Polypeptide compound and application thereof in preparation of product for inhibiting ATP enzyme hydrolysis activity

Abstract

The invention discloses a polypeptide compound and application thereof in preparation of a product for inhibiting ATP enzyme hydrolysis activity. The invention provides a polypeptide which is any one of the following: A1, the amino acid sequence of the polypeptide is sequence 1; a2, tag protein is added to the tail end of the polypeptide shown in A1, and the fused polypeptide is obtained. According to the method, multi-round progressive sequence optimization is performed by focusing a prototype peptide targeting 2C, simulating comprehensive utilization of a virtual neural network training model and actually-measured biochemical function evaluation in multiple aspects such as enzymatic inhibition and binding force evaluation, and a series of candidate peptides with relatively high comprehensive evaluation and shortened sequences are obtained; and a structural basis is provided for the design of a new round of antiviral peptidomimetic small molecules. Finally, candidate peptides are obtained through the research, and theoretical basis and technical support are provided for development of ATP enzyme inhibitors.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and relates to a polypeptide compound and application thereof in preparation of an inhibitor of ATPase hydrolysis activity. BACKGROUND

[0002] With the increasing threat of viruses worldwide, the research and development of antiviral drugs has become particularly important. Traditional drug development has a long cycle and high cost, while machine learning-based drug screening methods can greatly shorten this cycle. However, relying solely on machine learning prediction is not enough to ensure the actual effect of the drug, so combining biochemical verification has become a key step to ensure drug safety and effectiveness.

[0003] Foot and mouse disease (FMDV) belongs to the Aphthovirus of the Picornaviridae family, and is the earliest identified animal virus. Due to the high replication mutation rate, large population and short propagation cycle of the foot and mouse disease virus, the foot and mouse disease virus exists and evolves in the form of "quasispecies", and is considered to be one of the best models for studying species evolution. Through the study of the foot and mouse disease virus, it is helpful to study the evolution and variation of the picornavirus, and to crack the evolution mechanism and variation selection process of the picornavirus, which has important guiding significance for the development control theory of the picornavirus. Therefore, the foot and mouse disease virus is used as a model virus to study and explore the drug research and screening of the picornavirus.

[0004] FMDV 2C has ATPase hydrolysis activity, and forms a multimer through intermolecular interaction in cells to play a role in aggregating lipid droplets in host cells, and the hydrolysis activity and function-dependent multimer conformation of the target protein play an important regulatory role in viral transcription and replication, therefore, the design and development of allosteric drugs based on the target have become a research hotspot for targeting FMDV virus. SUMMARY

[0005] The technical problem solved by the application is to prepare a polypeptide required for the development of an FMDV virus inhibitor drug.

[0006] To solve the above technical problem, in a first aspect, the application provides a polypeptide, which is any one of the following:

[0007] A1) the amino acid sequence of the polypeptide is sequence 1;

[0008] A2) a tag protein is added to the end of the polypeptide in A1 to obtain a fusion polypeptide.

[0009] In a second aspect, the application provides the use of the polypeptide of the first aspect in the preparation of a product with any of the following functions:

[0010] B1 ) binds to FMDV 2C protein;

[0011] B2) reduces the thermal stability of FMDV 2C protein;

[0012] B3) inhibits ATPase activity.

[0013] In a third aspect, the present application provides a product comprising the polypeptide of the first aspect and a further pharmaceutically acceptable carrier.

[0014] The product described above has any one of the following functions:

[0015] B1 ) binds to FMDV 2C protein;

[0016] B2) reduces the thermal stability of FMDV 2C protein;

[0017] B3) inhibits ATPase activity.

[0018] The pharmaceutically acceptable carrier described above can be an excipient, a stabilizer, a suspending agent or a diluent, etc., which is well known to those skilled in the art.

