High-throughput screening method for inhibitor targeting E6AP-E6 protein-protein interaction

Abstract

The invention discloses a high-throughput screening method of an inhibitor targeting E6AP-E6 protein-protein interaction. The high-throughput screening method comprises the following steps: (1) incubating a solution of a compound to be detected with E6AP protein or E6 protein in vitro; (2) after incubation is completed, correspondingly adding E6 protein or E6AP protein into the mixed solution, uniformly mixing and then incubating; (3) after the incubation is completed, adding a fluorescence donor and a fluorescence receptor into the mixed solution, uniformly mixing and then incubating; and (4) after incubation is finished, detecting a fluorescence signal of the solution, and judging whether the compound to be detected can inhibit E6AP-E6 protein-protein interaction or not. The screening method has the advantages of convenience, rapidness, stability, high efficiency, low false positive rate, sample saving and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a high-throughput screening method for inhibitors targeting E6AP-E6 protein-protein interaction. Background Art

[0002] Cervical cancer is the fourth most common cancer among women worldwide. According to WHO data, there will be nearly 660,000 new cases and over 348,000 deaths from cervical cancer in 2022, making it a global health concern. The vast majority (over 95%) of cervical cancers are caused by human papillomavirus (HPV). Persistent infection with high-risk HPVs (such as HPV-16 and HPV-18) is closely associated with the development of cervical cancer. HPV is a spherical DNA virus. The early protein-coding (E) region of its genome encodes early proteins (E1, E2, E4, E5, E6, and E7) that play important roles in HPV replication, transcription, translation, and cell transformation. E6 and E7 are the main oncogenic proteins. HPV E6 hijacks the ubiquitin ligase E6AP to ubiquitinate substrates (including tumor suppressors such as p53, MAGI1, and DLG1), leading to their degradation. E6AP forms an extensive interaction interface (approximately 2,361 Ų) with the human papillomavirus early protein E6, with a binding index reaching the picomolar level. Inhibitors that effectively disrupt the E6AP-E6 protein-protein interaction (PPI) are expected to become lead compounds for the treatment of cervical cancer.

[0003] Current screening methods for E6AP-E6 PPI inhibitors are very limited. The vast majority rely on virtual screening based on the E6 structure. Virtual screening methods evaluate binding energy or drug-likeness based on the E6 structure, but cannot definitively prove that a compound disrupts the E6AP-E6 interaction, and further experimental verification is required after screening. Experimental screening methods are extremely rare, and in vitro screening methods based on disruption of the E6AP-E6 interaction all utilize E6AP peptides, which cannot directly demonstrate that a compound completely disrupts the E6AP-E6 interaction. These limitations in screening methods directly restrict the research on PPI inhibitors targeting E6AP-E6. Currently, no E6AP-E6 PPI inhibitors have entered the clinical stage. The number of investigated inhibitors is small, their activity is low, and their structure-activity relationships are unclear. New and efficient methods for screening PPI inhibitors targeting E6AP-E6 are urgently needed.

[0004] Homogeneous time-resolved fluorescence (HTRF) is a technique based on fluorescence resonance energy transfer. When the emission spectrum of one fluorescent group (donor) overlaps with the absorption spectrum of another group (acceptor), and the distance between the two fluorescent groups is appropriate (less than 100Å, 10nm), the donor energy is transferred to the acceptor. The fluorescent group structure is derived from a lanthanide complex and exhibits excellent performance in terms of stability and specificity, and is highly tolerant to most experimental conditions. The donor and acceptor fluorophores can be coupled to two interacting proteins. When the proteins are close enough, the fluorophores also move closer. The donor is excited by an energy source (such as a flash or laser), triggering energy transfer to the acceptor, which then emits a specific fluorescence at a given wavelength. HTRF combines standard fluorescence resonance energy transfer technology with time-resolved fluorescence to eliminate transient background fluorescence. A time delay of approximately 50 to 200µs is introduced between system excitation and fluorescence detection to eliminate all nonspecific, short-lived emission signals from the system. Because the HTRF donor is a lanthanide element with a long half-life, the HTRF acceptor accepts energy transfer from the donor fluorophore, emitting long-lived fluorescence. Therefore, the long-lived emission light detected after the time delay originates from energy transfer resulting from the proximity of biomolecules. HTRF technology eliminates the need for plate washing and can be detected by simply adding and incubating the sample, greatly increasing screening throughput. It offers the advantages of tolerance to most chemicals, multiple readouts, stable signals (up to hours and days), and low sample requirements.

