CN113577241A - Design and screening method of small blocking peptide and application of small blocking peptide in synthesizing medicament for treating fibrotic diseases - Google Patents

Abstract

The invention belongs to the technical field of blocking peptide synthesis, and particularly relates to a design and screening method of small blocking peptide and application of the small blocking peptide in synthesizing a medicament for treating a fibrotic disease. The beneficial effects are that: provides a new raw material of a medicine for treating the fibrotic diseases. Compared with the antibody used as the raw material of the traditional medicament for treating the fibrotic diseases, the technical scheme of the invention has excellent performances in the aspects of production cost and method, immune activation risk, off-target effect risk and the like.

Description

Design and screening method of small blocking peptide and application of small blocking peptide in synthesizing medicament for treating fibrotic diseases

Technical Field

The invention belongs to the technical field of blocking peptide synthesis, and relates to a design and screening method of small blocking peptide and application of the small blocking peptide in synthesizing a medicament for treating a fibrotic disease.

Background

Fibrosis is a disease process characterized by excessive and disorganized extracellular matrix (ECM) deposition, leading to disfigurement, organ dysfunction, and in some cases, organ failure and death.

The main cell types contributing to the onset of fibrogenesis and fibrosis are alpha-smooth muscle actin (alpha-SMA) stress fiber positive and contractile myofibroblasts. Myofibroblast-mediated progressive fibrosis can reduce to most organs and tissues, causing a variety of end-stage organ diseases including scleroderma, myocardial infarction, cirrhosis, lung disease, and chronic kidney disease.

Fibrotic ECM is composed of collagen I synthesized by a large number of activated fibroblasts and myofibroblasts. Alternative splice variants of Fibronectin (FN) contain the extra domain a of fibronectin (EDA-FN), glycosaminoglycans, hyaluronic acid. The classical view holds that the presence of transforming growth factor-beta 1 (TGF-beta 1) and EDA-FN-containing mechanohardened ECM is an essential prerequisite and requirement for the differentiation of activated fibroblasts into myofibroblasts.

It was found that the EDA-FN splice variant was expressed only in wound repair. EDA-FN is difficult to detect in normal adults, and is only transiently expressed during embryogenesis. Although intact adult EDA-FN deficient mice were indistinguishable from wild-type mice, the injured counterpart mice exhibited abnormal skin wound healing, ulceration and increased inflammation at the injured site.

A sustained accumulation of EDA-FN isoforms is observed in a range of human fibrotic diseases. The mouse knock-out EDA-FN-null model did not develop pulmonary fibrosis after bleomycin injury and showed a decrease in the number of activated fibroblasts to myofibroblast differentiation. These findings demonstrate that EDA-FN plays an essential role in the pathogenesis of fibrosis, and its expression allows activated fibroblasts to undergo myofibroblast differentiation.

The detailed cellular mechanisms by which EDA-FN promotes the fibrotic response have not been fully elucidated, but evidence suggests that interactions between potential TGF- β 1 activation, EDA-FN dependence increase matrix stress-strain properties and activation of certain mechanotransduction integrin receptors to trigger downstream signaling and transcriptional activity, accumulating to drive myofibroblast differentiation processes. In the context of fibrogenesis, the Shinde et al study and Kohan et al EDA-FN/integrin interaction suggest that fibroblast-expressed integrins α 4 β 1 and α 4 β 7, respectively, play distinct but related roles in mediating fibroblast formation into myofibroblasts.

Integrin α 4 β 7-mediated EDA-FN induces myofibroblast-associated stress fiber formation and thus contraction of mouse lung fibroblasts. Whereas activation of integrin α 4 β 1 by EDA-FN promotes fibrillating ECM synthesis and subsequent hardening of human dermal fibroblast matrix. Both are key events that ultimately lead to transdifferentiation of mature myofibroblasts. This study identified the integrin interaction with the EDGIHEL motif, which is located only within the polypeptide exposure loop of the EDA C' -C region, responsible for the downstream profibrotic response.

Antibodies IST-9 and F8 can target EDA-FN and mask most of the protein from its interaction site, essentially blocking functionality. Indeed, therapeutic vaccination against EDA-FN by stromal myofibroblasts can reduce the progression of metastatic breast cancer. Although antibodies have proven effective in preventing fibroblast differentiation into myofibroblasts, their use as a therapeutic option is limited in view of their cost and method of production, risk of immune activation and risk of extensive off-target effects.

Antibodies of quaternary structure and size (-150 kDa) interfere with key protein-protein interactions. Small blocking peptides offer an attractive alternative to design for binding and blocking protein regions with high specificity. Small peptides can also be synthesized at reduced cost, have long-term stability, and can increase the efficacy of a variety of therapeutic options. For example, small peptides can be readily incorporated into biological materials, or conjugated with biological imaging probes for more complex therapeutic uses.