[0019] Further, the carrier materials include, but are not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly water-soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate and carboxymethyl cellulose, etc.). Using these materials, various dosage forms can be prepared, including, but not limited to, tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, buccal tablets, suppositories, lyophilized powder injections, etc. They can be ordinary preparations, sustained-release preparations, controlled-release preparations, and various microparticle drug delivery systems. In order to prepare a unitary drug dosage form into a tablet, various carriers known in the art can be widely used. Examples of the carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, white clay, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders such as water, glycerol, polyethylene glycol, ethanol, propyl alcohol, starch paste, dextrin, sugar syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrants such as dry starch, alginate, agar powder, fucoidin, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glycerol tri-stearate, cocoa butter, hydrogenated oil, etc.; absorption accelerators such as quaternary ammonium salts, sodium dodecylsulfate, etc.; lubricants such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can be further prepared into coated tablets such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to prepare a unitary drug dosage form into a pill, various carriers known in the art can be widely used. Examples of the carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, talc, etc.; binders such as acacia, tragacanth gum, gelatin, ethanol, liquid sugar, rice paste or dough, etc.; disintegrants such as agar powder, dry starch, alginate, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc. In order to prepare a unitary drug dosage form into a suppository, various carriers known in the art can be widely used. Examples of the carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. In order to prepare a unitary drug dosage form into an injection preparation such as a solution, an emulsion, a lyophilized powder injection, and a suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid ester, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose, or glycerol can be added to the injection preparation, and in addition, a conventional co-solvent, a buffer, a pH adjustor, etc. can be added.In addition, coloring agents, preservatives, flavors, flavoring agents, sweeteners, or other materials can be added to the pharmaceutical preparation, if desired.

[0020] The product above can be a drug, a composition, a health product, a functional food, a food for special medical purposes, or other biological products.

[0021] Further, the product above can be an ATPase inhibitor.

[0022] The high-resolution FMDV 2C crystal structure was previously analyzed, and a target FMDV 2C protein antiviral prototype peptide was designed based on the structure, and the all-around intracellular antiviral activity verification was carried out. FMDV 2C has ATPase hydrolysis activity and the effect of mediating intracellular lipid droplet aggregation by forming intermolecular multimers, and the ATP binding site is adjacent to the multimerization site formed by the protein itself, so the allosteric polypeptide designed based on the multimerization site will break the multimer conformation and reduce its ATPase activity, and play a dual effect.

[0023] The present application focuses on the FMDV 2C targeted prototype peptide, and intends to comprehensively utilize the virtual neural network training model and the actual detection of enzymatic inhibition, combined with biochemical function evaluation such as force evaluation and thermal stability, to carry out multi-round progressive sequence optimization, obtain a series of candidate peptides with high comprehensive evaluation and shortened sequence, and provide a basis for the design of new antiviral peptidomimetic small molecules. Through the present research, the molecular inhibition mechanism of the candidate peptide is clarified, and theoretical basis and technical support are provided for the development of inhibitors targeting FMDV virus 2C protein. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The present application focuses on the FMDV 2C targeted prototype peptide, and intends to comprehensively utilize the virtual neural network training model and the actual detection of enzymatic inhibition, combined with biochemical function evaluation such as force evaluation and thermal stability, to carry out multi-round progressive sequence optimization, obtain a series of candidate peptides with high comprehensive evaluation and shortened sequence, and provide a basis for the design of new antiviral peptidomimetic small molecules. Through the present research, the molecular inhibition mechanism of the candidate peptide is clarified, and theoretical basis and technical support are provided for the development of inhibitors targeting FMDV virus 2C protein.

[0025] Figure 2 The present application focuses on the FMDV 2C targeted prototype peptide, and intends to comprehensively utilize the virtual neural network training model and the actual detection of enzymatic inhibition, combined with biochemical function evaluation such as force evaluation and thermal stability, to carry out multi-round progressive sequence optimization, obtain a series of candidate peptides with high comprehensive evaluation and shortened sequence, and provide a basis for the design of new antiviral peptidomimetic small molecules. Through the present research, the molecular inhibition mechanism of the candidate peptide is clarified, and theoretical basis and technical support are provided for the development of inhibitors targeting FMDV virus 2C protein.

[0026] Figure 3 The present application focuses on the FMDV 2C targeted prototype peptide, and intends to comprehensively utilize the virtual neural network training model and the actual detection of enzymatic inhibition, combined with biochemical function evaluation such as force evaluation and thermal stability, to carry out multi-round progressive sequence optimization, obtain a series of candidate peptides with high comprehensive evaluation and shortened sequence, and provide a basis for the design of new antiviral peptidomimetic small molecules. Through the present research, the molecular inhibition mechanism of the candidate peptide is clarified, and theoretical basis and technical support are provided for the development of inhibitors targeting FMDV virus 2C protein.

