]

[0011] Formula I

[0012] Further, the human papillomavirus (HPV) infection is human papillomavirus infection on the palms, soles, fingers, and toes.

[0013] Further, calycosin clears infected cells by activating the apoptosis pathway, and has a dual role of directly inhibiting virus replication and inducing apoptosis to fundamentally clear virus infection.

[0014] In the second aspect of the present invention, there is provided the use of calycosin in the preparation of a medicament for treating verruca plantaris.

[0015] Furthermore, calycosin has the effects of destroying HPV virus particles, inducing apoptosis, and inhibiting cell proliferation.

[0016] In the third aspect of the present invention, there is provided a medicament for treating verruca plantaris, which uses calycosin as the sole active ingredient.

[0017] Furthermore, the medicament is an external preparation. More specifically, the medicament further includes excipients commonly used in external preparations.

[0018] The present invention has no special limitation on the types and specific sources of excipients commonly used in external preparations, and common excipients in the field of external preparations can be adopted.

[0019] Furthermore, the effective dose of calycosin in the medicament is 85.35 μM.

[0020] The advantages and beneficial effects of the present invention are as follows:

[0021] Limited by the difficulty in obtaining verruca plantaris tissues after WRXYF intervention in clinical practice (the verruca usually falls off naturally), it is impossible to directly verify whether WRXYF exerts its antiviral effect through a specific pathway. In view of this limitation, the present invention innovatively constructs an organoid model of verruca plantaris, which is the first application of this model in the study of low-risk HPV infection. This organoid model provides an ideal platform for deeply analyzing the mechanism of action of WRXYF and its active ingredients, and lays a new foundation for the application research of traditional Chinese medicine compound in virus infection-related diseases.

[0022] In addition, the present invention identified the main chemical components of WRXYF by HPLC-MS analysis. Among them, calycosin showed strong binding affinity for HPV oncoproteins E6 and E7. To verify the core role of calycosin in the anti-HPV activity of WRXYF, we carried out in-depth research using the organoid model. Since verruca plantaris is related to low-risk HPV infection, organoid models of this type of disease are rarely constructed. It is worth noting that the present invention successfully established an organoid model of verruca plantaris for the first time and explored the pharmacological mechanism of traditional Chinese medicine WRXYF with this model. In the organoid model, calycosin disrupted the morphology of organoids induced by HPV infection. Transmission electron microscopy showed that the virus particles were fragmented and inactivated. In addition, calycosin significantly up-regulated the expression of pro-apoptotic genes Caspase 3, BAX, and P21, and down-regulated the anti-apoptotic gene BCL2, suggesting that it clears infected cells by activating the apoptotic pathway, and has the dual effects of directly inhibiting virus replication and inducing apoptosis to fundamentally clear virus infection.

[0023] Based on the above findings, the present invention constructed an E7-HIS overexpression cell model and further explored the molecular targets of calycosin through DARTs-MS and CESTA experiments, and identified FAM107A as its direct binding protein. FAM107A plays a key role in tumor cell growth, neuron survival, and spinal cord development. Studies have shown that FAM107A forms a novel nuclear complex with F-actin and COMMD1, participating in NF-κB degradation and cell cycle inhibition in neuroblastoma cells. Another study showed that miR-146b-3p directly targeted and regulated FAM107A expression induced by SP1 during the progression and metastasis of colorectal cancer. In addition, the inactivation of FAM107A caused by promoter methylation is a key mechanism for the continuous proliferation and metastasis of tumor cells. Molecular docking and molecular dynamics simulations showed that calycosin forms a stable interaction with FAM107A, and the complex maintains a stable structure during the simulation. The reduction of the solvent-accessible surface area (SASA) of key residues suggests that the binding of calycosin stabilizes the structure of FAM107A, possibly protecting it from degradation. Functional studies further confirmed that calycosin treatment led to an increase in the protein level of FAM107A but did not affect mRNA expression, indicating the existence of a post-translational regulation mechanism. Mechanistically, calycosin significantly reduced the ubiquitination level of FAM107A, thereby enhancing its stability. Knockout experiments verified the importance of FAM107A in the antiviral activity mediated by calycosin - the deletion of FAM107A could partially reverse the apoptosis, cell cycle arrest, and decreased adhesion ability of calycosin-induced HPV-infected cells. These findings established FAM107A as a key mediator of the antiviral and pro-apoptotic effects of calycosin. Brief Description of the Drawings

[0024] Figure 1 . Main components of WRXYF. (A) Total ion chromatograms of WRXYF in UHPLC-Q-Orbitrap-MS analysis (negative ion mode and positive ion mode). (B) Structures of 34 components identified in WRXYF.

[0025] Figure 2 . Molecular docking results of 34 identified components with E6, E7, TP53, and Rb proteins. (A) Heat map of the binding energies of 34 components with four target proteins; (B) Molecular docking results of the four components with the strongest binding affinity to the protein; (C) Effects of four compounds on the activity of HaCaT cells at the same concentration of 100 μM; (D) Survival rate of HaCaT cells treated with different concentrations of calycosin.

[0026] Figure 3 . Flow chart of the clinical trial.

[0027] Figure 4. Two patients (A) received WRXYF treatment. There were significant differences in the total score and DLQI score after 8 weeks of treatment compared with those at week 0 of treatment (B, C). The recurrence rate in the WRXYF group was significantly lower than that in the SAO group (D).

[0028] Figure 5 . Comparison of HPV types in PW tissue and normal tissue and evaluation of the antiviral effect of different WRXYF extracts on HPV particles. (A - B) FISH staining showed the DNA copy numbers of HPV2, HPV27, and HPV57 subtypes in normal tissue and PW tissue. (C) HE staining of normal human skin and verruca vulgaris skin tissue (magnification: 20×, scale bar = 50 μm). (D) mRNA expression levels of HPV2 and HPV27 in verruca plantaris tissue after intervention with different extracts.

