Small molecule polypeptide capable of being used as type 5 phosphodiesterase inhibitor and application of small molecule polypeptide
By designing the small molecule peptide FRPPAL, the problem of large side effects of existing PDE5 inhibitors has been solved, providing a safe and effective PDE5 inhibitor for the treatment and prevention of various diseases.
Patent Information
- Application Number
- CN202511264759.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-05
- Publication Date
- 2025-11-28
AI Technical Summary
Existing PDE5 inhibitors have significant side effects and numerous contraindications, making it difficult to develop natural and safe alternatives.
A small molecule polypeptide FRPPAL with the amino acid sequence FRPPAL was designed and synthesized. Polypeptides with high affinity for PDE5 protein were screened using molecular docking technology for the preparation of PDE5 inhibitors. The preferred dosage form is an oral formulation, and the administration routes include oral, spray inhalation, intravenous injection, intramuscular injection, rectal administration, or topical skin administration.
It achieves significant inhibition of PDE5, reduces side effects, and improves safety and applicability, making it suitable for the treatment and prevention of various diseases, including male erectile dysfunction and female sexual dysfunction.
Smart Images
Figure CN121021633A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biotechnology, in particular to a polypeptide which can be used as a phosphodiesterase type 5 inhibitor and application thereof. BACKGROUND
[0002] Phosphodiesterase type 5 (PDE5) is a member of the PDEs (phosphodiesterase) family, which can specifically hydrolyze cGMP and regulate the level of intracellular cGMP, and is involved in a variety of important biological and physiological processes in the body. PDE5 is widely distributed in the human lung, corpus cavernosum smooth muscle, and tissues such as blood vessels, internal organs, and airway smooth muscle, skeletal muscle, and platelets, and can well regulate the contractility of penile and pulmonary vascular smooth muscle, and plays an important role in the physiological process of penile erection. It can regulate Ca 2+ channels, and change the contraction state of penile smooth muscle. Nitric oxide (NO) is an important vasodilator, which enters the endothelial cells of smooth muscle to activate guanylate cyclase to catalyze the synthesis of the second messenger cGMP, activate calcium ion channels, and cause vasodilation and blood filling.
[0003] As an enzyme, PDE5 can catalyze the degradation of cGMP, and counteract the vasodilation caused by NO, thereby causing vasoconstriction. Therefore, PDE5 inhibitors can dilate blood vessels, thereby treating diseases related to vasoconstriction, or producing corresponding adverse reactions. Based on the above biological effects, PDE5 and its inhibitors were focused on cardiovascular treatment in the early stage of research and development, and were successfully used for the treatment of pulmonary hypertension. At the same time, during the development of sildenafil, it was accidentally found that it has a positive regulatory effect on cavernosal hyperemia, thereby successfully developing a market hot-selling sexual function treatment drug.
[0004] PDE5 inhibitors can inhibit PDE5 activity, maintain a high concentration of cGMP in the corpus cavernosum, and thus prolong the erection time of the penis, thereby treating male erectile dysfunction. Therefore, PDE5 is an ideal drug target for treating diseases related to cGMP levels, such as male erectile dysfunction (MED), pulmonary hypertension (PAH), etc. The currently clinically used PDE5 inhibitors are mainly chemical synthetic drugs of sildenafil and tadalafil, which have the disadvantages of large side effects, many contraindications, etc. Therefore, it is of great practical significance to develop natural and safe PDE5 inhibitors. SUMMARY
[0005] The present application provides a polypeptide which can be used as a phosphodiesterase type 5 inhibitor and application thereof.
[0006] The small molecule polypeptide as a phosphodiesterase type 5 inhibitor is a hexapeptide, and its amino acid sequence is FRPPAL. Phe is phenylalanine, Arg is arginine, Pro is proline, Ala is alanine, and Leu is leucine. The Phe end is the N-terminal (amino terminal), and the Leu end is the C-terminal (carboxyl terminal), so the sequence can also be represented as: and NH2-Phe-Arg-Pro-Pro-Arg-Leu-COOH.
[0007] The application further provides the use of the polypeptide in the preparation of a phosphodiesterase type 5 inhibitor for purposes other than disease treatment.