[0027] Figure 4 The present application focuses on the FMDV 2C targeted prototype peptide, and intends to comprehensively utilize the virtual neural network training model and the actual detection of enzymatic inhibition, combined with biochemical function evaluation such as force evaluation and thermal stability, to carry out multi-round progressive sequence optimization, obtain a series of candidate peptides with high comprehensive evaluation and shortened sequence, and provide a basis for the design of new antiviral peptidomimetic small molecules. Through the present research, the molecular inhibition mechanism of the candidate peptide is clarified, and theoretical basis and technical support are provided for the development of inhibitors targeting FMDV virus 2C protein. DETAILED DESCRIPTION

[0028] The application will be described in further detail below with specific reference being made to the detailed embodiments. The examples given are merely for the purpose of illustrating the application and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0029] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0030] Unless otherwise specified, the quantitative tests in the following examples are all set up with three repeated experiments, and the results are averaged.

[0031] Example 1, synthesis of polypeptide compounds

[0032] Figure 1 Based on the high-resolution structure of FMDV 2C protein, a polypeptide sequence design process is developed through multiple rounds of iterative optimization of machine learning and biochemical verification.

[0033] The process is as follows: first, a dataset of polypeptide and protein complex structure is collected, and a model capable of judging the binding ability of polypeptide and protein is obtained through model training by deep learning method. A large number of polypeptide sequences are randomly generated according to the amino acid composition, variable amino acid position and type of the original peptide. The randomly generated sequences and FMDV 2C protein are input into the model to obtain the prediction results of the binding strength of different sequences and are sorted. The top 200 polypeptide sequences are selected, and the complex structure is predicted by AlphaFold2, and the predicted complex structure is subjected to molecular dynamics simulation. According to the binding free energy parameters calculated by MMPBSA method, the complex structure is scored and evaluated, and the top 20 polypeptide sequences are obtained.

[0034] Twenty biotin-labeled polypeptide sequences are obtained by biosynthesis, and the binding activity of the 20 polypeptides and FMDV 2C protein is verified by using a biological membrane interference technology system. The results of each batch of wet experiment are input into the software cluster trained by the neural network in the form of parameters, and then the second round of optimized polypeptide sequences are obtained. Through four batches of iterative optimization, a series of optimized polypeptide sequences are finally obtained.

[0035] The high-score polypeptide sequence information of the four batches is shown in Tables 1-4 as follows:

[0036] Table 1 is the high-score polypeptide sequence information of the first batch

[0037]

[0038]

[0039] Table 2: High-scored polypeptide sequence information of batch 2

[0040] Batch Sequence Number of Amino Acids 2-1 EWLCVLDNQEYWIL 14 2-2 YFMPLGALYKF 11 2-3 IIDNMCPKFI 10 2-4 VDCVGVMGW 9 2-5 GSLDNMEVNFW 11 2-6 HVAEMMDVW 9 2-7 HMMGWGEVISMNLA 14 2-8 LGDVKVFIKM 10 2-9 MDIWALKRL 9 2-10 LWFFDISKLQLK 12 2-11 ITHLHVLWP 9 2-12 NQMISMCLIWF 11 2-13 WPVPLVLME 9 2-14 CNIMKPVQC 9 2-15 MCFVVLVKQF 10 2-16 VQTAIMMFI 9 2-17 VFFLVQPALVP 11 2-18 MMIIRLFHG 9 2-19 MVMFALVVQ 9 2-20 LRPGFADVCLIQ 12

[0041] Table 3: High-scored polypeptide sequence information of batch 3

[0042] Batch Sequence Number of Amino Acids 3-1 WLVIFIKRQDIKNM 14 3-2 MRFILWIFN 9 3-3 RVEWYICYLM 10 3-4 RLFHQKIIIIDVV 13 3-5 APMWFQRHCGVPA 13 3-6 MHYHCVQGYYLRFA 14 3-7 MMWMPNRCMPMVIW 14 3-8 MILIFMALESRHAK 14 3-9 IMYVYCAMKSA 11 3-10 WANKPYCAM 9 3-11 KWCGFVHELKSQ 12 3-12 RTSTIWHAVHMD 12 3-13 NMILYGIIRADCVH 14 3-14 WPCWEASMDHSHDP 14 3-15 QRYNPYDNFFLWL 13 3-16 FIFQLWPAPS 10 3-17 GTYFLMPAQGIWVH 14 3-18 LDSMSWARQFVGH 13 3-19 YMDMFENQECINIHV 15 3-20 RSVIELMPF 9