[0005] To date, no method based on homogeneous time-resolved fluorescence technology has been applied to the high-throughput screening of PPI inhibitors targeting E6AP-E6. Summary of the Invention

[0006] The present invention provides a high-throughput screening method for inhibitors targeting E6AP-E6 protein-protein interaction, which has the advantages of convenience, rapidity, stability, high efficiency, low false positive rate, and sample saving.

[0007] The technical solutions of the present invention are as follows: A high-throughput screening method for inhibitors targeting E6AP-E6 protein-protein interaction comprises the following steps: (1) Incubating the test compound solution with E6AP protein or E6 protein in vitro; (2) After the incubation is completed, E6 protein or E6AP protein is added to the mixed solution, mixed evenly, and then incubated; (3) After the incubation is completed, add the fluorescent donor and fluorescent acceptor to the mixed solution, mix well and incubate; (4) After the incubation, the fluorescence signal of the solution is detected to determine whether the compound to be tested can inhibit the E6AP-E6 protein-protein interaction.

[0008] The screening method of the present application is an in vitro screening method based on protein-protein interaction. E6AP and E6 protein with interaction are added in the system. The fluorescence donor and the fluorescence acceptor are close to each other through the interaction of the two, and then the fluorescence signal is generated under excitation light. The signal value of the sample with different compounds is analyzed. If the signal value is significantly reduced, it indicates that the compound corresponding to the sample is a potential E6AP-E6 PPI inhibitor.

[0009] In steps (1) and (2), the E6 protein and E6AP protein are full-length or truncated proteins expressed by eukaryotic or prokaryotic expression system.

[0010] In step (1), if the binding site of the compound to be tested is located on the E6AP protein, the compound is incubated with the E6AP protein; if the binding site of the compound to be tested is located on the E6 protein, the compound is incubated with the E6 protein; if the binding site of the compound to be tested is unknown, parallel experiments of incubating the compound to be tested with E6AP protein and E6 protein can be performed respectively.

[0011] In step (2), if the compound to be tested in step (1) is incubated with E6AP protein, E6 protein is added; if step (1) is incubated with E6 protein, E6AP protein is added.

[0012] The containers used for incubation in all the above steps can be PCR tubes or well plates.

[0013] Preferably, in steps (1)-(3), the compound to be tested, E6AP protein, E6 protein, fluorescence donor and fluorescence acceptor are diluted to a certain concentration using 1x reaction buffer.

[0014] Preferably, the components of the 1x reaction buffer include at least one of 4-hydroxyethyl piperazine ethanesulfonic acid buffer (HEPES), tris-hydroxymethyl aminomethane hydrochloric acid (Tris-HCl), citric acid / sodium citrate solution, 2-morpholinoethanesulfonic acid buffer (MES), and 3-morpholinopropanesulfonic acid buffer (MOPS); and further preferably 4-hydroxyethyl piperazine ethanesulfonic acid buffer.

[0015] Further preferably, the 1x reaction buffer further contains sodium chloride, BSA, and a detergent.

[0016] The detergent includes at least one of Triton X-100 (Triton X-100), ethylphenyl polyethylene glycol (NP-40), 3-[(3-cholaminylpropyl)dimethylamino]-2-hydroxy-1-propanesulfonic acid inner salt (CHAPSO), Tween-20 (Tween-20), and n-dodecyl β-D-maltoside (DDM); more preferably Triton X-100.

[0017] Preferably, the components in the 1× reaction buffer include 4-hydroxyethylazineethanesulfonic acid, sodium chloride, BSA, and Triton X-100; in the 1× reaction buffer, the concentration of 4-hydroxyethylazineethanesulfonic acid is 10 to 50 mmol / L, the pH is 7.0 to 8.0, and the concentration of sodium chloride is 100 to 500 mmol / L.

