Hsp70 protein-binding peptides, their screening methods and applications

Abstract

This invention discloses an Hsp70 protein-binding peptide, its screening method, and its uses. The Hsp70 protein-binding peptide has an amino acid sequence as shown in any one of SEQ ID No. 1 to SEQ ID No. 5. This invention also discloses a screening method for the Hsp70 protein-binding peptide. The screening method provided by this invention can improve the efficiency and success rate of obtaining Hsp70 protein-binding peptides in a single run. It is simple to operate, has a short experimental cycle, and is fast and efficient, increasing the possibility of obtaining peptide lead drugs. Simultaneously, the screened Hsp70 protein-binding peptide can regulate the ATPase activity of Hsp70 protein, and can be further applied as a peptide lead drug in the treatment of major diseases (pathogen prevention and control, cancer, Alzheimer's disease).

Description

Technical Field

[0001] This invention relates to an Hsp70 protein-binding polypeptide, and more particularly to an Hsp70 protein-binding polypeptide with regulated ATPase activity and its screening method, which is applied to polypeptide lead drugs and belongs to the field of biomedical technology. Background Technology

[0002] 70-kD heat shock proteins (Hsp70s) are a class of conserved stress-response molecular chaperones. They maintain cellular homeostasis by regulating protein folding, transport, assembly, and degradation. In lower organisms such as pathogenic microorganisms, abnormal function of Hsp70s can severely affect growth and even lead to death. In humans, Hsp70s are closely associated with neurodegenerative diseases (such as Alzheimer's disease) and cancer, and have become a promising new target protein for the treatment of these diseases.

[0003] Hsp70s mainly contains the following domains: a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD), the latter consisting of SBDα and SBDβ. It is generally believed that the substrate-binding pocket is located at the L of SBDβ. 1，2 and L 3，4 In the process of NBD binding ADP, the substrate-binding pocket closes, and the substrate peptide is trapped inside the pocket by binding to specific amino acid residues through hydrophobic interactions. When NBD binds ATP, it instantaneously triggers changes in the NBD, SBD / NBD interface, and SBDα structure, thereby completely altering the conformation of the SBDβ substrate-binding pocket, making it completely closed and releasing the previously bound substrate peptide. Then, with the help of Hsp40 (DnaJ), the substrate-binding pocket is reopened (to a greater extent than in the ADP state), allowing for extensive contact and capture of new substrate peptides requiring assistance (folding, assembly, transport, degradation, etc.). After the new substrate peptide is captured, ATP hydrolysis (ATPase activity) is accelerated, ultimately restoring the NBD, SBD / NBD interface, and SBD to their ADP-binding conformation, thus completing one working cycle of Hsp70. In summary, the essence of the efficient operation of Hsp70s is ATP-induced and highly efficient protein conformational coupling in cooperation with other factors (such as substrate peptides).

[0004] The emergence of the novel coronavirus has raised the bar for pathogen control. DnaK (Escherichia coli Hsp70 protein), due to its functional importance and relatively mature basic research, can serve as a key drug development target for such diseases. Currently, the screening of such drug molecules mainly focuses on discovering functional peptides that can competitively bind to the SBD substrate-binding pocket of DnaK. The screening principle is that the peptide must specifically bind to the SBD substrate-binding pocket of DnaK, with an affinity at least on the submicromolar level. This is necessary to effectively inhibit DnaK from further binding to intracellular peptides, disrupting the intracellular protein homeostasis of pathogenic microorganisms, and thus achieving an antibacterial effect. Currently, only one peptide drug has been proven to effectively inhibit the growth of pathogenic bacteria without attacking Hsp70s in human cells (Bikker et al., Chem Biol Drug Des, 2006, 68: 148-53). The clinical development progress of other potential peptides or small molecule drugs has been relatively slow.

[0005] Using human Hsp70 as a target protein, drug screening for intervention in diseases such as Alzheimer's and cancer mainly includes two categories: Hsp70 inducers and allosteric inhibitors. Peptides participate in the regulation of protein activity and functional cycles by directly binding to Hsp70, acting as both Hsp70 inducers and allosteric regulators. CHEC-7 is an active small peptide identified from the N-terminus of human Dermcidin protein (Stocki et al., J Biol Chem, 2011, 286: 12803-11). It mainly increases the survival rate of nerve cells by activating Hsp70 disaggregase activity to inhibit Aβ aggregation. It has been widely used in disease treatment experiments in animal models of neurodegeneration and has good clinical application prospects.