[0043] Table 4: High-scored polypeptide sequence information of batch 4

[0044]

[0045]

[0046] Example 2, biochemical verification of polypeptides

[0047] I. Measurement of the binding activity of polypeptides to FMDV 2C by biological membrane interference technology

[0048] The top 20 derivative polypeptides with high comprehensive evaluation scores were selected for chemical synthesis with reference to the titer of the FMDV 2C prototype peptide. The biological membrane interference (BLI) technology was used to comprehensively evaluate the binding dynamic characteristics of the derivative polypeptides to the target 2C protein. The kinetic parameters of the binding of the derivative polypeptides to the target were compared with reference to the prototype peptide, and the derivative polypeptide with high titer was found.

[0049] Four batches of high-scored polypeptides with N-terminal biotinylation (Tables 1-4) were synthesized, which were respectively fixed on the streptavidin biosensor (SA sensor), and the binding of the polypeptides in solution to FMDV 2C protein (sequence 2) was measured.

[0050] The Octet platform can be used for qualitative and quantitative analysis of intermolecular interactions. The probe biosensor is used for direct detection of samples, and the samples do not need to be labeled with any fluorescence or isotope. The instrument emits white light to the surface of the biosensor and collects the reflected light. Different frequencies of reflected light spectrum are affected by the thickness of the light film layer of the biosensor and form interference, which accurately quantitatively determines the intermolecular interaction process of the measured molecules. Through real-time monitoring, the system determines the association constant and dissociation constant, as well as the initial binding rate, and calculates the affinity information (K D ) by fitting.

[0051] The specific process is as follows:

[0052] 1. First, each polypeptide was configured into a 100 μM solution (solvent: 1 x PBS) as a detection stationary phase of biotin; FMDV 2C protein was configured into a 10 μM solution (solvent: 20 mM HEPES, 100 mM NaCl) as a detection mobile phase.

[0053] 2. Material preparation: Octet RED96 (Fortebio), SA sensor, 100 μM biotin-polypeptide, 10 μM FMDV 2C protein, buffer: 25 mM MES (PH = 5.5), 100 mM NaCl, 0.02% surfactant P20.

[0054] 3. Program setting: first, pre-wet the SA sensor with 200 μl water. Then baseline run for 60 seconds, immobilization for 200 seconds, binding for 50 seconds, dissociation for 150 seconds.

[0055] The affinity information (K D ) of each polypeptide to FMDV 2C was in the nanomolar range, and the K D value represents the binding force, the lower the value, the more stable the binding, (M) is molar, representing the unit.

[0056] The K D results of four batches of polypeptides with high binding activity are shown in Table X, wherein X-pep represents X-1 to X-20 polypeptides shown in Table X, and X value is 1-4. Figure 2

[0057] The prototype peptide K D data was used as a positive control, and was detected together with four batches of polypeptides, and the binding activity value A for comparison between batches was obtained by the formula K D (derived peptide)-K D (prototype peptide) as shown in Table 5. (A (M) = K D (derived peptide)-K D (prototype peptide)). Some polypeptide results are shown in Tables 5-8 as follows:

[0058] Table 5 is the polypeptide result shown in Table 1

[0059]

[0060]

[0061] Table 6 is the result of some polypeptides shown in Table 2

[0062] Polypeptide Name K D (M)]]> A(M) 2-17 3.09E-07 3.16E-08 Prototype Peptide 2.78E-07 0 2-2 2.06E-07 -7.20E-08 2-3 1.40E-07 -1.38E-07 2-4 5.53E-07 2.75E-07 2-5 2.31E-07 -4.66E-08 2-13 3.66E-07 8.86E-08 2-15 1.77E-07 -1.01E-07 2-7 3.08E-07 3.04E-08 2-9 2.85E-07 7.90E-09 2-6 4.69E-07 1.92E-07 2-8 6.67E-08 -2.11E-07 2-10 2.12E-07 -6.60E-08 2-11 1.32E-07 -1.46E-07 2-14 4.07E-07 1.29E-07 2-18 1.56E-07 -1.21E-07

[0063] Table 7 is the result of some polypeptides shown in Table 3 ​

[0064]

[0065]

[0066] Table 8 is part of the polypeptide results shown in Table 4

[0067]

[0068]

[0069] The above results can be seen that, compared with the prototype peptide, the high score polypeptide of four batches, K D value gradually decreased, and the binding force gradually increased. The K D value of the optimal 15 polypeptides is shown in Table 9: deducting based on the prototype peptide, the smaller the A value, the better. The top 15 with the best binding force are 4-2, 4-4, 4-5, 2-15, 2-18, 2-3, 3-5, 2-11, 3-18, 4-11, 2-8, 3-10, 3-8, 3-9, 3-11, respectively.