[0008] The application further provides the use of the polypeptide in the preparation of a drug for treating, relieving or preventing a phosphodiesterase type 5 disorder. Preferably, the phosphodiesterase type 5 disorder is male erectile dysfunction, male testicular ischemia, female sexual dysfunction, premature birth, dysmenorrhea, benign prostatic hyperplasia, bladder outlet obstruction, incontinence, stable, unstable and variant angina, hypertension, pulmonary arterial hypertension, Alzheimer's disease, congestive heart failure, arteriosclerosis, stroke, peripheral vascular disease, a disease of reducing vascular full opening, chronic asthma, bronchitis, allergic asthma, allergic rhinitis, glaucoma or a disease characterized by intestinal motility disorder.
[0009] The application further provides a drug for treating, relieving or preventing a phosphodiesterase type 5 disorder, and the active ingredient is the polypeptide.
[0010] Preferably, the drug is in the form of tablets, capsules, lozenges, sugar-coated pills, pills, granules, powders, solutions, emulsions, suspensions, dispersions, syrups, gels, aerosols. Preferably, it is an oral preparation, such as an oral tablet, capsule, etc.
[0011] The administration mode can be oral, spray inhalation, intravenous injection, intramuscular injection, rectal administration or local administration on the skin.
[0012] Preferably, it is oral.
[0013] Preferably, the drug is for treating, relieving or preventing a phosphodiesterase type 5 disorder, and the phosphodiesterase type 5 disorder is male erectile dysfunction, male testicular ischemia, female sexual dysfunction, premature birth, dysmenorrhea, benign prostatic hyperplasia, bladder outlet obstruction, incontinence, stable, unstable and variant angina, hypertension, pulmonary arterial hypertension, Alzheimer's disease, congestive heart failure, arteriosclerosis, stroke, peripheral vascular disease, a disease of reducing vascular full opening, chronic asthma, bronchitis, allergic asthma, allergic rhinitis, glaucoma or a disease characterized by intestinal motility disorder.
[0014] The application screens polypeptide sequences with high affinity (binding energy <-10.0 kcal / mol) to PDE5 protein by molecular docking technology. Through polypeptide design, polypeptide synthesis and in vitro activity determination, the active polypeptide Phe-Arg-Pro-Pro-Arg-Leu of the application is finally obtained. The preferred sequence is subjected to solid-phase synthesis and in vitro enzyme inhibition activity evaluation, and finally one candidate polypeptide with significant PDE5 inhibition activity is identified. When the mass concentration of FRPPAL is 100 μM, the inhibition rate of FRPPAL on PDE 5 is 31.81%.
[0015] Compared with small molecule compounds, polypeptides are easy to degrade in vivo and will not cause damage due to accumulation in vivo. In addition, small molecule polypeptides are easy to absorb and not easy to be degraded by gastrointestinal proteases, and can be taken orally. BRIEF DESCRIPTION OF DRAWINGS
[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.
[0017] Figure 1 Molecular docking diagram of small molecule peptide FRPPAL and PDE5;
[0018] Figure 2 cGMP ELISA detection results after HUVEC cells were treated with small molecule peptide FRPPAL;
[0019] Figure 3 PDE5 inhibition rate results of small molecule peptide FRPPAL;
[0020] Figure 4 NO detection kit detection results after HUVEC cells were treated with small molecule peptide FRPPAL;
[0021] Figure 5 Real-time PCR detection mRNA expression level results after HUVEC cells were treated with small molecule peptide FRPPAL;
[0022] Figure 6 Cell toxicity experiment diagram of small molecule peptide FRPPAL. DETAILED DESCRIPTION
[0023] Now a variety of exemplary embodiments of the present application will be described in detail, which should not be considered as a limitation of the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.
[0024] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, it is to be understood that each intervening value, to the upper or lower limit, is also specifically included within the scope of the present application. The upper and lower limits of these intervening values are also
[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the content of the present specification will control.
[0026] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.
[0027] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.
[0028] Example 1
[0029] (1) Molecular docking
[0030] PDE5 was selected as the protein receptor. First, its Entry ID was obtained through Uniprot, and the three-dimensional crystal structure of the protein receptor was downloaded from the protein data bank (PDB). Subsequently, the protein receptor structure was optimized using Pymol software, including removing water molecules and small molecule ligands. The three-dimensional structure of the polypeptide was generated through the RPBS online server. Next, the polypeptide and the protein receptor were hydrogenated, and the charge was calculated using AutoDockTools 1.5.6, and molecular docking was performed, and finally the binding energy of the protein receptor-polypeptide was calculated. The lower the binding energy, the better the binding effect. If the binding energy is <0 kcal / mol, it means that the small molecule ligand can spontaneously bind to the protein receptor. It was calculated that the molecular docking binding energy of the small molecule peptide FRPPAL (SEQ ID NO. 1) and PDE5 was -10.2 kcal / mol, as shown in Figure 1.Figure 1 as shown.