[0029] Figure 6 . Proteomic sequencing results of PW tissue before and after WRXYF treatment. (A) Heat map of differentially expressed proteins; (B) Volcano plot depicting the logarithm of the expression of differentially expressed proteins; (C) Subcellular localization analysis of up - and down - regulated proteins; (D) GO analysis of up - and down - regulated proteins; (E) KEGG analysis of up - and down - regulated proteins.

[0030] Figure 7 . Construction of verruca plantaris organ tissue and evaluation of the therapeutic effect of CAL. (A) Morphological changes of the verruca plantaris organism before and after intervention with calycin. (B) Morphological changes of verruca plantaris tissue attached to endothelial cells before and after intervention with calicheamicin. (C) DNA electrophoresis to identify E6 and E7 DNA in verruca plantaris organ tissue. (D) Electron microscopy observation of changes in virus particles in verruca plantaris organic tissue after intervention with calicin (magnification: 1500×; scale bar = 30 nm). (E) Expression of apoptotic protein calicin in the verruca plantaris organism after intervention with calicheamicin. (F) Statistical analysis of Western blot results.

[0031] Figure 8. CAL treatment can increase apoptosis and inhibit proliferation in the E7-HIS cell model. (A) DNA agarose gel electrophoresis confirmed the construction of E7-HIS. (B) Western blot analysis confirmed the overexpression of E7-HIS in the established cell line. (C) Morphological changes of E7-HIS cells observed under a light microscope. (D) IC50 value of CAL in E7-HIS cells. (E) Proliferation assay to evaluate the effect of CAL treatment on the growth of E7-HIS cells. (F) Flow cytometry analysis of cell apoptosis after CAL treatment. (G) EDU-555 assay to detect changes in DNA replication after CAL intervention. (H) TUNEL assay to evaluate the level of cell apoptosis after CAL treatment. (Magnification: 20×, scale bar = 50 µm).

[0032] Figure 9 . Identify and validate direct CAL targets using DARTS-MS. (A) Schematic diagram of DARTS-MS analysis. (B) Coomassie blue staining for DARTS detection. (C) DARTS-MS results of E7-HIS cells with and without CAL treatment. (D) Top 10 proteins identified by DARTS-MS. (E) Molecular docking analysis of the binding of CAL to FAM107A. (F) Radius of gyration (Rg) analysis of the CAL-FAM107A interaction. (G) Root mean square deviation analysis of the binding stability of CAL-FAM107A. (H) Root mean square fluctuation (RMSF) analysis of the dynamics of the CAL-FAM107A interaction. (I) Solvent accessible surface area (SASA) analysis of the CAL-FAM107A complex. (J) Cellular thermal shift assay (CETSA) to verify the binding of CAL-FAM107A. (K) Effect of increasing CAL concentration on FAM107A binding at a constant enzyme concentration. (L) Effect of increasing CAL concentration on FAM107A binding under constant temperature conditions.

[0033] Figure 10. CAL targets FAM107A ubiquitination and induces apoptosis in E7-HIS cells. (A) mRNA expression level of FAM107A after CAL treatment. (B) Protein expression level of FAM107A after CAL intervention. (C) CRISPR-Cas9-mediated knockout of the FAM107A gene. (D) mRNA expression of FAM107A after gene knockout. (E) Proliferation assay of FAM107A-KO cells treated with CAL. (F) DNA replication level of FAM107A-KO cells after CAL treatment (magnification: 20×, scale bar = 50 µm). (G) Cell adhesion assay of FAM107A-KO cells after CAL intervention. (H) Flow cytometry analysis of apoptosis in FAM107A-KO cells after CAL treatment. (I) Analysis of cell cycle distribution in FAM107A-KO cells after CAL treatment. (J) Co-IP analysis of the interaction between FAM107A and ubiquitin. Detailed implementation manners

[0034] The following detailed description of the specific implementation manners provided by the present invention will be given in combination with the embodiments.

[0035] Example 1:

[0036] I. Materials and methods

[0037] 1 Preparation of different WRXY extracts

[0038] WRXYF consists of eight traditional Chinese medicines as shown in Supplementary Table 1. The doses used in this study were determined according to the Chinese Pharmacopoeia (2015 Edition). The dry powder of the Chinese herbal medicine was extracted with water three times at room temperature, and the crude extract was obtained after removing the solvent. The extract was suspended in water and extracted three times with ethyl acetate and n-butanol in a 1:1 ratio to obtain three corresponding fractions (ethyl acetate fraction: EAE, n-butanol fraction: BA, water fraction: AE).

[0039] 2 Ultra-high performance liquid chromatography-quadrupole orbitrap mass spectrometry (UHPLC-Q Exactive Orbitrap-MS) determination

[0040] Liquid chromatography-mass spectrometry (LC-MS) and mass spectrometry-mass spectrometry (MS-MS) data were obtained by coupling an UHPLC Ultimate 3000 instrument with a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, California, USA). Chromatographic analysis was performed on an XBridge® BEH C18 (2.1 × 150 mm, 2.5 µm) column. The mobile phase consisted of A (water containing 0.1% formic acid) and B (acetonitrile containing 0.1% formic acid). The gradient elution program was as follows: 0–20 min, 20–50% B; 20–30 min, 50–80% B; 30–32 min, 80–95% B; 32–35 min, 95–20% B; 35–40 min, 20–20% B. The column temperature was 40°C and the flow rate was 0.3 mL / min. The injection volume was 5 µL, and the autosampler was heated to 15°C under constant temperature conditions. The mass spectrometer was equipped with a HESI probe, and the parameter settings were as follows: sheath gas pressure 45 arb, auxiliary gas pressure 10 arb, spray voltage 3.5 kV, capillary temperature 350°C, heater temperature 350°C. Glycosphingolipid compounds were identified using full scan and ddMS2 modes. The mass range was set to m / z 150 to 1500, in negative ion mode. Possible elemental compositions were obtained using Xcalibur 3.0 (Thermo Fisher Scientific, California, USA), and only molecular formulas with an error less than 5 ppm were used.