[0018] Most preferably, the 1× reaction buffer comprises: 20 mmol / L 4-hydroxyethylazineethanesulfonic acid buffer with a pH of 7.5, 100 mmol / L NaCl solution, 0.1% BSA solution, and 0.01% Triton X-100 solution.

[0019] Preferably, the fluorescent donor group includes at least one of europium, terbium, and fluorescein, and is further optimized to be terbium; the fluorescent acceptor group includes at least one of XL665, d2, and fluorescein, and is further optimized to be XL665.

[0020] XL665 is a modified allophycocyanin (APC) that has been conjugated to an APC subunit for increased stability. D2 is a second-generation fluorescent receptor developed after XL665. It is a synthetic protein with the same spectroscopic characteristics as XL665, but a smaller molecular weight of approximately 1 kDa.

[0021] Preferably, the ubiquitin ligase E6AP includes at least one of the wild type UBE3A (Uniprot ID No.Q05086) or a mutant in which cysteine ​​at position 843 is mutated to alanine; the human papillomavirus early protein E6 includes at least one of the wild type VE6_HPV16 (Uniprot ID No.P03126) or a mutant in which cysteine ​​at positions 87, 104, 118, and 147 are mutated to serine (4C / 4S), and the wild type VE6_HPV18 (Uniprot ID No.P06463).

[0022] Preferably, the E6AP protein is coupled to Strep tag II at the N-terminus or C-terminus, and the E6 protein is coupled to a 6×His tag at the N-terminus or C-terminus; further preferably, the E6AP protein is coupled to Strep tag II at the C-terminus, and the E6 protein is coupled to a 6×His tag at the N-terminus.

[0023] Preferably, in the incubation system, the E6AP protein concentration is 3.9 ~ 250nmoL / L, and the E6 protein concentration is 31.25 ~ 2000nmoL / L.

[0024] Further preferably, in the incubation system, the E6AP protein concentration is 3.9 ~ 50nmoL / L, and the E6 protein concentration is 31.25 ~ 50nmoL / L.

[0025] Most preferably, in the incubation system, the concentration of E6AP protein is 15.625 nmoL / L, and the concentration of E6 protein is 41.25 nmoL / L.

[0026] Compared with the prior art, the present invention has the following beneficial effects: Based on HTRF technology and the principle of protein-protein interaction, the present invention establishes a high-throughput screening method for PPI inhibitors, which is efficient, rapid, sample-saving, signal-stable, and easy to operate.

[0027] Furthermore, the screening method of the present invention directly reflects the ability of a compound to disrupt the in vitro interaction between the full-length E6AP protein and the full-length E6 protein. This method can eliminate false-positive hit compounds that only inhibit a portion of the interaction between the two regions but do not completely disrupt the interaction across all regions. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the principle of the HTRF-based high-throughput screening method for protein-protein interaction inhibitors; Figure 2 In vitro biomembrane interference (BLI) interaction analysis between wild-type E6AP, mutant E6AP C843A, and VE6_HPV16 (4C / 4S) protein; (a) wild-type E6AP, KD = 4.71 nM; (b) mutant E6AP C843A, KD = 4.79 nM; Figure 3 This is the HTRF reaction window result diagram corresponding to proteins with different label positions; Figure 4 This is the HTRF reaction window result diagram corresponding to using different reaction buffers; Figure 5The results of high-throughput screening for inhibitors targeting the protein-protein interaction between the human papillomavirus early protein E6 and the ubiquitin ligase E6AP are shown, where (a) is the preliminary screening result of the natural product library, and (b) is the structural formula of compound EP5-4; Figure 6 The results of compound EP5-4 inhibiting E6 hijacking E6AP to ubiquitinate and modify p53 in vitro, where (a) and (b) are the gel Coomassie brilliant blue staining and immunoblotting images of EP5-4 inhibiting p53 ubiquitination modification in vitro experiment, respectively; (c) is the quantitative analysis of unmodified p53 protein based on immunoblotting imaging to reflect the inhibitory effect of EP5-4. DETAILED DESCRIPTION

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and examples. It should be noted that the following examples are intended to facilitate understanding of the present invention and do not have any limiting effect on the present invention.