[0031] Example 2
[0032] (1) Inhibition rate of small molecule polypeptide on PDE5
[0033] Human umbilical vein endothelial cells HUVEC (purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China)) were inoculated in DMEM:F12 (1:1) complete culture medium containing 10% fetal bovine serum by volume. They were placed in a cell incubator at 37°C, 5% CO2, and observed daily for cell condition and timely medium replacement. When replacing the medium, the old medium was first discarded, washed three times with 37°C PBS, and then fresh complete culture medium was added. The cells were cultured to the logarithmic phase. The logarithmically growing cells were discarded from the culture supernatant, washed with PBS, and then 0.25% trypsin by mass volume was added for 3 min of digestion, which was terminated with complete culture medium. The cells were centrifuged at 800 rpm for 5 min at room temperature. The supernatant was discarded, and 1 mL of complete culture medium was used to blow the cells to prepare a cell suspension for counting. According to the counting results, the cell suspension was diluted to 1.58 x 10 5 cells / mL, inoculated into a 96-well plate at 100 μL per well, and cultured overnight in a 37°C incubator.
[0034] The small molecule peptide FRPPAL was synthesized by Nanjing Jiepeibio Technology Co., Ltd. using solid-phase chemical synthesis (SPPS) with a purity greater than 95%. The small molecule peptide FRPPAL was dissolved and diluted with complete culture medium to form 1 ml of working solution with a concentration of 100 μM and 10 μM. After overnight culture, the culture medium was removed from the cells, 100 μL of small molecule peptide solution with different concentrations was added to the experimental group, and 100 μL of complete culture medium was added to the control group. Each concentration was set in triplicate, and the cells were cultured in a 37°C incubator for 1 h. After 1 h of drug administration and culture, 0.1 M dilute hydrochloric acid was added for 1 min to lyse the cells. The extraction was centrifuged at 1500 x g for 10 min at 4°C, and the supernatant was used to detect cGMP ELISA KIT (catalog number MB-1568A) purchased from Jiangsu Meibiao Biotechnology (China) according to the manufacturer's instructions. The results are shown in Figure 2
[0035] (2) Inhibition of PDE5 activity by the sample, expressed as a percentage:
[0036] Inhibition rate % = (substrate concentration of sample tube - substrate concentration of control tube) / substrate concentration of control tube x 100%, wherein the substrate is cGMP, and the substrate concentrations of the sample tube and the control tube refer to the substrate concentrations after the reaction. It was determined that the inhibition rate of FRPPAL on PDE 5 was 31.81% when the mass concentration thereof was 100 μM; the inhibition rate of FRPPAL on PDE 5 was 17.31% when the mass concentration thereof was 10 μM, and the results are shown in Table 1. Figure 3
[0037] Example 3
[0038] (1) Determination of the effect of small molecule polypeptides on the NO-sGC-cGMP signal pathway of cells
[0039] The human umbilical vein endothelial cell HUVEC cell suspension was diluted to 1.58 x 10 5 cell / mL, and was inoculated into a 6-well plate at 2 ml of complete culture medium per well, and was cultured in a 37°C incubator overnight. The small molecule peptide FRPPAL was dissolved and diluted using the complete culture medium to form 20 ml of a working solution with a concentration of 100 μM. The culture solution of the cells after overnight culture was removed, 2 ml of small molecule peptide solution of different types was added to the experimental group, 2 ml of complete culture medium was added to the control group, 3 repeats were set for each concentration, and the cells were cultured in a 37°C incubator for 1 h. 50 μl of supernatant was taken and used in a NO detection kit (catalog number S0021S) purchased from Shanghai Biyun Tian Biotechnology Co., Ltd., 50 μl / well of room temperature Griess Reagent I was added to each well; 50 μl / well of room temperature Griess Reagent II was added to each well; the absorbance was determined at 540 nm, and the cells were used in subsequent real-time fluorescence quantitative experiments.