[0041] 3 Clinical research

[0042] 3.1 Participants

[0043] From April 2022 to January 2024, verruca plantaris patients who signed a written informed consent form and agreed to the publication of the study were recruited at Yueyang Hospital of Integrated Traditional Chinese and Western Medicine. Eligible subjects were aged 18–70 years and had not received relevant treatment for at least one month before treatment. Patients who were allergic to any component of the intervention drug or using drugs that might affect the treatment effect (such as immunosuppressants) were excluded. At the same time, patients with other skin diseases that might affect the evaluation of treatment outcomes were excluded.

[0044] 3.2 Study design and interventions

[0045] This study is a randomized, controlled, non-blind pilot study. The study has been approved by the Ethics Committee of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine (Ethical review approval number: 2022-008). In addition, this clinical trial has been registered at the Chinese Clinical Trial Registry (Registration number: ChiCTR2200058284) (https: / / www.chictr.org.cn / ). All participants signed a written informed consent form agreeing to participate in the study and publish the data. Patients were randomly assigned to receive topical salicylic acid ointment (SAO) (Shanghai Yunjian Huangpu Pharmaceutical Co., Ltd., Shanghai, China) (n = 55) twice daily or WRXYF (n = 57) once daily; the random number table in R software (version 4.2.0) was used to randomly assign patients to the treatment groups. All patients received 8 weeks of treatment. Patients who responded to the treatment were followed up for an additional 28 weeks.

[0046] 3.3 Outcome measures

[0047] The primary outcome was the total score, which consisted of the number of warts, the diameter of the warts, and the pain score. The secondary outcomes were the recurrence rate and the Dermatology Life Quality Index (DLQI). The total score and DLQI scores were recorded at weeks 0, 8, 12, 24, and 36. Recurrence was defined as an increase in the total score in patients with effective treatment.

[0048] 4 Mechanism study

[0049] 4.1 Construction of an organoid model from verruca plantaris tissues

[0050] Fresh verruca plantaris tissues were processed in a biosafety cabinet under aseptic conditions. The tissues were washed three times with PBS containing 1% penicillin-streptomycin to remove blood and impurities. After washing, the tissues were cut into small pieces of approximately 1 mm³ and transferred to a digestive solution containing collagenase IV (1 mg / mL), trypsin (0.25%), and DNase I (50 μg / mL). The volume of the digestive solution was three times the volume of the tissue sample. The mixture was incubated in a shaker at 37°C for 1–2 hours, and gently pipetted every 30 minutes to promote digestion. The reaction was terminated with PBS, and the digested tissues were filtered through a 70 μm cell strainer to collect the filtrate. The filtrate was centrifuged at 800 × g for 5 minutes, and the supernatant was discarded. The precipitate was resuspended in PBS, washed twice, and finally resuspended in the culture medium for subsequent use. Matrigel was thawed on ice, and an equal volume of cell suspension was mixed with Matrigel. Take 20–50 μL of the mixture and evenly drop it onto the bottom of a 24-well plate. The plate was placed at 37°C and incubated for 10 minutes to solidify Matrigel, and then the next step was carried out.

[0051] The components of the organoid culture medium are as follows: DMEM / F12 basal medium, B27 supplement (1×), N2 supplement (1×), EGF (50 ng / mL), FGF10 (10 ng / mL), Noggin (100 ng / mL), and ROCK inhibitor Y-27632 (10 μM, used to prevent cell apoptosis). The medium is changed every 2–3 days, and the growth of organoids is monitored under a microscope. The culture plates are placed in an incubator at 37°C and 5% CO2. The formation and morphological changes of organoids are observed regularly, and obvious organoid structures are usually visible after 7–10 days of culture.

[0052] 4.2 Tandem mass tag (TMT)-based quantitative proteomics analysis

[0053] Proteins were extracted from tissue samples and sonicated. The proteins were digested with trypsin and labeled with TMT according to the manufacturer's instructions. The resulting peptides were analyzed by LC-MS using an EASY-nLC 1200 (Thermo Fisher Scientific, Waltham, MA, USA). The raw data were processed by Proteome Discover 2.4 (Thermo Fisher Scientific) and compared with the UniProt database.

[0054] 4.3 Pathway enrichment analysis

[0055] Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using Metascape (http: / / www.Metascape.org / ). Functions and pathways were ranked according to their nominal p-values, with a cutoff value of 0.05.

[0056] 4.4 Molecular docking

[0057] 3D structure information was downloaded from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). Candidate targets were searched in the UniProt database (https: / / www.uniprot.org / ) and obtained from the Protein Data Bank (http: / / www.rcsb.org / ). The Autodock tool 1.5.7 (http: / / autodock.Scripps.Edu / resources / tools) was used for dehydration, hydrogenation, Gasteiger charge calculation, and target storage. The bioactive components were presented in the "pdbqt" format. The binding conformation was visualized using PyMOL 2.4.0.

