Compounds as TGF-β receptor agonist peptides and their uses

Abstract

This invention belongs to the field of peptide medicine, specifically relating to a compound as a TGF-β receptor agonist peptide and its uses. The amino acid sequence of the peptide is shown in SEQ ID No. 1. The peptide of this invention has a significant agonistic effect on the TGF-β receptor and can be used in the preparation of drugs for the prevention and / or treatment of TGF-β-related diseases, particularly for the prevention of hair loss and / or the promotion of hair growth.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide medicine, specifically relating to a compound that is a TGF-β receptor agonist peptide and its uses. Background Technology

[0002] Transforming growth factor beta (TGF-β) is a member of the TGF-β superfamily, which includes three isoforms: TGF-β1, TGF-β2, and TGF-β3. TGF-β is a multifunctional protein, and numerous studies have shown that it has different functions in different cell types, and these functions are environment-dependent. Normally, cell survival, metabolism, growth, proliferation, differentiation, adhesion, migration, and death are all regulated by TGF-β. Normal TGF-β signaling is essential for normal function and homeostasis in a healthy body, while abnormal TGF-β signaling can lead to various diseases.

[0003] TGF-β has a high-affinity receptor (TGF-β R) on the cell surface, which is divided into three subtypes: TGF-β RI (also known as ALK5), TGF-β RII, and TGF-β RIII. TGF-β forms a complex by binding to TGF-β RII, which has serine / threonine kinase activity on the cell membrane surface, and then forms a complex with TGF-β RI (also known as ALK5). This process transduces signals and activates the corresponding signaling pathways, thereby regulating cell proliferation, differentiation, and apoptosis.

[0004] Currently, TGF-β remains a challenging therapeutic target due to its diverse functions, variable biological behavior, and toxicity to organs such as the heart. Despite these challenges, recent advancements in the development of therapies targeting the TGF-β pathway have led to significant progress in recent years, including the emergence of concepts such as small molecule inhibitors, fusion molecules, monoclonal antibodies, "TGF-β traps," vaccines, and antisense oligonucleotides.

[0005] The prior art CN104902916A discloses a mutant polypeptide of the TGF-β molecule, whose primary sequence is highly homologous to the human TGF-β sequence. The mutant polypeptide loses the ability to interact with TGF-β RI (also known as ALK5) but retains the interaction with receptors TGF-β RII and TGF-β RIII (see paragraphs [0015-0017] of the specification). Existing TGF-β RI inhibitors, including SM16 and GW788388, have been shown to have anti-fibrotic effects. In addition, drugs such as Galunisertib, PF-06952229, and LY2109761 specifically bind to TGF-βR1, blocking TGF-βR1-mediated signal transduction, thereby being used for anti-tumor therapy.

[0006] A team led by Qixuan Wang at the University of California, Riverside, identified how TGF-β controls the division and formation of new cells (including stem cells) in hair follicles, or controls their own death, ultimately leading to the death of the entire hair follicle (A probabilistic Boolean model on hair follicle cell fate regulation by TGF-β, Biophys J. 2022 Jul 5;121(13):2638-2652.). The results indicate that transforming growth factor β (TGF-β) plays a dual role in the regulation of hair follicle cell fate, acting as both an anti-apoptotic and pro-apoptotic agent. Activating the TGF-β signaling pathway helps activate hair follicle cells to generate new life, thereby activating hair follicle stem cells and stimulating hair growth. Summary of the Invention

[0007] Through in-depth research and creative work, this application has discovered a group of peptides that can effectively activate TGF-β receptors. These peptides can be artificially intervened to activate hair follicle stem cells and stimulate hair growth. Complete wound healing also requires hair follicle regeneration, thus possessing the potential as a therapeutic or adjuvant treatment. Therefore, the following invention is provided:

[0008] One aspect of the present invention relates to a polypeptide, the amino acid sequence of which is shown in SEQ ID No. 1.

[0009] SEQ ID No. 1:

[0010] ALX3TNYCFSSTETNCCVRPLYIDFEKDLGWKWIHEPKGYYANFCLGPCPPGWSLX 55 TQYSKVLALYHAHNPSSSAKPCCVPX 81 X 82LAPLTIX 89 YYKGX 94 KX 96 VTX 99 QLSNMIVX 107 SCX 110 CS

[0011] in,

[0012] X3 represents D or Y;

[0013] X 55 Indicates R or E;

[0014] X 81 Indicates E or R;

[0015] X 82 Indicates K or E;

[0016] X 89 Indicates K or T;

[0017] X 94 Indicates N or R;

[0018] X 96 Indicates L or K;

[0019] X 99 Indicates K or E;

[0020] X 107 Indicates K or E;

[0021] X 110 It represents R or V.