[0070] Table 9 is the top 15 polypeptides with the best binding force and their results

[0071] sample A(M) Prototype Peptide 0 4-2 -7.96E-08 4-4 -8.43E-08 4-5 -9.24E-08 2-15 -1.01E-07 2-18 -1.21E-07 2-3 -1.38E-07 3-5 -1.38E-07 2-11 -1.46E-07 3-18 -1.59E-07 4-11 -1.62E-07 2-8 -2.11E-07 3-10 -2.21E-07 3-8 -2.41E-07 3-9 -2.58E-07 3-11 -2.85E-07

[0072] II. Differential scanning fluorimetry to detect the thermal stability of each batch of polypeptide and FMDV 2C

[0073] Differential scanning fluorimetry (DSF) is a method of slowly heating samples on a fluorescence quantitative PCR instrument, detecting the amount of fluorescent dye combined with the structure changed protein during heating, to evaluate the thermal stability of the protein. DSF can monitor the change of protein conformation during heating by fluorescence dye or endogenous fluorescence signal to calculate the melting temperature Tm (the temperature when the folded protein is equal to the unfolded protein). The most commonly used dye method DSF is SYPRO Orange dye, which is an environmentally sensitive hydrophobic dye. When the temperature rises, the protein unfolds and the hydrophobic part is exposed. The dye specifically binds to the hydrophobic part of the protein, and the fluorescence is enhanced. In the presence of specific compounds or ligands, the stability of the protein will increase, which is manifested as an increase in melting temperature.

[0074] FMDV 2C protein was incubated with four batches of derivative polypeptides shown in Table 1-4 respectively at a molar ratio of 1:10 in buffer (20 mM HEPES, 100 mM NaCl) at low temperature (4°C) overnight, and the next day, SYPRO Orange fluorescent dye was added at a ratio of 1:100 for differential scanning fluorimetry detection. FMDV 2C protein without polypeptide was used as a control.

[0075] The details are as follows:

[0076] 1. Take 96-well sharp white plates, 30 μl of reaction system per well, at least 3 replicate wells per well.

[0077] 2. 30 μl of reaction system for each condition: final concentration of 8 μM FMDV 2C protein, final concentration of 80 μM of each polypeptide, 1:100 of fluorescent dye SYPRO Orange, and the rest is buffer (20 mM HEPES, 100 mM NaCl).

[0078] 3. Use 8-well row gun for sample addition, be careful not to have bubbles, and pay attention to light protection operation.

[0079] The results are shown in Figure 3 , Tables 10-13.

[0080] Table 10 is the Tm value of the polypeptide shown in Table 1

[0081]

[0082]

[0083] Table 11 is the Tm value of the polypeptide shown in Table 2

[0084] sample Melting Temperature (°C) 2-1 43 2-17 44 2-2 45 2-3 45 2-4 45 2-5 45 2-13 45 2-15 45 2-7 45 2-9 45 2-20 46 2-6 46 2-8 46 2-10 46 2-11 46 2-14 46 2-18 46

[0085] Table 12 is the Tm value of the polypeptide shown in Table 3

[0086]

[0087]

[0088] Table 13 is the Tm value of the polypeptide shown in Table 4

[0089]

[0090]

[0091] The above results show that the Tm value of the prototype peptide and FMDV 2C protein binding decreases compared with the FMDV 2C protein control; the Tm value of the high-scored polypeptides of the four batches shown in Tables 1-4 and FMDV 2C protein binding generally shows a decreasing level, indicating that the binding of the polypeptide to the protein will affect the thermal stability of the FMDV 2C protein, and thus can affect the FMDV virus.