[0058] 4.5 Western blotting

[0059] Proteins were extracted using RIPA150 lysis buffer containing 1× protease inhibitor (Sigma-Aldrich, Missouri, USA). The total proteins extracted were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Bio-Rad, California, USA). The membrane was incubated with the primary antibody overnight at 4°C and then with the corresponding secondary antibody. The antibodies used included: anti-caspase-3 rabbit polyclonal antibody (GB11767C-100), anti-p21 rabbit polyclonal antibody (GB11153-100), recombinant anti-CDKN2A / p16INK4a antibody (mouse monoclonal antibody, GB151605-100), recombinant anti-β-actin antibody (mouse monoclonal antibody, GB15001-100), recombinant anti-Bcl-2 antibody (rabbit monoclonal antibody, GB154380-100), anti-Bak rabbit polyclonal antibody (GB11324-100), and recombinant anti-Bax antibody (rabbit monoclonal antibody, GB154122-100), all purchased from Servicebio (Wuhan, China).

[0060] 4.6 Sample collection and real-time quantitative PCR

[0061] Human verruca vulgaris tissue samples were collected at Yueyang Hospital of Integrated Traditional Chinese and Western Medicine. Informed consent and approval were obtained from the Ethics Committee of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine (2022-008). HPV RNA was isolated from HaCaT cells infected with verruca vulgaris tissue. Total RNA was extracted using TRIzol reagent (Invitrogen, Massachusetts, USA). cDNA was synthesized using the PrimeScript RT kit (TaKaRa, Beijing, China). qPCR was performed using the SYBR Prime Script RT-PCR kit (TaKaRa, Beijing, China) according to the manufacturer's protocol.

[0062] 4.7 FISH staining

[0063] The paraffin pretreatment II kit (including pretreatment solution and protease buffer), HPV2, HPV27, and HPV57 probe kits, and rubber cement were all purchased from Servicebio. The paraffin sections were baked overnight at 56°C. The conventional dewaxing treatment included placing the slides in xylene three times for 10 minutes each; then placing them in 100% absolute ethanol three times for 10 minutes each. After air drying, the tissue sections were covered with a coverslip and sealed with rubber cement. The slides were placed in a hybridization instrument, denatured at 73°C for 5 minutes, and then hybridized overnight at 37°C. The next day, after removing the rubber cement, they were washed in an elution solution (2× SSC / 0.3% NP-40) at 73°C for 2 minutes, and then quickly rinsed in the elution solution at room temperature for 5 seconds and air dried naturally. Finally, nuclear staining was performed with DAPI (4′,6-diamidino-2-phenylindole), and the results were observed under a fluorescence microscope.

[0064] 4.8 Transmission electron microscopy (TEM)

[0065] Briefly, tissue samples were first fixed in 1% glutaraldehyde (Sigma-Aldrich, St. Louis, USA) for 4 hours, and then post-fixed in 1% osmium tetroxide (Sigma-Aldrich) for 1 hour. After dehydration in a gradient of acetone, the samples were embedded in Resin 12 resin (Ted Pella, USA). Ultrathin sections (70 nm thick) were placed on copper grids, stained with 1% uranyl acetate and 1% lead citrate (both purchased from Sigma-Aldrich), and then observed and photographed using a JEM-1230 transmission electron microscope (JEOL, Japan).

[0066] 4.9 Agarose gel electrophoresis

[0067] 1% agarose gel was prepared by dissolving 1 g of nuclease-free agarose in 100 ml of TAE electrophoresis buffer and poured into a gel cassette to cool and solidify. For Northern blot experiments, agarose gels were prepared using 1×NorthernMax™-Gly electrophoresis buffer. DNA samples were mixed with sample buffer containing glyoxal at a volume ratio of 1:1 and denatured at 50 °C for 30 minutes, then cooled on ice for at least 3 minutes. The denatured DNA was loaded into the gel wells and electrophoresed at 50 V at room temperature until the bromophenol blue dye front ran to the edge of the gel (about 80 minutes). After electrophoresis, the gel was post-stained with SYBR Safe dye (diluted 1:10,000 in 1×NorthernMax™-Gly buffer). Images were acquired using the "SYBR-Safe" mode of the Bio-Rad Gel Doc XR imaging system and Image Lab 5.2 software.

[0068] 4.10 Construction of PcDNA3.1-E7-6xHis cells and construction of px459-FAM107A plasmid

[0069] HaCaT cells were cultured until in good condition and in the logarithmic growth phase, digested with 0.05% trypsin and suspended in complete medium to make a single-cell suspension. The cells were counted and seeded into 6-well plates at a density of 5×10^5 cells per well. The pcDNA3.1-E7-6xHis overexpression plasmid with MOI = 20 was transfected into HaCaT cells, and after thorough mixing, it was incubated in a 5% CO2 incubator for 4 hours. Fresh complete medium was replaced and the cells were cultured for another 48 hours. Antibiotic screening was started according to the cell status, and the cell status was observed daily during the screening period; the antibiotic screening cycle depends on the cell status. Successfully screened stably transfected cells can be cultured routinely or cryopreserved.

[0070]

[0071] 4.11 Cell viability

[0072] E6-HIS cells were seeded into 96-well plates at a density of 5,000 cells per well, with at least 6 replicates in each group. After the cells adhered, they were treated with different concentrations of CAL (final concentrations: 10 μM, 25 μM, 50 μM, 75 μM, 100 μM, and 150 μM). The negative control group was added with an equal volume of DMSO (final concentration <0.1%). After incubation for 48 hours, subsequent analysis was performed.

[0073] 4.12 CCK-8 assay

[0074] Add 10 μL of CCK-8 reagent (Beyotime) to each well, and then incubate for 4 hours at 37°C under 5% CO2. Measure the absorbance (OD450) at a wavelength of 450 nm using a microplate reader.