[0022] In some instances, the amino acid sequence of the polypeptide is as shown in SEQ ID No. 2, SEQ ID No. 3, or SEQ ID No. 4.

[0023] SEQ ID No. 2:

[0024] ALDTNYCFSSTETNCCVRPLYIDFEKDLGWKWIHEPKGYYANFCLGPCPPGWSLRTQYSKVLALYHAHNPSSSAKPCCVPRELAPLTIKYYKGNKLVTEQLSNMIVKSCVCS,

[0025] SEQ ID No. 3:

[0026] ALYTNYCFSSTETNCCVRPLYIDFEKDLGWKWIHEPKGYYANFCLGPCPPGWSLETQYSKVLALYHAHNPSSSAKPCCVPEKLAPLTITYYKGRKKVTKQLSNMIVESCRCS,

[0027] SEQ ID No.4:

[0028] ALDTNYCFSSTETNCCVRPLYIDFEKDLGWKWIHEPKGYYANFCLGPCPPGWSLETQYSKVLALYHAHNPSSSAKPCCVPEKLAPLTITYYKGRKKVTKQLSNMIVESCRCS.

[0029] In this invention, site-directed mutagenesis is used to identify amino acids that play a key role in the activity of the peptide of this invention, thereby determining the amino acids that play a key role in the activity of the polypeptide molecule.

[0030] Site-directed mutagenesis techniques, such as alanine scanning, introduce mutations into every residue of the polypeptide molecule and examine the biological activity of the resulting polypeptide molecule to identify the amino acid residues that play a key role in the activity of the polypeptide molecule; PCR site-directed mutagenesis techniques, such as designing primers containing non-specific paired bases, then introducing the mutation site into the product through PCR.

[0031] Using site-directed mutagenesis, it was determined that position 25E of the polypeptide of the present invention is an essential amino acid for activity. Furthermore, positions 20-30, "YIDFEKDLGW", are essential amino acids for activity. Even further, positions 13-39, "TNCCVRPLYIDFEKDLGWKWIHEPKGY", are essential amino acids for activity. Even further, positions 3-54, "TNYCFSSTETNCCVRPLYIDFEKDLGWKWIHEPKGYYANFCLGPCPPGWSL", and positions 56-80, "TQYSKVLALYHAHNPSSSAKPCCVP", are essential amino acids for activity.

[0032] On the other hand, the present invention provides a recombinant polypeptide, wherein the recombinant polypeptide is obtained by coupling a cell-penetrating peptide and the polypeptide shown in SEQ ID No. 1 through a linker, wherein the coupling is a covalent linking via an amide bond.

[0033] In some instances, the linker contains, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.

[0034] In some instances, the connector is selected from one of G, GG, GGG, S, SS, SSS, GS, GSGS, GSGSGS, GGS, GGSGGS, GGSGGSGG, GGGS, GGGSGGGSGGS, GGGSGGGSGGGS, GSS, GSSGSS, GSSGSSGSS, GGSS, GGSSGGSS and GGSSGGSSGGSS.

[0035] Preferably, the connector is selected from GSGS.

[0036] In some instances, the cell-penetrating peptide is selected from one or more of the following: TAT peptide of HIV-1 viral reverse transcription activator protein, D-Tat, transcription factor Antp peptide of Drosophila antennal homologous protein, Pep-1 peptide, Pep-2 peptide, MPG peptide, and RGD peptide.

[0037] In some instances, the cell-penetrating peptide is selected from the group consisting of:

[0038]

[0039] Preferably, in some instances, the cell-penetrating peptide is selected from GRKKRRQRRRPQ.

[0040] In some instances, the recombinant polypeptide is obtained by coupling a cell-penetrating peptide and the polypeptide shown in SEQ ID No. 1 via a linker.

[0041] In some instances, the recombinant polypeptide is obtained by coupling a cell-penetrating peptide and the polypeptide shown in SEQ ID No. 2 via a linker.

[0042] In some instances, the recombinant polypeptide is obtained by coupling a cell-penetrating peptide and the polypeptide represented by SEQ ID No. 3 via a linker.

[0043] In some instances, the cell-penetrating peptide is attached to the N-terminus or C-terminus of the aforementioned polypeptide via a linker.

[0044] In some instances, the cell-penetrating peptide is located at the N-terminus of the recombinant polypeptide.