[0030] like Figure 1 As shown, the high-throughput screening method for PPI inhibitors based on HTRF technology and the principle of protein-protein interaction of the present invention includes: (1) incubating the compound to be screened with E6AP or E6 protein; (2) After the incubation is completed, E6 protein or E6AP protein is added to the mixed solution and mixed and incubated; (3) After the incubation is completed, the fluorescent donor and the fluorescent acceptor are added to the mixed solution and mixed and incubated; (4) After the incubation, the fluorescence signal of the solution is detected to determine whether the compound can inhibit the E6AP-E6 protein-protein interaction.

[0031] Example 1: Effects of E6AP and E6 proteins with different tag positions on the HTRF reaction window The HTRF signal is based on the proximity of the fluorescent donor and fluorescent acceptor; the closer the distance, the longer the reaction window. In the present invention, the fluorescent donor and fluorescent acceptor are attached to the N-terminus or C-terminus of E6AP and E6 protein via affinity tags. In this example, combinations of E6AP and E6 protein with different tag positions were tested according to the experimental method of the present invention. By comparing the HTRF signal windows of different combinations, the impact on the screening system was analyzed to confirm the feasibility of the experimental method of the present invention.

[0032] (1) Material preparation: E6AP C843A protein (N-terminal Strep tag II or C-terminal Strep tag II), VE6_HPV16 4C / 4S protein (N-terminal 6×His tag or C-terminal 6×His tag), HTRF Strep-Tactin-XL665 (Revvity, 610STXLA), HTRF mAb Anti-6His Tb-Conjugate (Revvity, 61HISTLB), 1× reaction buffer (20 mM Hepes pH=8.0, 150 mM NaCl, 0.1% BSA, 0.01% TritonX-100), 384-well plate (Revvity, 6007290), and microplate reader (Perkin Elmer).

[0033] (2) Operation steps: ① Dilute E6AP protein (N-terminal Strep tag II), E6AP protein (C-terminal Strep tag II), E6 protein (N-terminal 6×His tag), and E6 protein (C-terminal 6×His tag) to 62.5 nM, 62.5 nM, 165 nM, and 165 nM, respectively, using 1× reaction buffer. Set up four experimental groups: E6AP protein (N-terminal Strep tag II) + E6 protein (N-terminal 6×His tag), E6AP protein (N-terminal Strep tag II) + E6 protein (C-terminal 6×His tag), E6AP protein (C-terminal Strep tag II) + E6 protein (N-terminal 6×His tag), and E6AP protein (C-terminal Strep tag II) + E6 protein (C-terminal 6×His tag). Also set up a control group without protein addition. Set up three replicate wells for each group. In the experimental group, 5 μL E6AP protein and 5 μL E6 protein were mixed in each well, while in the control group, an equal volume of 1× reaction buffer was used instead and incubated at 25°C for 1 hour.

[0034] ② Dilute HTRF Strep-Tactin-XL665 and HTRF mAb Anti-6His Tb-Conjugate 100-fold in 1× reaction buffer. Mix 5 μL of HTRF Strep-Tactin-XL665 and 5 μL of HTRF mAb Anti-6His Tb-Conjugate in each well and incubate at 25°C for 0.5 hours. After incubation, read the signal using a microplate reader.

[0035] (3) Analysis of experimental results: To confirm that the E6AP C843A mutation does not affect the binding of E6AP to E6, the affinity of E6AP wild type and E6AP C843A mutant to E6 protein was identified by biomembrane interference experiment, as shown in Figure 2. Figure 2 As shown, the mutant and wild type have basically the same affinity for E6. Figure 3 As shown, the reaction windows of the E6AP protein (N-terminal Strep tag II) + E6 protein (C-terminal 6×His tag) and E6AP protein (C-terminal Strep tag II) + E6 protein (C-terminal 6×His tag) groups were both less than 1-fold, while the reaction window of the E6AP protein (N-terminal Strep tag II) + E6 protein (N-terminal 6×His tag) group reached 4-fold, and the reaction window of the E6AP protein (C-terminal Strep tag II) + E6 protein (N-terminal 6×His tag) group reached the highest of 5.3-fold. Therefore, in order to achieve high sensitivity and easy to distinguish results, it is recommended to use the E6AP protein (C-terminal Strep tag II) + E6 protein (N-terminal 6×His tag) combination for the remaining screening experiments. In addition, this example confirms the sensitivity and feasibility of this method.