[0075] 4.13 Detection of cell apoptosis by Annexin V-FITC / PI staining flow cytometry

[0076] Seed E7-HIS cells in a 6-well plate at a density of 2 × 10 5 cells / well. After the cells adhere to the wall, add different concentrations of CAL for treatment. After incubating for 24 hours, collect the cells. Collect the suspended cells in the medium and the adherent cells digested with trypsin together. Centrifuge the cell suspension at 4°C and 1000 rpm for 5 minutes, and wash twice with cold PBS. Perform apoptosis detection according to the instructions of the Annexin V-FITC / PI apoptosis detection kit (Beyotime). Resuspend the cells in 100 μL of 1× binding buffer, with a final concentration of approximately 1 × 10 6 cells / mL. Add 5 μL of Annexin V-FITC and 5 μL of PI to each sample, and incubate in the dark at room temperature for 15 minutes. Then add 400 μL of binding buffer, gently vortex and mix well, and then analyze. Use a BD FACSCanto II flow cytometer for detection, with an excitation wavelength of 488 nm, and detect the FITC (green fluorescence) and PI (red fluorescence) signals respectively.

[0077] 4.14 Detection of cell proliferation by EdU-555 staining

[0078] Seed the cells in a 12-well plate (with coverslips) or a glass-bottom culture dish, and culture until the cell density reaches 30–50%. Treat the cells with CAL (85 μM) for 24 hours, with the final concentration as described above, and set up a control group treated with DMSO. Add EdU-555 to a final concentration of 5 μM, and continue to incubate for 2 hours. Then fix the cells with 4% paraformaldehyde for 15 minutes, and wash three times with PBS. Permeabilize with 0.5% Triton X-100 for 10 minutes. Perform the Click reaction according to the instructions to label EdU-positive cells. Finally, add DAPI staining solution (1 μg / mL), incubate at room temperature for 5 minutes, and then image.

[0079] 4.15 Analysis of cell cycle by flow cytometry

[0080] Seed the cells at 2 × 10 5Cells were seeded in 6-well plates at a density of cells / well and incubated overnight. Cells were treated with CAL at final concentrations of 10 μM and 20 μM for 24 or 48 h, and the control group was added with 0.1% DMSO. After treatment, cells were digested with trypsin, centrifuged to collect the cells, and the supernatant was discarded. The cell pellet was resuspended in 500 μL of PBS, and then 1.5 mL of pre-cooled 70% ethanol was added dropwise while gently vortexing to prevent cell aggregation. The cells were fixed at 4°C overnight to ensure complete fixation. The next day, the cells were centrifuged at 1200 rpm for 5 min, and the ethanol was discarded. The pellet was resuspended in 200 μL of RNase A solution (100 μg / mL, dissolved in PBS) and incubated at 37°C for 30 min to degrade RNA and prevent interference with PI staining. Subsequently, 300 μL of PI solution (50 μg / mL, dissolved in PBS) was added and gently vortexed. The samples were incubated at room temperature in the dark for 30 min to ensure sufficient staining. Before analysis, the cell suspension was passed through a 40-μm filter to remove cell clumps and debris. Detection was performed using a BD FACSCalibur flow cytometer with an excitation wavelength of 488 nm, and PI fluorescence was detected through the FL2 channel (bandpass filter: 585 / 42 nm). Data were analyzed using FlowJo or ModFit LT software, and the percentages of cells in the G1, S, and G2 / M phases were calculated.

[0081] 4.16 DARTS assay

[0082] The total protein lysates of the treated samples were divided into two groups: CAL-treated group: CAL was added to a final concentration of 10 μM (pre-dissolved in DMSO and diluted). Control group: An equal volume of DMSO (<0.1%) was added. After thorough mixing, the samples were incubated at room temperature for 30 min to ensure sufficient binding of CAL to the proteins. Subsequently, different concentrations of proteinase K were added to each sample and vortexed. The samples were incubated at room temperature for 10 min for partial protein digestion. After the reaction, 5× SDS sample buffer was added to each sample to ensure complete dissolution of the proteins. The samples were heated at 95°C for 5 min to denature the proteins. When performing SDS-PAGE, 10–20 μg of protein was loaded into each well onto a 12% SDS-PAGE gel. Electrophoresis was carried out under standard conditions (voltage: 80–120 V) until the dye front approached the bottom of the gel. After electrophoresis, protein bands specifically enriched or with enhanced resistance in the CAL-treated group were identified. These bands were cut out with a sterile scalpel and transferred to 1.5-mL microcentrifuge tubes for subsequent LC-MS analysis.

[0083] 4.17 Immunoprecipitation (Co-IP) assay

[0084] Mix the antibody with Protein A / G agarose beads at a ratio of 1:1. Incubate the mixture by gentle rotation at 4°C for 1 hour or overnight. Wash the agarose beads with protein-free buffer (such as PBS or immunoprecipitation buffer) to remove unbound antibodies. Add the pre-incubated antibody-agarose bead complex to the protein lysate (add 10 μg antibody and 30 μL agarose beads per 100 μg protein). Incubate the mixture by gentle rotation at 4°C for 4 hours or overnight to ensure sufficient binding. After incubation, centrifuge the agarose beads at 3000 rpm for 5 minutes to collect the beads and discard the supernatant. Wash the agarose beads four times with immunoprecipitation buffer for 10 minutes each to remove non-specifically bound proteins. Add SDS sample buffer to the agarose beads to elute the target protein of immunoprecipitation. Heat the eluted protein sample at 95°C for 5 minutes to terminate the reaction. The eluted proteins can be analyzed by SDS-PAGE, Western blotting, or mass spectrometry. The antibodies used include: polyclonal antibody against FAM107A (Cat No.12176-1-AP, Proteintech, Wuhan) and polyclonal antibody against ubiquitin (Cat No. 10201-2-AP, Proteintech, Wuhan).