[0045] In some instances, the cell-penetrating peptide is located at the C-terminus of the recombinant polypeptide.

[0046] In some instances, the amino acid sequence of the recombinant polypeptide is shown in SEQ ID No. 5, SEQ ID No. 5:

[0047] GRKKRRQRRRPQGSGSALX3TNYCFSSTETNCCVRPLYIDFEKDLGWKWIHEPKGYYANFCLGPCPPGWSLX55 TQYSKVLALYHAHNPSSSAKPCCVPX 81 X 82 LAPLTIX 89 YYKGX 94 KX 96 VTX 99 QLSNMIVX 107 SCX 110 CS

[0048] in,

[0049] X3 represents D or Y;

[0050] X 55 Indicates R or E;

[0051] X 81 Indicates E or R;

[0052] X 82 Indicates K or E;

[0053] X 89 Indicates K or T;

[0054] X 94 Indicates N or R;

[0055] X 96 Indicates L or K;

[0056] X 99 Indicates K or E;

[0057] X 107 Indicates K or E;

[0058] X 110 It represents R or V.

[0059] In some instances, the amino acid sequence of the recombinant polypeptide is as shown in SEQ ID No. 6, SEQ ID No. 7, or SEQ ID No. 8.

[0060] SEQ ID No. 6:

[0061] GRKKRRQRRRPQGSGSALDTNYCFSSTETNCCVRPLYIDFEKDLGWKWIHEPKGYYANFCLGPCPPGWSLRTQYSKVLALYHAHNPSSSAKPCCVPRELAPLTIKYYKGNKLVTEQLSNMIVKSCVCS,

[0062] SEQ ID No. 7:

[0063] GRKKRRQRRRPQGSGSALYTNYCFSSTETNCCVRPLYIDFEKDLGWKWIHEPKGYYANFCLGPCPPGWSLETQYSKVLALYHAHNPSSSAKPCCVPEKLAPLTITYYKGRKKVTKQLSNMIVESCRCS,

[0064] SEQ ID No. 8:

[0065] GRKKRRQRRRPQGSGSALDTNYCFSSTETNCCVRPLYIDFEKDLGWKWIHEPKGYYANFCLGPCPPGWSLETQYSKVLALYHAHNPSSSAKPCCVPEKLAPLTITYYKGRKKVTKQLSNMIVESCRCS.

[0066] On the other hand, the present invention provides a composition comprising excipients and an active ingredient, wherein the active ingredient comprises the aforementioned polypeptide or recombinant polypeptide; the excipients comprise at least one of a diluent, filler, binder, humectant, absorption enhancer, surfactant, lubricant, and stabilizer.

[0067] On the other hand, the present invention provides the use of the above-mentioned polypeptide or recombinant polypeptide or composition in the prevention and / or treatment of diseases related to TGF-β.

[0068] In some instances, the application is for preventing hair loss and / or promoting hair growth.

[0069] In some instances, the present invention provides a cosmetic composition for preventing hair loss or promoting hair growth, said composition being selected from one or more of the following: hair conditioner, hair serum, hair liquid, shampoo, hair conditioner, hair cream, hair wax, hair aerosol, hair mask, hair nourishing pack, shampoo bar, shampoo foam, hair oil, hair conditioner, hair dye, hair curling agent, and hair gel.

[0070] The term "natural amino acid" refers to an amino acid encoded by the organism's own genetic code. In some preferred embodiments, "natural amino acid" is defined as the group consisting of alanine (A), arginine (R), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), histidine (H), isoleucine (I), glycine (G), aspartic acid (N), leucine (L), lysine (K), methionine (M), phenylalanine (F), co-amino acid (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

[0071] Site-directed mutagenesis is a molecular biology technique used to intentionally alter specific amino acids in a protein sequence to study their effects on protein structure, function, or interactions with other molecules. Those skilled in the art understand that the function of small peptides is closely related to their amino acid sequence, and alterations to sequence length, amino acid types, etc., can affect their function and efficacy. In such cases, those skilled in the art cannot predict the impact of site-directed mutagenesis on the function of small peptide sequences, nor can they predict the impact of site-directed mutagenesis on the activity level of small peptide sequences, as illustrated in CN106822865A and CN115385827A. Therefore, not all site-directed mutagenesis-derived TGFβ peptides can achieve the same effects as the peptides in this application; that is, not all site-directed mutagenesis-derived TGFβ peptides can achieve the same effective effect in preventing hair loss and / or promoting hair growth as the peptides in this application.