Thus, the present invention is directed to the use of computer molecular docking analysis to identify EDA-FN C-C' loop binding regions on a newly generated integrin α 4 β 1 receptor model and a resolved α 4 β 7 receptor model. The new affinity and contact data were then used to design small peptides that mimic the receptor binding site and block any EDA-FN C-C' loop interaction with its cellular receptor. The resulting small blocking peptides with the highest binding affinity were evaluated for their potential to attenuate TGF- β 1 stimulated myofibroblast formation, block integrin α 4 β 1 signaling and prevent subsequent induction of the profibrotic gene. The advantage of using blocking peptides in this manner is to allow for the interference of ligand site-specific interactions with the receptor without disrupting many of the other delivery functions of EDA-FN and FN.

Disclosure of Invention

The invention aims to solve the first technical problem of providing the application of the small blocking peptide in synthesizing medicaments for treating the fibrotic diseases.

The second technical problem to be solved by the invention is to provide a design and screening method of small blocking peptides, so as to quickly screen out effective small blocking peptides.

The invention discloses application of small blocking peptide in synthesizing a medicament for treating a fibrotic disease.

Further, the invention also discloses a design screening method of the small blocking peptide, which comprises the following steps:

step 1, generating an integrin alpha 4 beta 1 protein model;

step 2, generating a plurality of blocking peptide models by using a molecular docking means and screening;

and 3, carrying out cell culture on the screened blocking peptide model.

The invention has the beneficial effects that:

1. provides a new raw material of a medicine for treating the fibrotic diseases.

2. Compared with the antibody used as the raw material of the traditional medicament for treating the fibrotic diseases, the technical scheme of the invention has excellent performances in the aspects of production cost and method, immune activation risk, off-target effect risk and the like.

Drawings

FIG. 1 incubation of TGF-. beta.1 treated mouse dermal fibroblasts with peptides over a range of TGF10 dilutions;

FIG. 2 incubation of TGF-. beta.1 treated human lung fibroblasts with peptides over a range of TGF10 dilutions;

FIG. 3 detection of signal proteins ERK1/2 and FAK using Western blot analysis;

FIG. 4 uses gelatinase spectroscopy to assess gelatinase activity in conditioned media taken from peptide-treated cells;

FIG. 5 is a graph of hydroxyproline assay data;

FIG. 6 is a graph of immunocytochemistry data for α -SMA stress fiber formation;

FIG. 7 is a graph of expression of genes associated with a profibrotic myofibroblast phenotype and indicative fibrogenesis;

figure 8 genetic data plots for integrin α 4 β 1 activation by peptide treatment.

Detailed Description

The following examples are given to illustrate the technical examples of the present invention more clearly and should not be construed as limiting the scope of the present invention.

The materials used in the examples of the invention are as follows:

cell culture reagents and plastic ware were purchased from Sigma-Aldrich (Poole, Dorset, UK).

Peptides were synthesized by ChinaPeptides (shanghai, china).

Human lung fibroblast (HFL 1; ATCC CCL-153, Manassas, Va., USA) cells were kindly supplied by professor Wen Ning (university of south Kelvin).

Mouse skin fibroblasts (NIH/3T 3; ATTC CRL-1658) were supplied by professor Zhao Qiang (university of south Ken).

TGF-. beta.1 was purchased from R & D Systems (Shanghai, China).

All antibodies were purchased from Abcam (Cambridge, UK).

Example 1

A synthetic method of a design screening method of small blocking peptide comprises the following steps:

step 1, generating an integrin alpha 4 beta 1 protein model;

since integrin beta 1(ITB1_ human) has 42.112% amino acid sequence homology with integrin beta 7(ITB7_ human). Thus, using integrin α 4 β 7 as a template, integrin β 1 subunit is mapped onto the template to generate a working integrin α 4 β 1 model.

First, an integrin α 4 β 1 model was created using the Chimera v1.13.1(UCSF, San Francisco, CA, USA) and MODELLER v9.21(UCSF) software suite; specific manipulations the resolved crystal structure of the α 4 β 7 head domain (3V 4V; RCSB PDB) and the complete ITB7_ human sequence were used as a reference for structural mapping and overlay of ITB1_ human on the 3V4V crystal structure and to mimic binding to the integrin α 4 subunit;

then comparing the structure direction of integrin beta 1 subunit between the newly generated alpha 4 beta 1 model and the analytic crystal structure of integrin alpha 5 beta 1(4WK 2; RCSB PDB); the similarity between the sequence and head region alignments was used to validate the mapping of integrins; as a further validation control for the predicted binding site;

integrin α 4(ITA4_ human) was then mapped to the 4WK2 isolated model using integrin α 5(ITA5_ human) sequence as a reference template;

this was followed by examination and assurance of the quality of the receptor model using PROCHECK (EMBL-EBI, Cambridge, UK) and Verify3D (UCLA-DOE Institute, Los Angeles, Calif., USA).