[0036] Example 2: Effects of different reaction buffers on the HTRF reaction window The HTRF signal originates from the emission of a fluorescent donor, which further excites a fluorescent acceptor, causing the acceptor to emit light. The signal is calculated as the ratio of the acceptor's emission to the donor's emission. Different buffer components may affect the binding of the donor and acceptor to the protein, as well as the intensity of the fluorescent signal. In this example, reaction buffers with different compositions were used to conduct tests according to the experimental method of the present invention. By comparing the HTRF signal windows of different experimental groups, the impact on the screening system was analyzed to confirm the feasibility of the experimental method of the present invention.

[0037] (1) Material preparation: E6AP protein (C-terminal Strep tag II), VE6_HPV16 4C / 4S protein (N-terminal 6×His tag), HTRFStrep-Tactin-XL665 (Revvity, 610STXLA), HTRF mAb Anti-6His Tb-Conjugate (Revvity, 61HISTLB), 1× reaction buffer 1 (20 mM Hepes pH = 7.0, 150 mM NaCl, 0.1% BSA, 0.01% TritonX-100), 1× reaction buffer 2 (20 mM Hepes pH = 7.5, 150 mM NaCl, 0.1% BSA, 0.01% TritonX-100), 1× reaction buffer 3 (20 mM Hepes pH = 8.0, 150 mM NaCl, 0.1% BSA, 0.01% TritonX-100), 1× reaction buffer 2-1 (20 mM Hepes pH = 7.0, 150 mM NaCl, 0.1% BSA, 0.01% TritonX-100), : 1× reaction buffer 2-2 (20 mM Hepes pH = 7.5, 200 mM NaCl, 0.1% BSA, 0.01% TritonX-100), 1× reaction buffer 2-3 (20 mM Hepes pH = 7.5, 250 mM NaCl, 0.1% BSA, 0.01% TritonX-100), 1× reaction buffer 2-4 (20 mM Hepes pH = 7.5, 500 mM NaCl, 0.1% BSA, 0.01% TritonX-100), 1× reaction buffer 2-1-1 (20 mM Hepes pH = 7.5, 100 mM NaCl, 0.1% BSA, 0.01% NP-40), 1× reaction buffer 2-1-2 (20 mM Hepes pH = 7.5, 250 mM NaCl, 0.1% BSA, 0.01% TritonX-100), pH = 7.5, 100 mM NaCl, 0.1% BSA, 0.01% CHAPSO), 384-well plate (Revvity, 6007290), and microplate reader (PerkinElmer).

[0038] (2) Operation steps: ① Using different 1× reaction buffers in (1), dilute E6AP protein (C-terminal Strep tag Ⅱ) and E6 protein (N-terminal 6× His tag) to 62.5nM and 165nM respectively. Each 1× reaction buffer was used to set up an experimental group and a control group without protein, with three replicates in each group. In each well of the experimental group, 5μL of E6AP protein and 5μL of E6 protein were mixed, while the control group used an equal volume of 1× reaction buffer instead and incubated at 25℃ for 1 hour.

[0039] ② Using different 1× reaction buffers in (1), dilute HTRF Strep-Tactin-XL665 and HTRF mAb Anti-6His Tb-Conjugate 100-fold, respectively. Mix 5 μL of HTRF Strep-Tactin-XL665 and 5 μL of HTRF mAb Anti-6His Tb-Conjugate in each well and incubate at 25°C for 0.5 h. After incubation, read the signal using a microplate reader.