[0072] The term "cell penetrating peptide (CPP)," also known as "cell-penetrating peptide," "protein translocation domain (PTD)," "Trojan horsepeptides," or "transduction peptide," refers to peptides that can promote the permeation of various molecules (e.g., various macromolecules including proteins or nucleic acids). Examples of CPPs include, but are not limited to: (i) protein-derived CPPs: such as sequences derived from genes controlling antennae (e.g., pAntp (43-58); sequences derived from HIV-1, such as Tat-derived peptides, such as amino acid residues 37-72, 37-60, 48-60, or 49-57 from TAT; (ii) model peptides: such as VT5; MAP; or arginine stretch sequences, etc.; (iii) designed CPPs: such as MPG; Transportan; Transportan 10; Pep-1; peptides selected from KALA; or peptides selected from Bulforin2, etc.

[0073] Furthermore, the CPP used in the conjugates of the present invention may also be selected from polypeptide sequences having approximately 60, 70, 80, 90, 95, 99% or 100% sequence identity with any polypeptide sequence as described above, provided that the polypeptide sequence still retains its biological activity, i.e., promoting cellular uptake of the isolated polypeptide (or its variants) of the present invention and / or promoting the isolated polypeptide (or its variants) of the present invention across the blood-brain barrier.

[0074] Those skilled in the art can easily mutate the polypeptides of the present invention using known methods, such as site-directed mutagenesis or directed evolution. Those artificially modified sequences that have 90% or more identity with the polypeptides of the present invention, and have the same function as the polypeptides of the present invention, are all derived from and equivalent to the nucleotide sequences of the present invention.

[0075] The term "alanine scan mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085, refers to a useful method for identifying residues or regions of a peptide that can be targeted for mutagenesis. In this method, residues or target residue groups (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the peptide and its receptor is affected. Additional substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the peptide-receptor complex can be used to identify the contact points between the peptide and its receptor. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they possess the desired properties. Attached Figure Description

[0076] Figure 1 The image shows the HPLC detection results of the peptide shown in SEQ ID No. 2.

[0077] Figure 2 The image shows the mass spectrometry detection results of the peptide shown in SEQ ID No. 2.

[0078] Figure 3 The image shows the mass spectrometry detection results of the peptide shown in SEQ ID No. 3;

[0079] Figure 4 A graph showing the cell proliferation-promoting effect of second-generation human follicular dermal papillary cells (HFDPC);

[0080] Figure 5 The figure shows the cell proliferation-promoting effect of fourth-generation human follicular dermal papillary cells (HFDPC). Detailed Implementation

[0081] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the following embodiments, unless otherwise specified, all raw materials used are commercially available.

[0082] Polypeptide biological expression process

[0083] Example 1 Synthesis of Polypeptides

[0084] Polypeptide biological expression process

[0085] 1. Gene Construction

[0086] The target polypeptide DNA sequence was designed using overlapping oligonucleotide primers and synthesized. The full-length target DNA sequence was obtained through PCR and ligated into the vector pET15b-sumo. The polypeptide sequence was located downstream of the SUMO protein and fused with it for expression. The ligation vector was transformed into E. coli culture, plasmids were extracted and sequenced, and the sequencing results were analyzed to ensure consistency with the designed target sequence.

[0087] 2. Peptide expression

[0088] Construct the correct bacterial culture and inoculate it into SB medium. Add 2 / 1000 ampicillin (stock solution concentration: 200 mg / mL), and incubate at 37°C with shaking for 4 hours. Then adjust the temperature to 17°C and continue to incubate with shaking for 1 hour. Then add IPTG to the final concentration of 1 mM and incubate overnight with shaking at 200 rpm to induce expression.

[0089] 3. Peptide purification

[0090] The overnight induced bacterial culture was dissolved in 288g of urea in a 60℃ water bath for 5 min, then 6M NaOH was added for ultrasonic disruption (3 s disruption, 8 s interval, 3 min, 60% power). 17.7g of 6M HCl was added for neutralization, followed by incubation with 25mL of nickel magnetic beads for 1 hour. The supernatant was removed by strong magnetic adsorption, and the culture was resuspended. After washing three times, 500 mM imidazole was used as the eluent. SUMO protein was added to the eluent and incubated overnight at 4℃ for shearing. 8M guanidine hydrochloride was added to the shearing buffer, and the culture was loaded onto a pre-equilibrated C18 reversed-phase column (600mg). The culture was washed with 1% acetonitrile and 1% acetic acid, followed by elution with 50% acetonitrile. The eluent was analyzed by SDS-PAGE electrophoresis.