Step 2, generating a plurality of blocking peptide models by using a molecular docking means and screening;

EDA-FN ligand (YSSPEDGHIEL) and its flanking amino acid sequences were isolated from the NMR structure (1J 8K; RCSB PDB) and the structure was minimized using AMBER protocol (AmberTools18, UCSF). Integrin α 4 β 1 (internally generated) and integrin α 4 β 7 (from 3V4V) receptor docking compatibility models, as well as EDA-FN peptide docking ligand models, were generated using the Chimera V1.13.1(UCSF) software suite. Molecular docking simulations were performed using AutoDock Tools and AutoDock Vina (Scripps Research, San Diego, CA, USA).

The specific operation is as follows: a total of five grids were selected to examine the entire integrin receptor surface. Each grid was analyzed separately to simulate the EDA-FN peptide ligands and AutoDockMolecular docking to add polarity in each assay, the first 7 binding modes from each assay were at PyMOL v2.2.3: (b)

Tokyo, Japan) and staining based on affinity scores. Using PyMOL v2.2.3

The built-in script assesses the electrostatic potential of the integrin receptor surface, hydrogen bond prediction and polar contacts between receptor and ligand. The PEP-SiteFinder tool (RPBS, Paris, France) was used to enhance the prediction of candidate amino acid binding regions.

Amino acids of integrin receptors that have polar contact with ligand ≦ 3 are listed and predicted to form hydrogen bond donors. A series of 10 peptides modeled from integrin receptor sites was generated based on the native receptor sequence and key predicted interacting amino acid regions. Peptides were constructed manually and placed at PyMOL v2.2.3

Initial folding simulation was performed. Peptides were exported to Chimera v1.13.1(UCSF) and minimized by amber (UCSF) protocol. The peptides were further examined and narrowed to 5 selections according to AMBER (UCSF), PROCHECK (EMBL-EBI) and RaptorX Contact Prediction (University Chicago, Chicago, IL, USA) scores. The 5 best peptide derivatives were uploaded to PEP-FOLD3(RPBS) and evaluated for each best folded conformation, followed by 5 best binding conformations, which had the highest docking affinity. EDA-FN peptide from AutoDock Vina (Scripps Research) was used (YSSPEDGHIEL). The peptide with the overall highest binding score was selected for synthesis.

Step 3, carrying out cell culture on the screened blocking peptide model;

HFL1 cells were cultured to pH7 in Kaighn's F-12(F-12K) modified Ham's F-12 (Sigma-Aldrich) medium containing NaHCO 3 and HEPES. Cells were grown to confluence in T75 flasks in the presence of 10% fetal bovine serum. (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Using 0.05% trypsin at 1: 3 ratio HFL1 cells were subcultured. NIH/3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone, Thermo Fisher Scientific) containing 100U/mL penicillin and 100. mu.g/mL streptomycin. Cells were grown to confluence in the presence of 10% FBS in T75 culture flasks. Using 0.05% trypsin/EDTA at 1: 3 ratio of passage NIH/3T3 cells. Cells were maintained at 37 ℃ and 5% CO 2, and growth medium was supplemented every 3 days until the experiment. Cells were arrested for 48 hours in serum-free medium before the experiment, and all experiments were performed under serum-free conditions using cells between passages 3-8.

To assess whether blocking peptides have anti-fibrotic function or cytotoxicity, we designed the following experiment: TGF- β 1 treated mouse dermal fibroblasts (figure 1) or human lung fibroblasts (figure 2) were incubated with peptides in TGF10 dilution range.

From FIG. 1, it can be seen that TGF-. beta.1 induced the maximum mRNA expression of the myofibroblast marker a-SMA over the course of 72 hours at the 48 hour time point in 3T3 cells. Treatment with blocking peptide significantly attenuated gene induction at 48 hours at all concentrations tested. At the 72 hour time point, α -SMA mRNA expression remained elevated in the untreated control group. However, only peptide treatment at a concentration of 10. mu.g/mL was sufficient to significantly attenuate the upregulation of α -SMA mRNA, suggesting that peptide concentrations of 0.1-1. mu.g/mL might only inhibit the differentiation response of a. The cytotoxicity of the peptides was assessed to ensure that the observed effect did not indicate apoptosis of 3T3 cells. Indeed, cells maintain a consistent growth pattern in response to TGF- β 1 stimulation, and the results indicate that treatment with either concentration of peptide does not interfere with cell proliferation nor lead to cell death.