[0040] It was determined that the NO release amount of the FRPPAL treatment group was 7.44 μM when the mass concentration thereof was 100 μM, which was 459.67% of the control group, and the results are shown in Table 2. Figure 4
[0041] (2) Real-time fluorescence quantitative PCR (Real-time PCR) detection of mRNA level expression
[0042] Total RNA extraction, reverse transcription was performed for each RNA sample using Primescript™ RT Master Mix (Takara, China). QRT-PCR was performed using QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher, USA) to perform QRT-PCR based on SYBR Green PCR Master Mix (Takara, China). The mRNA expression level of the test gene was standardized to the mRNA expression level of PDE5 and PKG. The relative mRNA expression level was calculated using 2 -ΔΔCT The relative mRNA expression level was calculated using 2 Figure 5 -ΔCt method. The results are shown in FIG. 4.
[0043] Example 4
[0044] Small molecule peptide cytotoxicity assay
[0045] The cytotoxicity experiment of the antimicrobial peptide was performed using human umbilical vein endothelial cells HUVEC. The above cells were inoculated in DMEM:F12 (1:1) complete culture medium containing 10% fetal bovine serum by volume. They were placed in a cell incubator at 37°C, 5% CO2, and observed daily for cell status and timely medium replacement. The old medium was discarded, and the cells were washed three times with 37°C PBS before adding fresh complete culture medium. The cells were cultured to the logarithmic phase. The logarithmic phase cells were discarded, washed with PBS, and then 0.25% trypsin was added for 3 min. The digestion was terminated with complete culture medium, and the cells were centrifuged at 800 rpm for 5 min at room temperature. The supernatant was discarded, and the cells were counted by blowing the cells with 1 mL of complete culture medium to prepare a cell suspension. According to the counting results, the cell suspension was diluted to 1.58 x 10 5 cells / mL, inoculated into a 96-well plate at 100 μL per well, and cultured overnight in a 37°C incubator.
[0046] The small molecule peptide FRPPAL was dissolved and diluted with complete culture medium to form 500 μL of working solution at concentrations of 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM. The culture medium of the cells after overnight culture was removed, 100 μL of antimicrobial peptide solution at different concentrations was added to the experimental group, and 100 μL of complete culture medium was added to the control group. Three replicates were set for each concentration, and the cells were cultured in a 37°C incubator for 24 h. After 24 h of drug culture, 10 μL of WST-8 reagent was added to each well, and the cells were incubated in a 37°C incubator for 1 h. The plate was read at a wavelength of 450 nm on an enzyme marker, and the results are shown in FIG. 5. Figure 6
[0047] The cytotoxicity of the small molecule peptide FRPPAL was detected. When the concentration of the small molecule peptide FRPPAL reached 200 μM, the survival rate of HUVEC cells was 97.16%, and no obvious cytotoxicity was observed at this concentration, indicating that the small molecule peptide FRPPAL has excellent medical treatment potential.
[0048] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.
Claims
1. A PDE5 inhibitor, characterized in that, The active ingredient of the PDE5 inhibitor includes a small molecule polypeptide with an amino acid sequence as shown in SEQ ID NO.
1.
2. The use of the PDE5 inhibitor according to claim 1 in the preparation of a type 5 phosphodiesterase inhibitor drug.
3. The application according to claim 2, characterized in that, The drug is used to treat or prevent diseases associated with abnormal PDE5 activity.
4. The application according to claim 3, characterized in that, The diseases associated with abnormal PDE5 activity include erectile dysfunction, pulmonary hypertension, benign prostatic hyperplasia, heart failure, or renal dysfunction.
5. The application according to claim 2, characterized in that, The drug enhances the activity of the nitric oxide / cGMP signaling pathway by inhibiting PDE5 activity.
6. The application according to claim 2, characterized in that, The dosage forms of the drug include oral preparations, sublingual preparations, injections, transdermal preparations, or sustained-release preparations.
7. The application according to claim 2, characterized in that, The effective concentration of the small molecule polypeptide with the amino acid sequence shown in SEQ ID NO.1 when the drug is used is 6.25-200 μM.
8. A type 5 phosphodiesterase inhibitor drug, characterized in that, The drug comprises the PDE5 inhibitor of claim 1 and a pharmaceutically acceptable carrier or excipient.