[0092] III. Enzymatic evaluation of ATPase activity of protein and polypeptide binding

[0093] The potency of the derived polypeptide in inhibiting the hydrolysis of ATP by FMDV 2C was evaluated using ATPase assay. FMDV 2C protein has an ATPase activity center, and the prototype peptide can inhibit ATPase activity, and the hydrolysis activity plays an important role in the regulation of viral transcription replication, so further screening can be carried out to obtain the best derived peptide sequence by analyzing the effect of the derived peptide on the hydrolysis activity of FMDV 2C protein.

[0094] ATPase activity was detected using the quantitative ATPase / GTPase assay kit of the biological detection system QuantiChromTM ATPase / GTPase Assay Kit (DATG-200). ATPases and GTPases catalyze the decomposition of ATP or GTP into ADP or GDP and free phosphate ions. These enzymes play a key role in transport, signal transduction, protein biosynthesis and cell differentiation.

[0095] The quantitative ATPase / GTPase assay kit of the biological detection system provides a highly sensitive microplate format for determining ATPase / GTPase activity. The unique formula is characterized by a single reagent that accurately measures enzyme activity at room temperature in 30 min. The improved malachite green reagent forms a stable deep green color with the released phosphate, which is measured on a Greiner 96-well transparent bottom enzyme plate (620 nm). The enzyme activity value is calculated according to the formula: Enzyme Activity (U / L) = [phosphate concentration (μM) * reaction volume (μL)] ÷ [protein volume μL) * time (min)].

[0096] Enzyme Activity represents ATPase activity, and the lower the value, the more inhibited the ATPase activity.

[0097] The negative control group is only different from the detection group in that no polypeptide is added.

[0098] Detection group: The above 15 top-ranked derived polypeptides of the four batches of screening binding activity (shown in Table 14) were used as the detection group, and the specific steps were as follows.

[0099] 1. Experimental equipment and consumables: QuantiChrom™ ATPase / GTPase Assay Kit (DATG-200), Greiner 96-well clear-bottom microplate, SpectraMax iD5 multi-functional microplate reader.

[0100] 2. The reaction volume for each well is 120 μl. The reaction system consists of: 10 μl Assay buffer, 5 μl 16 mM ATP, 2.7 μl FMDV 2C protein, 1.2 μl peptide, 1.1 μl buffer (20 mM HEPES, 100 mM NaCl), and 100 μl Reagent.

[0101] 3. First, add the Assay buffer and ATP from the kit to the wells in sequence. Then, using an 8-well pipette, add FMDV 2C protein, peptide, and buffer respectively. After reacting at room temperature for 5 minutes, add the Reagent solution from the kit and allow it to stand at room temperature for 30 minutes. Finally, place the 96-well plate in a multi-plate reader for detection and calculate the Enzyme Activity value.

[0102] The control group consisted of the reaction between the original peptide and FMDV 2C protein, with the sequence NLHEKVASQPIFKQ, and the procedure was the same as that for the test group. The original peptide is abbreviated as WT below. A lower Enzyme Activity value indicates a better inhibition of enzyme activity.

[0103] The results are as follows Figure 4 As shown in Table 14, WT serves as the control group. It can be seen that among the top 15 peptides with the best binding affinity in the four batches, 4-11 has the best inhibition of ATPase activity and its sequence is the shortest, consisting of 9 amino acids, with the sequence: KTHTGLHIC. Compared with the original peptide WT, it has 5 fewer amino acids. Therefore, it was selected as the optimal candidate peptide sequence for the design and optimization of peptide-like small molecule compound skeletons targeting FMDV 2C protein based on this sequence.

[0104] Table 14 shows the results of ATPase activity.

[0105]

[0106]

[0107] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a specific example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. A polypeptide, which is any one of the following: The amino acid sequence of the polypeptide shown in A1) is sequence 1; A2) Add a tag protein to the end of the polypeptide shown in A1 to obtain a fusion polypeptide.

2. The use of the polypeptide of claim 1 in the preparation of products having any of the following functions: B1) binds to FMDV 2C protein; B2) Reduces the thermal stability of FMDV 2C protein; B3) Inhibits ATPase activity.

3. A product comprising the polypeptide of claim 1 and other pharmaceutically acceptable carriers.

4. The product according to claim 3, characterized in that: The product has any of the following functions: B1) binds to FMDV 2C protein; B2) Reduces the thermal stability of FMDV 2C protein; B3) Inhibits ATPase activity.

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