[0006] However, research on screening protein-binding peptides targeting Hsp70 to develop lead drugs for treating major diseases (pathogenic microbial diseases, cancer, Alzheimer's disease) is still in its early stages. Therefore, the techniques are relatively limited and the screening efficiency is low. Based on previous literature, those skilled in the art often choose random peptide libraries to screen for short peptides binding to the Hsp70 protein, such as phage display libraries. Admittedly, phage display libraries are constructed using molecular biology and other techniques, and their library size (10...) 9 It has certain advantages in terms of quantity (order of magnitude). However, the screening results show that, under the premise of large library capacity, the positive rate is not ideal (≤0.3%). (Gragerov et al., J Mol Biol, 1994, 235: 848-854; Ru...) ..(Digger et al., EMBO J, 1997, 16: 1501-1507). Furthermore, not all sequences can be ideally expressed and displayed in phages, therefore the screening method is relatively passive and lacks target selection prioritization and bias.

[0007] In summary, efficient screening of antimicrobial peptides or activity-regulating peptides (inducers and allosteric agents) targeting Hsp70 through other approaches is of great practical significance for accelerating the discovery of peptide lead drugs with potential clinical applications that can treat the aforementioned major diseases, thereby contributing to human health. Summary of the Invention

[0008] The main objective of this invention is to provide an Hsp70 protein-binding polypeptide and a screening method thereof, so as to overcome the shortcomings of the prior art.

[0009] Another object of the present invention is to provide the use of the Hsp70 protein-binding polypeptide.

[0010] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0011] This invention provides an Hsp70 protein-binding polypeptide having an amino acid sequence as shown in any of SEQ ID No. 1 to SEQ ID No. 5, or a sequence that is more than 70% identical to the full-length sequence of any of SEQ ID No. 1 to SEQ ID No. 5 and is capable of binding to the Hsp70 protein, or a sequence derived from any of SEQ ID No. 1 to SEQ ID No. 5 that is capable of binding to the Hsp70 protein.

[0012] Furthermore, the Hsp70 protein-binding polypeptide can inhibit luciferase renaturation.

[0013] Furthermore, the Hsp70 protein-binding polypeptide can inhibit the growth of Escherichia coli.

[0014] Furthermore, the Hsp70 protein-binding polypeptide can regulate the ATPase activity of the Hsp70 protein.

[0015] This invention also provides a method for screening Hsp70 protein-binding peptides, comprising:

[0016] Select polypeptide sequences that meet the chosen criteria and fabricate polypeptide chips;

[0017] The Hsp70 protein with a histidine (His) fusion tag is incubated with a peptide chip to capture the Hsp70 protein-bound peptides conjugated to the peptide chip; and,

[0018] Repeat the above screening steps at least three times to obtain the aforementioned Hsp70 protein-binding peptide.

[0019] This invention also provides the use of the aforementioned functional material Hsp70 protein-binding peptide in the preparation of peptide lead drugs.

[0020] Furthermore, the polypeptide lead drug includes any one of the following: drugs for the prevention and treatment of pathogenic microorganisms, drugs for the treatment of cancer, and drugs for the treatment of Alzheimer's disease.

[0021] Accordingly, embodiments of the present invention also provide a polypeptide lead drug, which includes the Hsp70 protein-binding polypeptide.

[0022] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0023] The screening method for Hsp70 protein-binding peptides provided by this invention can improve the screening efficiency and success rate of obtaining Hsp70 protein-binding peptides in a single operation. It is simple to operate, has a short experimental cycle, and is fast and efficient, which can increase the possibility of obtaining peptide lead drugs. At the same time, the screened Hsp70 protein-binding peptides can regulate the ATPase activity of Hsp70 protein, and can be further applied as peptide lead drugs in the treatment of major diseases (pathogen prevention and control, cancer, Alzheimer's disease). Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a schematic diagram of the screening process for Hsp70 protein-binding peptides in a typical embodiment of the present invention;

[0026] Figure 2 This is a schematic diagram of the 6x6 polypeptide matrix used to detect the concentration of the HRP-labeled Anti-His tag in Embodiment 1 of the present invention.

[0027] Figures 3A-3C This is a graph showing the peptide chip screening results in Example 1 of the present invention;

[0028] Figures 4A-4E This is a diagram showing the regulatory effect of the peptides screened in Example 1 of this invention on the activity of DnaK protein;

[0029] Figure 5This is a graph showing the test results of the effect of the peptide (VP-3) screened in Example 1 of this invention on the growth of Escherichia coli;

[0030] Figures 6A-6B This is a graph showing the results of the peptides screened in Example 1 of this invention regulating the ATPase activity of human Hsp70 protein. Detailed Implementation

[0031] In view of the shortcomings of the existing technology, the inventors of this invention, through long-term research and extensive practice, have proposed the technical solution of this invention. Its main function is to provide a new screening platform that improves the screening efficiency and success rate of obtaining Hsp70 protein-binding peptides in a single run, thereby increasing the possibility of obtaining peptide lead drugs. The following will further explain and illustrate this technical solution, its implementation process, and its principles.