[0085] 4.18 Cellular Thermal Shift Assay (CETSA)

[0086] Treat E7-HIS cells (1.5 × 10 6 cells / dish) with 85 μM CAL or an equal concentration of DMSO at room temperature for 30 minutes. Subsequently, divide the lysate into five equal parts and heat them at 52°C, 57°C, 62°C, 67°C, and 72°C for 3 minutes respectively, and then stabilize at room temperature for 5 minutes. After heating, centrifuge the lysate at 4°C for 20 minutes to extract the supernatant, and then analyze it by Western blot.

[0087] 4.19 Molecular Dynamics Simulations (MDs)

[0088] Molecular dynamics (MD) simulations were performed using the GROMACS software and the Amber99 force field. First, the energy of the system was minimized by the steepest descent method. Subsequently, the system was placed in a cubic water box with a buffer distance of 2 Å between the solute and the box walls. Counterions were added to neutralize the system charge. The system was equilibrated in two stages: 100 ps in the NVT ensemble (constant volume and temperature) and then another 100 ps in the NPT ensemble (constant pressure and temperature). Productive MD simulations of 2 ns were carried out under periodic boundary conditions with a time step of 2 fs. The particle mesh Ewald (PME) method was used to handle long-range electrostatic interactions, and the LINCS algorithm was used to constrain bond lengths. Trajectory analysis, including root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), and solvent accessible surface area (SASA), was performed using standard GROMACS tools.

[0089] 5 Statistical analysis

[0090] All data were presented as mean ± standard deviation (SD) or proportion, as appropriate. Statistical analysis was performed using Student’s t-test. The log-rank test was used to analyze the Kaplan–Meier curves of non-recurrent patients. Statistical significance was defined as P < 0.05.

[0091] II. Results

[0092] 1 High-performance liquid chromatography–mass spectrometry (HPLC–MS) and molecular docking for the analysis of the components in the ethyl acetate fraction of WRXYF and the screening of active ingredients

[0093] To deeply explore the small molecule compounds of WRXYF with antiviral effects against HPV, we used HPLC-QExactive Orbitrap-MS technology to qualitatively analyze its main chemical components. Based on the accurate m / z information of the quasi-molecular ion peaks and secondary fragment ions provided by high-resolution mass spectrometry, the structures, compositions, and characteristic fragments of the relevant substances were confirmed ( Figure 1 A). Combining database retrieval and relevant literature, a total of 34 compounds were identified, namely: gentiobiose, protocatechuic acid, protocatechualdehyde, caffeic acid, vanillin, p-coumaric acid, curcumol A, ferulic acid, calycosin-7-O-β-D-glucoside, lithospermic acid, bisdemethoxycurcumin, atractylenolide III, rosmarinic acid, azelaic acid, ononin, salvianolic acid B, atractylenolide II, triterpenone C, quercetin, salvianolic acid A, calycosin, neo-curcumol, gingerenol, (15Z)-9,12,13-trihydroxy-15-octadecenoic acid, atractylenolide I, curcumol, isocurcumol, atractylone, dihydrotanshinone I, curdione, cryptotanshinone, astragaloside I, isoastragaloside I, and tanshinone IIA (Table 1), and their structures are as Figure 1As shown in B.

[0094] Table 1 The 34 main components of WRXYF

[0095]

[0096]

[0097] Meanwhile, we performed molecular docking of these 34 main compounds with HPV-E6, HPV-E7, Rb, and TP53 proteins to predict their binding affinities ( Figure 2 A). We found that azelaic acid, calycosin, triterpenone C, and vanillin had the strongest binding affinities with these four proteins ( Figure 2 B), indicating that these four compounds might be the key substances for WRXYF to inhibit the proliferation of HPV-infected cells. Based on the results of molecular docking screening, we further screened azelaic acid, calycosin, triterpenone C, and vanillin at a concentration of 50 μM. Since the cells infected with HPV are usually keratinocytes, we used the HaCaT cell line as a drug screening model. The results showed that calycosin had the most significant inhibitory effect on E6-E7 cells ( Figure 2 C). The non-toxic concentration of calycosin was approximately 20 μM ( Figure 2 D).

[0098] 2 Clinical research results

[0099] 2.1 WRXYF significantly alleviates the skin lesions of verruca plantaris

[0100] To evaluate the efficacy and safety of WRXYF in the treatment of patients with verruca plantaris (PWs), we conducted a prospective, single-center pilot study. A total of 112 participants were successfully enrolled in the clinical trial, including 57 in the WRXYF group and 55 in the SAO group. The detailed screening process is as Figure 3 shown. The baseline data and results of the two groups are shown in Table 2. Only one patient with a history of varicose veins in the lower extremities had an adverse reaction (lower extremity swelling) during the treatment with WRXYF, and the symptoms disappeared after drug withdrawal. No other drug adverse reactions occurred during the treatment. Figure 4 A shows the comparison images of two patients before and after 8 weeks of treatment with WRXYF. The verrucae shrank and flattened, and the pain of the patients was relieved. Before the intervention, the total scores and baseline scores of the dermatology life quality index (DLQI) of the two groups were basically similar, but after 8 weeks of treatment, the total score of the WRXYF group decreased significantly (Table 2) ( Figure 4 B-C).