[0091] 4. Polypeptide quality inspection

[0092] The molecular weight of the peptide was determined by mass spectrometry, and the purity of the peptide was determined by HPLC.

[0093] All peptides of this invention can be synthesized using the above-described biological expression methods. The HPLC and mass spectrometry detection results of the peptides shown in SEQ ID No. 2 and SEQ ID No. 3 are as follows: Figures 1 to 3 As shown.

[0094] 1. Experimental materials:

[0095]

[0096] 2. Experimental steps:

[0097] The day before the assay, TGF-β Reporter HEK293 cells were seeded at 6000 cells / well, 20 μl / well, into 384-well white translucent cell culture plates and incubated overnight for at least 16 hours. The next day, the fully adhered cell plates were removed, and the samples were diluted with loading buffer containing 0.1% BSA. The assay sample, TGF-β1 protein, and buffer were added at 5 μL / well to the corresponding 384-well plates (TGF-β1 protein as a positive control, 0.1% BSA loading buffer as a negative control), and incubated for 6 hours or overnight (approximately 20 hours). After incubation, the cell plates were removed and chemiluminescence immunoassay was performed using a multifunctional cell imaging microplate reader. The activating activity of the sample was calculated with the TGF-β1 protein activation rate set at 100%, using the following formula:

[0098] Activation rate = 100% × (sample signal - buffer signal) / (TGF-β1 signal - buffer signal).

[0099] Plot the concentration-response curve of the peptide of the present invention to the activation of TGF-β receptor, and the EC50 of the peptide of the present invention. 50 The results are shown in Table 1. The peptides shown in SEQ ID No. 2 and SEQ ID No. 3 showed the strongest activation of the TGF-β receptor.

[0100] Table 1. Results of peptide concentration response in this invention

[0101]

[0102] 1. Experimental materials and reagents:

[0103]

[0104]

[0105] 2. Experimental Procedure

[0106] Human dermal papilla cells from the first four generations were seeded into clear 96-well plates coated with poly-L-lysine at a density of 10,000 cells / well and incubated at 37°C in a 5% CO2 incubator for 24 h. After cell adhesion, the original culture medium was discarded, and DMEM / F12 basal culture medium was added as a negative control. The test sample prepared in DMEM / F12 basal culture medium and TGF-β1 prepared in DMEM / F12 basal culture medium were added as positive controls. The volume of culture medium or drug per well was 100 μL. After drug addition, the cells were incubated at 37°C in a 5% CO2 incubator for 72 h. 10 μL of CCK8 reagent was added to each well and incubated at 37°C in a 5% CO2 incubator for 1 h. OD was measured using a microplate reader. 450 Absorbance value.

[0107] Promotion rate calculation: Promotion rate (%) = ((OD) 450 EXP -OD 450 BLANK ) / (OD 450 阴性对照 -OD 450 BLANK )-1)×100%.

[0108] 3. Experimental Results

[0109] The polypeptides of this invention showed no toxicity or inhibitory effect on HFDPC cells. The proliferation-promoting rate was as follows: Figure 4 , Figure 5 As shown in Table 2, some calculation results are presented. Compared with the blank group, the peptide of the present invention can significantly promote the proliferation of HFDPC cells; in the first four generations, the proliferation rate of each sample is relatively stable, and the proliferation rate at higher concentrations is 40%-70%. The peptide shown in SEQ ID No. 2 has a relatively good concentration gradient effect.

[0110] Table 2 Calculation results of proliferation rate for each sample

[0111]

[0112] The in vitro hair loss prevention experiment in mice also confirmed the role of the polypeptides of this invention in promoting hair growth and preventing or treating hair loss in vivo.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No.

4.

2. A recombinant polypeptide, characterized in that, The amino acid sequence of the recombinant polypeptide is shown in SEQ ID No. 6 or SEQ ID No.

7.

3. A composition comprising an adjuvant and an active ingredient, characterized in that, The active ingredient is the polypeptide of claim 1 or the recombinant polypeptide of claim 2; and the adjuvant comprises at least one of a diluent, a filler, a binder, a humectant, an absorption enhancer, a surfactant, a lubricant and a stabilizer.

4. Use of the polypeptide according to claim 1, the recombinant polypeptide according to claim 2 or the composition according to claim 3 for the manufacture of a medicament for the prevention and / or treatment of a disease associated with TGF-β, characterized in that, The disease is alopecia.

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