[0032] One aspect of this invention provides an Hsp70 protein-binding polypeptide having an amino acid sequence as shown in any one of SEQ ID No. 1 to SEQ ID No. 5.

[0033] Furthermore, the sequence of the Hsp70 protein-binding polypeptide can be IIVKLLPNLPKD (SEQ ID No. 1).

[0034] Furthermore, the sequence of the Hsp70 protein-binding polypeptide can be TEKQAKLALLINQVG (SEQ ID No. 2).

[0035] Furthermore, the sequence of the Hsp70 protein-binding polypeptide can be RLRSLAPRKLVIISK (SEQ ID No. 3).

[0036] Furthermore, the sequence of the Hsp70 protein-binding polypeptide can be KRGILSPAQLLSFSK (SEQ ID No. 4).

[0037] Furthermore, the sequence of the Hsp70 protein-binding polypeptide can be VSYQTKVNLLSAIKSPCQRE (SEQ ID No. 5).

[0038] Another aspect of the present invention provides an Hsp70 protein-binding polypeptide having a sequence that is more than 70% identical to the full-length sequence of any one of SEQ ID No. 1 to SEQ ID No. 5 and is capable of binding to the Hsp70 protein.

[0039] Furthermore, the Hsp70 protein-binding polypeptide has a sequence that is more than 80% identical to the full-length sequence of any one of SEQ ID No. 1 to SEQ ID No. 5 and is capable of binding to the Hsp70 protein.

[0040] Another aspect of the present invention provides an Hsp70 protein-binding polypeptide having a sequence derived from any one of SEQ ID No. 1 to SEQ ID No. 5 that is capable of binding to the Hsp70 protein.

[0041] The five short peptide sequences obtained in this invention, or short peptide sequences with similar amino acid sequences (homology of 80% or more, similarity of 70% or more), can all bind to DnaK protein with high affinity. Moreover, their sequences are specific, and the amino acid sequence lengths range from 12 to 20 AA.

[0042] Furthermore, the Hsp70 protein-binding polypeptide can inhibit luciferase renaturation.

[0043] Furthermore, the Hsp70 protein-binding polypeptide can inhibit the growth of Escherichia coli.

[0044] Furthermore, the Hsp70 protein-binding polypeptide can regulate the ATPase activity of the Hsp70 protein.

[0045] The above five short peptides are derived from viral structural proteins (VP-1), important human autoimmune disease-associated antigen proteins (VP-2, VP-3, VP-4), and tumor necrosis factor (VP-5), respectively.

[0046] Another aspect of this invention provides a method for screening Hsp70 protein-binding peptides, comprising:

[0047] Select polypeptide sequences that meet the chosen criteria and fabricate polypeptide chips;

[0048] The Hsp70 protein with a histidine (His) fusion tag is incubated with a peptide chip to capture the Hsp70 protein-bound peptides conjugated to the peptide chip; and,

[0049] Repeat the above screening steps at least three times to obtain the aforementioned Hsp70 protein-binding peptide.

[0050] In some embodiments, the screening method further includes: detecting Hsp70 protein captured by chip-positive peptides using a commercially available antibody that specifically binds to histidine tags (Anti-His) and is coupled with horseradish peroxidase (HRP), and imaging it with chemiluminescence.

[0051] In some embodiments, the peptide sequences meeting the selected criteria include at least one of the following: peptides with a high proportion of hydrophobic amino acids, peptides derived from proteins that interact with Hsp70, peptides derived from proteins associated with Hsp70, and peptides with a length of 7–20 amino acids. This invention utilizes the iPDMS nanofilm peptide chip technology, an in-house laboratory platform, to perform a pre-selective selection based on an existing laboratory peptide library: ① peptides with a high proportion of hydrophobic amino acids; ② short peptides derived from proteins that theoretically interact with Hsp70; ③ short peptides derived from other important proteins related to major diseases. This pre-selection greatly increases the likelihood of obtaining Hsp70 protein-binding peptides.

[0052] Please see Figure 1 As shown, in some more preferred embodiments, the present invention uses an indirect ELISA method to screen Hsp70 protein-binding peptides, which mainly includes three steps: ① Assigning a fusion tag to the Hsp70 protein—a histidine (His) tag: The nucleotide sequence encoding histidine is synthesized together with the target protein nucleotide sequence through gene synthesis (completed by Suzhou Genewise Biotechnology Co., Ltd.), expressed in BL21(DE3) and purified by affinity chromatography; ② Incubating the Hsp70 protein with the His fusion tag with a peptide chip to capture the protein-binding short peptides coupled to the chip; ③ Detecting and imaging positive peptide signal points using His tag-specific binding monoclonal antibody (HPR label): Chemiluminescence imaging is performed under the action of HRP substrate.