[0101] Table 2 The main outcomes and recurrence rates of the two groups

[0102]

[0103] *P < 0.05, compared with week 0. ** Patients with effective treatment.

[0104] 2.2 WRXYF has the effect of preventing recurrence

[0105] During the 28-week follow-up period, no serious adverse events were reported. The follow-up data ( Figure 4 D) showed that the recurrence rate in the WRXYF intervention group was significantly lower than that in the SAO group (22.43%, 18%, 40.43%, P = 0.0149), and this result was clinically significant. These findings indicate that local application of WRXYF can be an effective measure for preventing the recurrence of verruca plantaris.

[0106] 3 Mechanism study

[0107] 3.1 Proteomic analysis of HPV-induced verruca plantaris

[0108] Previous studies have shown that verruca plantaris infection is mainly associated with HPV types 2, 27, and 57. Fluorescence in situ hybridization (FISH) results showed that in the patients of our study, the DNA copy number of HPV type 2 was significantly higher than that of HPV types 27 and 57 ( Figure 5 A - B). These findings indicate that HPV type 2 is the main strain of verruca plantaris infection in the Shanghai area. Histological examination by hematoxylin - eosin (H&E) staining found obvious parakeratosis and granulomatous hyperplasia in the verruca tissue, with many keratinocytes vacuolized, the nucleus shrank and a perinuclear halo appeared ( Figure 5 C).

[0109] To further study the active ingredients in WRXYF, we obtained different extracts of WRXYF, divided into aqueous phase (AE), ethyl acetate phase (EAE), and n-butanol phase (BA). qPCR results showed that EAE had the strongest inhibitory effect on the mRNA expression of HPV2 and HPV27 ( Figure 5 D).

[0110] To explore the effect of HPV infection on verruca plantaris tissue at the proteomic level, we used TMT-based quantitative proteomic analysis to compare verruca tissue with normal skin tissue of healthy people. A total of 1,209 differentially expressed genes (DEGs) were identified, among which 521 genes were up-regulated and 688 genes were down-regulated. The heat map and volcano plot are shown as Figure 6 A and 6B. Further analysis of the localization of these DEGs found that they were mainly located in the membrane, extracellular region, and cytoplasm ( Figure 6 C).

[0111] To gain in-depth understanding of the functions of these genes and the signaling pathways they are involved in, we performed GO and KEGG analyses. GO analysis showed that the upregulated genes were mainly related to biological processes such as nucleic acid metabolism and cellular aromatic compound metabolism, mainly localized in the nuclear lumen and nucleoplasm, and were related to molecular functions such as protein binding and RNA binding. In contrast, the downregulated genes were mainly related to biological processes such as complement activation and humoral immune response, and the extracellular matrix and extracellular region were the main enriched cellular components, and were related to molecular functions such as extracellular matrix structural components and antigen binding ( Figure 6 D). KEGG analysis showed that the upregulated genes were mainly involved in pathways such as splicing and cellular senescence, while the downregulated genes were mainly related to focal adhesion, complement, and coagulation cascades ( Figure 6 E).

[0112] 3.2 CAL Treats Verruca Plantaris Organoids by Inducing Apoptosis

[0113] We cultured organoids from verruca plantaris tissues of HPV-infected patients and treated them with calycosin. Figure 7 A shows the status of the organoids before and after CAL treatment. We also inoculated the organoids onto the surface of endothelial cells, and the results showed that CAL disrupted the morphology of verruca plantaris organoids ( Figure 7 B).

[0114] DNA agarose gel electrophoresis showed that HPV2-E6 and HPV2-E7 DNA expressions were present in the verruca organoids, but not detected in normal tissues ( Figure 7 C). Transmission electron microscopy observations found that there were no viral particles in the normal group, while the viral particles in the CAL treatment group were fragmented and lost their original structure, indicating that CAL has the ability to disrupt HPV particles ( Figure 7 D). To verify whether calycosin treats verruca plantaris by inducing apoptosis, we detected the protein expressions of apoptosis-related genes. Western blot analysis showed that after CAL intervention, the expressions of pro-apoptotic genes such as Caspase 3, P21, P16, BAX, and BAK were significantly increased, while the expression of the anti-apoptotic gene BCL2 was significantly decreased ( Figure 7 E-F). These results indicate that calycosin, as the key active ingredient of WRXYF, can treat verruca plantaris by inducing apoptosis.

[0115] 3.3 CAL Promotes Apoptosis and Inhibits Proliferation of E7-HIS Overexpressing HaCaT Cells

[0116] E7 is a key pathogenic factor in HPV-related palmar and plantar warts. Increasing evidence indicates that it plays important roles in regulating host immunity, promoting cell proliferation, and reducing cell death. To explore the effect of CAL on HPV-infected cells, we overexpressed E7-HIS protein in HaCaT cells ( Figure 8 A). Western blot verified the successful overexpression of E7-HIS ( Figure 8 B). Under the microscope, HaCaT cells transfected with the empty vector pcDNA3.1 still maintained a typical "cobblestone-like" morphology with smooth boundaries; while E7-HIS overexpressing cells showed protrusion-like extensions ( Figure 8 C). By measuring the IC50 value of CAL in E7-HIS cells to be 85.35 μM ( Figure 8 D), it was found that CAL significantly inhibited cell proliferation after 72 hours of treatment ( Figure 8 E). Flow cytometry showed that CAL intervention increased apoptosis by approximately 3% ( Figure 8 F). EdU-555 staining indicated that CAL treatment inhibited DNA replication ( Figure 8 G), and TUNEL staining further confirmed a significant increase in apoptotic cells after CAL exposure ( Figure 8 H).