It can be seen from figure 2 that similar results were observed when evaluating the potential of HFL1 cell blocking peptides to attenuate α -SMA expression. The upregulation of α -SMA mRNA expression by TGF-. beta.1 was significantly reduced by 0.1-10. mu.g/mL peptide at 48 hours, and only 10. mu.g/mL at 72 hours was sufficient to inhibit expression. Evaluation of peptide toxicity on HFL1 cells indicated that the blocking effect was not detrimental to cell numbers.

Further, we designed experiments to evaluate peptide inhibition of integrin α 4 β 1 signaling and profibrotic cell activity

We next attempted to investigate which events downstream of EDA-FN/integrin α 4 β 1 binding could be attenuated by our blocking peptides. We used Western blot analysis to detect the signal proteins ERK1/2 and FAK (FIG. 3), which are believed to be downstream of integrin beta 1 activation. Activation of extracellular mitogen-activated kinases 1 and 2(ERK1/2) was not affected by 24 hour peptide and TGF-. beta.1 stimulation, as may be explained by the Epidermal Growth Factor Receptor (EGFR) and TGF. beta.RI/II pathways, and may also be signaled by ERK 1. Phosphorylation of Focal Adhesion Kinase (FAK) protein showed significantly less activation, indicating inhibition of integrin/FAK signaling pathway by blocking peptides. At the same time, blocking peptide also slightly reduced the total protein level of FAK, indicating that its inhibitory effect on the FAK pathway was doubled; reduced activation and reduced total protein synthesis.

Matrix Metalloproteinase (MMP) gelatinase release and activity is associated with integrin α 4 β 1 and other fibronectin-dependent activations of β 1-containing integrins. We used gelatinase spectroscopy to assess gelatinase activity in conditioned media taken from peptide-treated cells (figure 4). The results indicate that both pro-MMP9 and pro-MMP2 gelatinase activities are significantly reduced, and both MMPs have activity levels that are inhibited to similar levels observed in resting fibroblasts. EDA-FN binding to integrins α 4 β 1 and α 4 β 7 was shown to be responsible for mechanical transduction and the production of the resulting collagen-rich ECM. Total collagen synthesis as measured by the hydroxyproline assay (figure 5) demonstrated a reduction in total collagen synthesis to approximately 60% that observed in cells treated with TGF- β 1 alone, similar to collagen production by resting fibroblasts. The immunocytochemistry used for α -SMA stress fiber formation confirmed the results shown by mRNA analysis (fig. 6 top). When TGF-. beta.1 treated 3T3 cells were also treated with 10. mu.g/mL blocking peptide, protein expression of a-SMA was inhibited, indicating the absence of a myofibroblast phenotype. Activated fibroblasts reassemble their cytoskeleton to form long F-actin fibers, extending the length of the cell. Therefore, we tried to assess whether this process was prevented in peptide-treated cells (figure 6 bottom). This indicates that although myofibroblast maturation may be attenuated, fibroblasts still progress to the activated state. Usually associated with a synthetic fibroblast phenotype (or myofibroblast).

Subsequent analysis of the expression of genes associated with the profibrotic myofibroblast phenotype and indicative of fibrogenesis (FIG. 7) revealed upregulation of mRNA expression by TGF- β 1 on collagen type I and III, fibronectin and EDA-FN splice variants. When blocking peptides were also incubated with fibroblasts, all were inhibited. Genes indicating integrin α 4 β 1 activation downstream of TGF- β 1 stimulation were also attenuated by peptide treatment (fig. 8). These include mRNA expression of MMP9, TGF- β 1, latent TGF- β binding protein 1(LTBP1), TGF- β 1-induced transcript 1(HIC5), and cardioxin-related transcription factor a (mrtfa), which provides strong evidence that peptides have successfully blocked the interaction between EDA-FN and integrin α 4 β 1, preventing subsequent upregulation of these integrin activation-dependent genes.

Overall, the data indicate that blocking peptides interfere with EDA-FN/integrin binding and downstream gene expression induction, attenuate FAK-dependent signaling, reduce production and activation of MMP2 and MMP9, and prevent type I and type III collagen enrichment. An ECM; while bringing the fibroblasts into an activated state, but not allowing the mature myofibroblast phenotype to progress.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (2)

1. An application of small blocking peptide in synthesizing the medicines for treating fibrotic diseases is disclosed.

2. A method for designing and screening small blocking peptides is characterized by comprising the following steps:

step 1, generating an integrin alpha 4 beta 1 protein model;

step 2, generating a plurality of blocking peptide models by using a molecular docking means and screening;

and 3, carrying out cell culture on the screened blocking peptide model.

Images