[0053] Another aspect of the present invention provides the use of the aforementioned Hsp70 protein-binding peptide in the preparation of peptide lead drugs.

[0054] Furthermore, the polypeptide lead drug has antibacterial properties.

[0055] Furthermore, the peptide lead drug can regulate the ATPase activity of the Hsp70 protein.

[0056] Furthermore, the peptide lead drug includes, but is not limited to, any of the following: pathogen control drugs, cancer treatment drugs, and Alzheimer's disease treatment drugs. The short peptides or sequence-similar short peptides obtained by this invention can be further applied as peptide lead drugs in the treatment of major diseases (pathogen control, cancer, Alzheimer's disease).

[0057] Another aspect of the present invention provides a polypeptide lead drug comprising the Hsp70 protein-binding polypeptide.

[0058] By means of the above technical solution, the screening method for Hsp70 protein-binding peptides provided by the present invention can improve the screening efficiency and success rate of obtaining Hsp70 protein-binding peptides in a single operation. It is simple to operate, has a short experimental cycle, and is fast and efficient, which can increase the possibility of obtaining peptide lead drugs. At the same time, the Hsp70 protein-binding peptides obtained by screening can regulate the ATPase activity of Hsp70 protein.

[0059] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be further described in detail below with reference to the accompanying drawings and several preferred embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Test methods in the following embodiments that do not specify specific conditions are all performed under conventional conditions. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0060] The reagents and raw materials used in the following examples are all commercially available, and the test methods that do not specify specific conditions are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers.

[0061] Example 1

[0062] Please see Figure 1 As shown, the peptide library constructed using chemical synthesis in this embodiment has a capacity of approximately 15,000 entries (>100 protein sequences). To improve the efficiency of screening for Hsp70-binding peptides, before customizing the Hsp70 screening chip, the inventors first classified the peptides in the peptide library and selected peptides containing the following characteristics ( Figure 1 Part A): ① Peptides with a high proportion of hydrophobic amino acids; or ② Short peptides from proteins that theoretically interact with Hsp70; or ③ Short peptides from other important proteins related to major diseases (associated with Hsp70); ④ Other short peptides of varying lengths (7-20 AA) selected randomly. Ultimately, the inventors selected 2645 short peptides (synthesized at Nanjing Genscript Biotech Co., Ltd.) containing the above characteristics and customized them into the peptide chip of this invention. Figure 1 Part B). In this embodiment, the peptide chip base film is made of iPDMS nanomaterials, which has near-zero background and provides a guarantee for further improving screening efficiency. Figure 1 Part C).

[0063] In this embodiment, Hsp70 protein-binding peptides were screened using an indirect ELISA method. Figure 1 Part D of the process mainly includes three steps: ① Assigning a fusion tag to the Hsp70 protein—a histidine (His) tag: The nucleotide sequence encoding histidine is synthesized together with the target protein nucleotide sequence through gene synthesis (completed by Suzhou Genewise Biotechnology Co., Ltd.), expressed in BL21(DE3) and purified by affinity chromatography; ② Incubating the Hsp70 protein with the His fusion tag with a polypeptide chip to capture protein-binding short peptides coupled to the chip; ③ Detecting and imaging positive polypeptide signal points using His tag-specific binding monoclonal antibody (HPR label): Chemiluminescence imaging is performed under the action of HRP substrate.

[0064] This embodiment will focus on the experimental methods described above, obtaining target short peptides for subsequent research through at least three rounds of screening. The experimental design process includes ( Figure 1 Part E): By using a single protein concentration, all chips containing 2645 short peptides are screened to identify target short peptides; the target short peptides identified in the first round of screening are then customized into new chips (the number of chips will be significantly reduced compared to the first round of screening). The newly customized chips are screened using a series of Hsp70 protein concentrations, and repeated experiments are performed to complete the re-validation of Hsp70 protein binding to short peptides; the function of Hsp70 binding short peptides obtained through the first two rounds of screening is tested (in vitro and in vivo experiments).

[0065] Specifically, in this embodiment, the key parameters for implementing the indirect ELISA method are as follows:

[0066] All reagents used in the reaction buffer solution of the experimental system were purchased from Beyotime Biotechnology Co., Ltd.