[0040] (3) Analysis of experimental results: like Figure 4 As shown. For 1× reaction buffers 1 to 3, the difference lies in the pH values ​​of 7.0, 7.5, and 8.0, respectively. 1× reaction buffer 2 achieved the highest window value. Therefore, based on 1× reaction buffer 2, 1× reaction buffers 2-1 to 2-4 were prepared, with the salt concentrations of the solutions controlled at 100mM, 200mM, 250mM, and 500mM, respectively. 1× reaction buffer 2-1 achieved the highest window value. Therefore, based on 1× reaction buffer 2-1, 1× reaction buffers 2-1-1 and 2-1-2 were prepared, using NP-40 and CHAPSO as detergents, respectively. The group with the highest window value still belonged to 1× reaction buffer 2-1. Therefore, to achieve high sensitivity and easy-to-interpret results, it is recommended to use a 1× reaction buffer composed of 20mM Hepes pH=7.5, 100mM NaCl, 0.1% BSA, and 0.01% Triton X-100 for the remaining screening experiments. Furthermore, this example demonstrates the sensitivity and feasibility of this method.

[0041] Example 3: High-throughput screening of inhibitors targeting the protein-protein interaction between human papillomavirus early protein E6 and ubiquitin ligase E6AP The method of the present invention and the laboratory natural product library were used to screen PPI inhibitors targeting E6AP-E6.

[0042] (1) Material preparation: E6AP protein (C-terminal Strep tag II), VE6_HPV16 4C / 4S protein (N-terminal 6×His tag), HTRFStrep-Tactin-XL665 (Revvity, 610STXLA), HTRF mAb Anti-6His Tb-Conjugate (Revvity, 61HISTLB), 1× reaction buffer (20mM Hepes pH=7.5, 100mM NaCl, 0.1%BSA, 0.01%TritonX-100), laboratory compound library (320, 10mM, 100% DMSO), DMSO (Sigma-Aldrich, D8418), 384-well plate (Revvity, 6007290), and microplate reader (Perkin Elmer).

[0043] (2) Operation steps: ① Dilute E6AP protein (C-terminal Strep tag II) and E6 protein (N-terminal 6× His tag) to 44.6 nM and 412.5 nM, respectively, in 1× reaction buffer. Dilute each compound to 142.9 μM in 1× reaction buffer, and dilute DMSO to 1.43%. In the experimental group, mix 7 μL of E6AP protein with 7 μL of compound. In the positive control group, mix 7 μL of E6AP protein with 7 μL of 1.43% DMSO. In the negative control group, mix 7 μL of 1× reaction buffer with 7 μL of 1.43% DMSO. Incubate at 25°C for 1 hour. After incubation, mix 2 μL of E6 protein in each well of the experimental and positive control groups. Replace the negative control well with 2 μL of 1× reaction buffer and incubate at 25°C for 1 hour.

[0044] ② Dilute HTRF Strep-Tactin-XL665 and HTRF mAb Anti-6His Tb-Conjugate 40-fold in 1× reaction buffer. Mix 2 μL of HTRF Strep-Tactin-XL665 and 2 μL of HTRF mAb Anti-6His Tb-Conjugate in each well and incubate at 25°C for 0.5 hours. After incubation, read the signal using a microplate reader.

[0045] (3) Analysis of experimental results: like Figure 5 As shown in Figure 2, at a final concentration of 50 μM, compound EP5-4 exhibited an inhibition rate of over 50%, indicating that compound EP5-4 may be a potential E6AP-E6 PPI inhibitor. In vitro substrate ubiquitination experiments demonstrated that compound EP5-4 can significantly inhibit E6 hijacking E6AP ubiquitination modification of p53 in vitro (e.g. Figure 6 ). Figure 6 (a) and Figure 6 Middle (b) shows the Coomassie brilliant blue staining and immunoblotting images of the protein gel in the in vitro ubiquitination experiment. It can be observed that compared with the control group, the amount of unmodified p53 in the experimental group with the addition of EP5-4 was significantly increased at each time point, and the ubiquitination band was significantly reduced. Figure 6 Middle (c) is a quantitative analysis of the amount of unmodified p53 protein based on immunoblotting results. The amount of unmodified p53 in the experimental group with the addition of EP5-4 was significantly increased at each time point.