[0117] In summary, we successfully mimicked the cell state after HPV infection through E7-HIS overexpression. Although E7 has an anti-apoptotic effect, CAL can still significantly induce apoptosis in E7-HIS-expressing cells, highlighting its potential as a therapeutic drug.

[0118] 3.4 Identification of FAM107A as a direct target of CAL based on target fishing technology

[0119] To clarify the mechanism by which CAL promotes apoptosis in E7-HIS overexpressing cells, drug affinity responsive target stability mass spectrometry (DARTS-MS) was used to screen for potential direct binding targets of CAL ( Figure 9 A). E7-HIS cells were treated with different concentrations of CAL (12.5, 25, 50, 100, 200 μM) and protease (0.1, 0.25, 0.5, 1, 2 μg) ( Figure 9 B), and 100 μM CAL and 1 μg protease were selected for LC-MS analysis ( Figure 9 C). Among the differential proteins, the abundance difference of FAM107A between the E7-HIS group and the CAL treatment group was the most significant ( Figure 9 D), making it the primary target for verification.

[0120] Molecular docking showed that the binding energy between CAL and FAM107A reached -5.3 kcal / mol ( Figure 9E). The radius of gyration (Rg) analysis indicated that the overall conformation of the complex changed slightly during the 5000 ps trajectory. The slight decrease at 2000 ps suggested that the protein underwent conformational adjustment to accommodate CAL binding ( Figure 9 F). Molecular dynamics simulations further verified the binding stability: The RMSD analysis showed that the FAM107A-CAL complex tended to be stable after 350 ps (fluctuation range 0.13 nm), and no obvious dissociation was observed ( Figure 9 G); The RMSF analysis showed low conformational flexibility at the binding interface ( Figure 9 H); The solvent accessible surface area (SASA) analysis showed significant fluctuations in residues 0 - 50, stable binding interfaces formed by residues 50 - 120, and increased exposure of residues after 120 (especially residue 150) ( Figure 9 I).

[0121] To experimentally verify the molecular docking and kinetic results, we performed CESTA assays. The results showed that FAM107A in the CAL treatment group remained stable even when the temperature was increased from 52 °C to 72 °C ( Figure 9 J). In the DARTs experiment, under the conditions of fixed protease concentration and temperature, decreasing the CAL concentration (from 100 μM to 1 μM) led to a corresponding decrease in the expression level of FAM107A ( Figure 9 K - L). These findings confirmed that FAM107A is a direct binding target of CAL.

[0122] To further explore the role of FAM107A in the CAL-mediated effects on E7-HIS cells, we detected its expression level. The qRT-PCR results showed that CAL did not change the FAM107A mRNA expression ( Figure 10 A), but the WB results indicated that CAL treatment significantly increased the FAM107A protein level ( Figure 10 B). To determine whether FAM107A is a key mediator of CAL-induced apoptosis in E7-HIS cells, we constructed a FAM107A knockout cell line (PX459-FAM107A). The WB and qRT-PCR results confirmed successful knockout, with a significant decrease in FAM107A mRNA and only trace amounts of protein remaining ( Figure 10 C - D). After 72 hours of CAL treatment, the FAM107A-KO cells partially recovered their proliferative ability ( Figure 10 E), and EdU-555 staining showed that the inhibitory effect of CAL on DNA replication was weakened in the absence of FAM107A ( Figure 10 F). The cell adhesion assay showed that CAL treatment significantly reduced the adhesion ability of E7-HIS cells, but this effect was reversed in FAM107A-KO cells ( Figure 10G). Flow cytometry analysis showed that FAM107A depletion reduced CAL-induced apoptosis ( Figure 10 H). Cell cycle analysis indicated that CAL-induced G1 phase arrest was significantly alleviated in FAM107A-KO cells ( Figure 10 I). Given that CAL did not affect FAM107A mRNA levels but increased its protein expression, we speculated that CAL stabilized FAM107A by inhibiting its degradation. Co-IP results showed that CAL treatment significantly reduced the ubiquitination level of FAM107A ( Figure 10 J), indicating that CAL stabilized FAM107A by preventing ubiquitination-mediated degradation, thereby promoting apoptosis in E7-HIS cells.

[0123] In summary, the present invention confirmed that CAL directly binds to FAM107A and reduces its ubiquitination degradation, resulting in increased protein levels and enhanced apoptosis in E7-HIS cells. These findings established FAM107A as a key target of CAL and provided new insights into potential therapeutic strategies for HPV-related diseases.

[0124] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art can also make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Application of calycosin in the preparation of a drug for anti-human papillomavirus infection.

2. Use of calycosin according to claim 1 in the preparation of a medicament for preventing human papillomavirus infection, characterized in that, The human papillomavirus infection described is the human papillomavirus infection on palms, soles, fingers and toes.

3. Use of calycosin according to claim 1 in the preparation of a drug for preventing human papillomavirus infection, characterized in that, Calycosin clears infected cells by activating the apoptosis pathway, and has the dual effects of directly inhibiting virus replication and inducing apoptosis to fundamentally clear virus infection.

4. Application of calycosin in the preparation of a drug for treating verruca plantaris.

5. A drug for treating verruca palmaris et plantaris, characterized in that, The drug for treating verruca plantaris uses calycosin as the only active ingredient.

6. The medicament for treating palmar and plantar warts according to claim 5, wherein, The drug for treating verruca plantaris is an external preparation.

7. The medicament for treating palmar and plantar warts according to claim 6, wherein, The drug for treating verruca plantaris also includes excipients commonly used in external preparations.

8. The medicament for treating palmar and plantar warts according to claim 5, wherein The effective dose of calycosin in the drug for treating verruca plantaris is 85.35 μM.

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