[0067] 1) Determination of the concentration of the anti-His-tagged HRP-labeled detection antibody (Abclonal, AE028):

[0068] Six chips were randomly selected (6x6 size, each with a different peptide probe sequence, such as...). Figure 2 As shown in the figure); after the chip was dynamically incubated with reaction buffer 1 (25mM Hepes-KOH, pH 7.5; 100mM KCl, 10mM Mg(OAc)2, 1mM DTT, 10% glycerol) for a certain period of time, the reaction buffer was completely discarded; detection antibodies of different dilutions were incubated with the chip again for 20 minutes, and then the antibodies were washed away. Chemiluminescence detection was performed using HRP substrate (performed using a GE ImageQuant LAS4000mini chemical imaging analyzer). Finally, the appropriate detection antibody concentration in this embodiment was determined to be 1:5000 to 1:6000.

[0069] 2) Determination of Hsp70 protein concentration (taking DnaK as an example) in the screening system:

[0070] Six chips were randomly selected (as above); the chips were dynamically incubated with different concentrations of DnaK protein (diluted 1 part reaction buffer) for a certain period of time, and then the reaction buffer was completely discarded; the chips were incubated again with the detection antibody, and then the detection antibody was washed away, and chemiluminescence detection was performed using HRP substrate. Finally, it was determined that the optimal initial detection concentration of DnaK protein in this embodiment was 10 μM.

[0071] 3) Determination of the reaction time between DnaK protein and the microarray in the screening system:

[0072] Six chips were randomly selected (as above); after dynamic incubation of the chips with 10 μM DnaK protein (diluted with reaction buffer 1) for different times, the reaction buffer was completely discarded; a 1:6000 detection antibody was used to incubate the chips again, and then the antibody was washed away. Chemiluminescence detection was performed using HRP substrate. The interaction time between the DnaK protein and the chip was ultimately determined to be 1 hour in this embodiment.

[0073] 4) Determination of incubation speed between DnaK protein and the microarray in the screening system:

[0074] Six chips were randomly selected (as above); the chips were incubated with 10 μM DnaK protein (diluted in reaction buffer 1) at different reaction speeds (100-400 rpm) for 1 hour, and the reaction buffer was completely discarded; a 1:6000 detection antibody was used to incubate the chips again, and then the antibody was washed away, and chemiluminescence detection was performed using HRP substrate. Finally, in this embodiment, the incubation speed of DnaK protein with the chips was determined to be 200 rpm.

[0075] according to Figure 1 The technical solution process, through initial screening, shows that the inventors in this case were able to increase the response rate of peptides to proteins to 5.2% (e.g., Figure 3A As shown), the threshold for judging positive signal values ​​(au = 2000) is consistent with the literature. The peptides that responded in the first round of screening were re-fabricated into a chip and reacted with different concentrations of DnaK protein (e.g., ...). Figure 3B (As shown). The inventors of this case discovered that the response of some peptides to proteins is a random signal—the response signal value and protein concentration are not positively correlated. After excluding these randomly responding peptides, peptides that truly bind to the DnaK protein were obtained. The response signal values ​​of these peptides to DnaK continuously increase with the increase of the concentration of the protein participating in the reaction; the number of responding peptides also increases with the increase of protein concentration (e.g., ...). Figure 3C (As shown).

[0076] The sequence contents of the five polypeptides obtained in this invention are shown in Table 1 below:

[0077] Table 1. Sequences of 5 polypeptides

[0078]

[0079] Among them, (1) Newcastle disease virus (NDV) fusion protein; (2) Auto-Immune Thyroid Disease (AITD); (3) Sodium / iodide symporter (NIS); and (4) Thyroid peroxidase (TPO).

[0080] The five short peptide sequences obtained in this embodiment, or short peptide sequences with similar amino acid sequences (homology of 80% or more, similarity of 70% or more), can all bind to the DnaK protein with high affinity. Furthermore, they are sequence-specific, with amino acid sequence lengths ranging from 12 to 20 amino acids. These five short peptides are derived from viral structural proteins (VP-1), important human autoimmune disease-associated antigen proteins (VP-2, VP-3, VP-4), and tumor necrosis factor (VP-5), respectively.