[0046] The embodiments described above provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements and equivalent substitutions made within the scope of the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A high-throughput screening method for inhibitors targeting E6AP-E6 protein-protein interaction, characterized in that: The following steps are involved: (1) Incubating the test compound solution with E6AP protein or E6 protein in vitro; (2) After the incubation is completed, E6 protein or E6AP protein is added to the mixed solution, mixed evenly, and then incubated; (3) After the incubation is completed, add the fluorescent donor and fluorescent acceptor to the mixed solution, mix well and incubate; (4) After the incubation, the fluorescence signal of the solution is detected to determine whether the test compound can inhibit the E6AP-E6 protein-protein interaction.

2. The high-throughput screening method for inhibitors targeting E6AP-E6 protein-protein interaction according to claim 1, characterized in that: In steps (1)-(3), the test compound, E6AP protein, E6 protein, fluorescent donor, and fluorescent acceptor are diluted with 1× reaction buffer.

3. The high-throughput screening method for inhibitors targeting E6AP-E6 protein-protein interaction according to claim 2, characterized in that: The components of the 1× reaction buffer include at least one of 4-hydroxyethylazineethanesulfonic acid buffer, tris(hydroxymethylaminomethane) hydrochloric acid, citric acid / sodium citrate solution, 2-morpholineethanesulfonic acid buffer, and 3-morpholinepropanesulfonic acid buffer.

4. The high-throughput screening method for inhibitors targeting E6AP-E6 protein-protein interaction according to claim 3, characterized in that: The 1× reaction buffer further comprises sodium chloride, BSA, and a detergent; the detergent comprises at least one of Triton X-100, ethylphenyl polyethylene glycol, 3-[(3-cholaminylpropyl)dimethylamino]-2-hydroxy-1-propanesulfonic acid inner salt, Tween-20, and n-dodecyl β-D-maltoside.

5. The high-throughput screening method for inhibitors targeting E6AP-E6 protein-protein interaction according to claim 4, characterized in that: The components of the 1× reaction buffer include 4-hydroxyethylazineethanesulfonic acid, sodium chloride, BSA, and Triton X-100; in the 1× reaction buffer, the concentration of 4-hydroxyethylazineethanesulfonic acid is 10 to 50 mmol / L, the pH is 7.0 to 8.0, and the concentration of sodium chloride is 100 to 500 mmol / L.

6. The high-throughput screening method for inhibitors targeting E6AP-E6 protein-protein interaction according to claim 1, characterized in that: The fluorescent donor group includes at least one of europium, terbium and fluorescein; the fluorescent acceptor group includes at least one of XL665, d2 and fluorescein.

7. The high-throughput screening method for inhibitors targeting E6AP-E6 protein-protein interaction according to claim 1, characterized in that: The ubiquitin ligase E6AP includes at least one of the UBE3A wild type or a mutant in which cysteine ​​at position 843 is mutated to alanine; the human papillomavirus early protein E6 includes at least one of the VE6_HPV16 wild type or a mutant in which cysteine ​​at positions 87, 104, 118, and 147 are mutated to serine, and the VE6_HPV18 wild type.

8. The high-throughput screening method for inhibitors targeting E6AP-E6 protein-protein interaction according to claim 1, characterized in that: The E6AP protein was coupled to Strep tag II at the N-terminus or C-terminus, and the E6 protein was coupled to 6×His tag at the N-terminus or C-terminus.

9. The high-throughput screening method for inhibitors targeting E6AP-E6 protein-protein interaction according to claim 8, characterized in that: The E6AP protein was coupled to Strep tag II at the C-terminus, and the E6 protein was coupled to a 6×His tag at the N-terminus.

10. The high-throughput screening method for inhibitors targeting E6AP-E6 protein-protein interaction according to claim 1, characterized in that: In the incubation system, the E6AP protein concentration was 3.9 ~ 250nmoL / L, and the E6 protein concentration was 31.25 ~2000nmoL / L.

Images