[0081] Based on the conclusions of the chip system, the inventors of this case further verified the affinity (Kd) of the five short peptides for DnaK in liquid solution through fluorescence polarization experiments. Consistent with the chip results, all five short peptides could bind to DnaK with high affinity, as shown in Table 2. Among them, VP-1 and VP-3 showed better affinity for DnaK than NR (NRLLLTG, the model short peptide). Through kinetic detection, the inventors of this case also found that VP-3 bound to DnaK protein at a faster rate; and that under ATP and APO conditions, the time required for the short peptides to reach equilibrium with DnaK (gray: 10 μM; pink: 1.25 μM) was similar (e.g., ...). Figure 4A and Figure 4B (As shown). In the APO state, when VP-3 reaches equilibrium with the protein, the addition of ATP does not significantly induce a decrease in signal intensity, indicating that VP-3 may disrupt or inhibit ATP-induced protein conformational coupling (e.g., ...). Figure 4C (As shown). Through denaturing luciferase renaturation experiments, the inventors of this case discovered that the five short peptides obtained all exhibited significantly stronger inhibitory effects on the DnaK renatured protein activity than the NR short peptide, with VP-2 being the most potent (e.g. Figure 4D (As shown). Compared to NR short peptides, except for VP-3 short peptides, other short peptides promote the hydrolysis of ATP by DnaK at a slightly lower rate (e.g., Figure 4E (As shown).

[0082] Table 2. Results of the regulation of DnaK activity by five short peptides.

[0083] VP-1 0.37±0.002 VP-4 1.46±0.114 VP-2 1.31±0.330 VP-5 2.10±0.136 VP-3 0.43±0.012 NR (Control) 0.44±0.021

[0084] In summary, through luciferase renaturation assays, the inventors discovered that five short peptides significantly inhibited luciferase renaturation, demonstrating their potential as lead peptides in antibacterial activity, with VP-2 and VP-3 showing particularly strong performance. Through fluorescence polarization experiments and peptide-protein binding kinetics experiments, the inventors further learned that the VP-3 short peptide has a strong binding affinity to DnaK, a rapid protein binding rate, and inhibits protein conformational coupling to a certain extent, indicating its potential for development and promising applications. Furthermore, the five short peptides exhibit a consistent regulatory trend on ATP hydrolysis, an ability associated with important human diseases. Therefore, further testing on the human homologous Hsp70 protein is needed to confirm the application potential of these five peptides (lead drug development for human diseases).

[0085] The three detection systems used in this embodiment are as follows:

[0086] I. Fluorescence polarization series (short peptide binding affinity, kinetics, and short peptide release assays) experimental conditions are as follows:

[0087] 1. Reaction buffer 1 (formulation as above).

[0088] 2. FITC-labeled peptides: In this embodiment, FITC was applied to the N-terminus of the peptides, which were synthesized by Nanjing Genscript Biotech Co., Ltd. and Jier Biochemical (Shanghai) Co., Ltd. The purity of both was greater than 95%.

[0089] Experimental procedure:

[0090] 3. Using reaction buffer 1, Hsp70 protein was serially diluted 2-fold from a concentration of 40 μM until a final concentration of 5 nM was reached. Then, the protein concentrations in each tube were uniformly mixed with the FITC-labeled short peptides (all peptides had a final concentration of 10 nM), transferred to a 384-well plate (Corning 3575), and incubated at room temperature in the dark for a specified time (the incubation time varied depending on the binding rate of different short peptides to different Hsp70 proteins) until the reaction between the short peptide and the protein reached equilibrium. Finally, the static fluorescence polarization signal in each well was detected using a Cytation 3 (BioTek) multi-function imager. The obtained signal values ​​were analyzed using PRISM 7.0 (GraphPad), and the data were fitted and plotted using the One site-specific binding program to calculate the binding affinity between the short peptide and the protein.

[0091] 4. The binding kinetics of the short peptide to the protein were detected, similar to the method described above. The difference was that once the protein and the FITC-labeled short peptide were mixed, no incubation was required; the mixture was immediately transferred to a 384-well plate. The dynamic changes in fluorescence polarization signals in each well over equal time intervals (10 s) were detected using a Cytation 3 (BioTek) multi-function imager. The obtained signal values ​​were analyzed using PRISM 7.0 (GraphPad), and data fitting and plotting were performed using the Binding-Kinetics program.

[0092] 5. To test the release of FITC-labeled short peptides from proteins after the addition of ATP (Beijing Solarbio Biotechnology Co., Ltd., A8270), the following procedure must be followed: After mixing and incubating the specified concentration of protein with the FITC-labeled short peptide (both peptides have a final concentration of 10 nM) to reach reaction equilibrium, the stability of the fluorescence polarization signal within the well (kinetic changes over 10 minutes) is detected using a Cytation 3 (BioTek) multifunction imager. Then, ATP is added to the well to a final concentration of 2 mM, and the mixture is incubated for 2 minutes. Subsequently, the dynamic changes of the fluorescence polarization signal within the well over equal time intervals (10 s) are detected using a Cytation 3 (BioTek) multifunction imager. The obtained signal values ​​are analyzed and plotted using PRISM 7.0 (GraphPad).

[0093] II. The conditions for the denaturing luciferase renaturation experiment are as follows:

[0094] 1. Reaction buffer 2: 25mM Hepes-KOH, pH 7.5; 100mM KCl, 10mM Mg(OAc)2, 1mM DTT, 3mM ATP.

[0095] 2. Required auxiliary proteins for the reaction: DnaJ and GrpE (both were purified in the laboratory); luciferase protein was purchased from Beijing Solarbio Biotechnology Co., Ltd., 61970-00-1.

[0096] 3. Luciferase detection system: purchased from Promega, E1500.

[0097] Experimental procedure:

[0098] In a buffer solution, a certain concentration of high-purity luciferase protein was mixed with 3 μM DnaK protein, and the mixture was heated at 42 °C to denature the luciferase protein. The denatured luciferase protein was then mixed with the refolding system, which consisted of DnaK, DnaJ, and GrpE, to induce luciferase protein refolding. At regular intervals, 2 μL of sample was taken from the refolding reaction system and reacted with the protein substrate of the luciferase detection system. Readings were measured using a Cytation 3 multi-functional imaging system. To detect the effect of other peptides on the refolding ability of DnaK, a series of peptide molecules at different concentrations were added to the refolding system beforehand, followed by the same subsequent procedures as described above.

[0099] III. The experimental conditions for the DnaK protein ATPase activity assay are as follows:

[0100] 1. Reaction buffer 1 (formulation as above).

[0101] 2. ATP reagent: purchased from Beijing Solarbio Biotechnology Co., Ltd., A8270.

[0102] 3. ATP assay kits: CytoPhos Phosphate Assay BioChem Kit (Cytoskeleton, BK054), ADP-Glo ​​Kinase Assay (Promega, PAV9101).

[0103] Experimental procedure:

[0104] In this embodiment, two kits were used to detect protein ATPase: since peptide VP-1 can bind to the substrate in the CytoPhosPhosphate Assay BioChem Kit, the ADP-Glo ​​Kinase Assay Kit was selected; the remaining peptides were tested using the CytoPhos Phosphate Assay BioChem Kit.

[0105] The CytoPhos Phosphate Assay BioChem Kit uses the ATPase endpoint assay to determine protein ATPase activity by measuring the amount of inorganic phosphate (Pi) produced during ATPase hydrolysis. The procedure should be followed according to the kit's instructions. Simply put, the standard concentration is plotted against (A) using the inorganic phosphate standard provided in the kit. 650nmThe correlation curve of absorbed light signal values ​​was then generated. The protein was then diluted to an appropriate concentration with reaction buffer 1 (divided into 10 equal tubes), and 0.1 mM ATP was added to each tube to initiate the reaction. At different time points, 70 μL of CytoPhos reagent was added to each protein tube to terminate the reaction. After incubation at room temperature for 10 minutes, A was read. 650nm The absorbed light signal value was used to calculate the actual concentration of inorganic phosphate generated in each tube. Finally, the data was analyzed and plotted using PRISM 7.0 (GraphPad).

[0106] The ADP-Glo ​​Kinase Assay kit measures protein ATPase activity by calculating the percentage of ATP converted to ADP. The initial experimental procedures are the same as those described above. The difference lies in the addition of ADP-Glo ​​at different time points. TM The reagent terminates the ATP hydrolysis of protein in each tube, and then Kinase Detection Reagent is added to convert the hydrolyzed ADP into ATP. Finally, the luminescence signal values ​​in different tubes are measured using a Cytation 3 multi-functional imager. Based on the signal values, the inventors can obtain the percentage of ADP converted to ATP at different time points, and then analyze and plot the ATP hydrolysis rate using PRISM 7.0 (GraphPad).

[0107] When detecting the effect of short peptides on protein ATPase activity, Hsp70 should be incubated with a certain concentration of short peptides beforehand, and then the experiment should be carried out as described above.

[0108] The inventors in this case conducted antibacterial (wild-type Escherichia coli, ATCC 25922) experiments on short peptides such as VP-3. Figure 5 As shown in the experimental results, under high-temperature heat shock conditions, VP-3 has a significantly stronger inhibitory effect on the growth of wild-type Escherichia coli than NR short peptide.

[0109] Experimental methods:

[0110] A single colony of wild-type Escherichia coli (ATCC 25922) was inoculated into LB broth and incubated overnight at 37°C and 200 rpm. The next day, the overnight culture was diluted with fresh LB broth (approximately 300-fold dilution, to OD200). 600(Readings were 0.08–0.1 μL). The diluted bacterial solution was then inoculated into 96-well plates at 200 μL per well for further incubation. Before continuing incubation, peptide solutions of different dilutions were slowly added to the *E. coli* dilution solution and mixed thoroughly with a pipette. After mixing the *E. coli* and peptide mixture, the 96-well plate was transferred to a 42°C biochemical incubator and incubated for 12–16 hours. After incubation, the OD values ​​in each well were measured. 600 reading.

[0111] The inventors of this case investigated the regulation of protein activity by five short peptides targeting two human homologous Hsp70 proteins. As shown in Table 3, contrary to the results of the DnaK protein test, VP-3 showed low binding affinity to human Hsp70 (hHsp70, NM_005345.6) and Hsc70 (NM_006597.6), resulting in no detectable values ​​(Not Determined, ND). Among all peptides, these two proteins showed the highest binding affinity to VP-5 (Figure 6-A). Based on this, the inventors further investigated the regulation of ATPase activity of the two proteins by the peptides. Compared with the NR short peptide, VP-1 inhibited the ATPase activity of hHsp70 to a certain extent; while VP-5 increased the protein ATPase activity by more than four times (e.g., NR peptide ). Figure 6A (As shown). For Hsc70 protein, both VP-4 and VP-5 can increase protein activity by 2-4 times (e.g., Figure 6B (As shown). Given that VP-1, VP-4, and VP-5 regulate the ATPase activity of human Hsp70, they are likely to be used as short peptides, or even peptide lead drug molecules, for the treatment of cancer or neurological diseases (Alzheimer's disease).

[0112] Table 3. Short peptides regulate the ATPase activity of human Hsp70 protein.

[0113]

[0114] In this embodiment, the above screening scheme and process are implemented according to a typical scheme. However, peptide chip screening systems, ELISA, etc., based on other materials are also within the protection scope of this invention.

[0115] The above screening of Hsp70 protein-binding peptides was conducted according to the peptide bias selection scheme provided by this invention for peptide library construction. However, experimental schemes based on other screening systems are also within the scope of protection of this invention.

[0116] In the above screening of Hsp70 protein-binding peptides, the target protein uses other tags, but has a similar function to the His tag in this embodiment; other experimental systems that involve reaction temperature, reaction speed, incubation time of target protein and chip, etc., and whose screening schemes are determined according to the parameters provided by this invention are also within the protection scope of this invention.

[0117] In the above screening of Hsp70 protein-binding peptides, the target protein is not tagged. However, other experimental systems that determine the screening scheme according to the parameters provided in this invention, such as reaction temperature, reaction speed, and incubation time between the target protein and the chip, are also within the scope of protection of this invention.

[0118] Furthermore, the peptides used in this embodiment are all original sequences of natural proteins, which can further increase the possibility of obtaining effective peptide molecules compared with phage display libraries (randomly synthesized amino acid sequences).

[0119] Furthermore, this embodiment uses iPDMS nanomembrane peptide chip technology. Based on the zero background characteristic of the chip substrate, it can achieve the goal of high-throughput screening in a single run with only a small amount of Hsp70 protein (1 micromolar). Compared with phage display technology, immunoprecipitation and other technologies, it is simple to operate, has a short experimental cycle, and is fast and efficient.

[0120] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims. sequence list <110> Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences <120> Hsp70 protein-binding peptides, their screening methods and applications <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 12 <212> PRT <213> Artificial sequence() <400> 1 Ile Ile Val Lys Leu Leu Pro Asn Leu Pro Lys Asp 1 5 10 <210> 2 <211> 15 <212> PRT <213> Artificial sequence() <400> 2 Thr Glu Lys Gln Ala Lys Leu Ala Leu Leu Ile Asn Gln Val Gly 1 5 10 15 <210> 3 <211> 15 <212> PRT <213> Artificial sequence() <400> 3 Arg Leu Arg Ser Leu Ala Pro Arg Lys Leu Val Ile Ile Ser Lys 1 5 10 15 <210> 4 <211> 15 <212> PRT <213> Artificial sequence() <400> 4 Lys Arg Gly Ile Leu Ser Pro Ala Gln Leu Leu Ser Phe Ser Lys 1 5 10 15 <210> 5 <211> 20 <212> PRT <213> Artificial sequence() <400> 5 Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro 1 5 10 15 Cys Gln Arg Glu 20

Claims

1. An Hsp70 protein-binding polypeptide, characterized in that, Its amino acid sequence is shown in any one of SEQ ID No. 1, SEQ ID No. 4, or SEQ ID No.

5.

2. A polypeptide lead drug, characterized in that... Includes the Hsp70 protein-binding polypeptide as described in claim 1.

3. The polypeptide lead drug according to claim 2, characterized in that: The polypeptide lead drug is selected from any one of the following: drugs for the prevention and treatment of pathogenic microorganisms, drugs for the treatment of cancer, and drugs for the treatment of Alzheimer's disease.

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