CN116003530B

CN116003530B - D-protein inhibitor for interleukin-6 and application thereof

Info

Publication number: CN116003530B
Application number: CN202211684805.3A
Authority: CN
Inventors: 朱雁磊; 孙科; 王通越; 刘磊; 卢培龙
Original assignee: Tsinghua University; Westlake University
Current assignee: Tsinghua University; Westlake University
Priority date: 2022-12-27
Filing date: 2022-12-27
Publication date: 2023-12-15
Anticipated expiration: 2042-12-27
Also published as: CN116003530A

Abstract

The application relates to a D-protein inhibitor aiming at interleukin-6 and application thereof. The D-protein inhibitor illustratively has the amino acid sequence shown in SEQ ID No. 1, with the constituent amino acids thereof all being in the D configuration except glycine. The D-protein inhibitor is not easy to hydrolyze by protease in vivo, has better thermal stability, simultaneously shows better inhibiting activity on interleukin-6, and can be developed into a therapeutic drug for related diseases mediated by interleukin-6.

Description

D-protein inhibitor for interleukin-6 and application thereof

Technical Field

The application belongs to the field of biochemistry, and particularly relates to a D-protein inhibitor aiming at interleukin-6 and application thereof.

Background

Compared with small molecular medicines, the biological macromolecular medicines such as polypeptide, antibody and the like have better bioactivity and target protein specificity, and especially when the target protein has large and flat hydrophobic surface, the small molecules are difficult to prepare medicines and can only be inhibited by the protein medicines. The polypeptide and protein medicine is composed of L-amino acid, and has various limitations, such as easy hydrolysis by protease, easy immune reaction, etc. There is a need for a protein drug that is resistant to hydrolysis and does not elicit an immune response to fill the gap of L-protein drugs.

The D-protein medicine is a protein which is natural and chiral and symmetrical with the L-protein medicine, has the characteristics of difficult immune reaction, incapability of being degraded by protease in vivo, easy chemical modification and the like, is focused in recent years, and a plurality of D-protein medicines are marketed in clinical trials at present.

Since interleukin-6 was first discovered in 1973, recent 50 years of research have shown that interleukin-6 is involved in a variety of biological events, such as immunomodulation, tumor growth, hematopoiesis, emergency response, and the like. Disruption of the hexamer formation of interleukin-6 with interleukin-6 receptor (IL-6 receptor) and gp130 is critical to inhibiting interleukin-6 signaling. However, the binding interface of such hexamers is too complex and extensive, making it very challenging to inhibit their formation with small molecules. Interleukin-6 has three sites involved in the formation of hexamer complexes, site I, site IIa and site IIIa, respectively. Wherein site I is the site where interleukin-6 binds to interleukin-6 receptor, which consists of two alpha-helices A and C, site IIa is the junction surface consisting of two alpha-helices A and C of interleukin-6, responsible for binding to gp130, site IIIa is the plane of the top of the alpha-helix bundle of interleukin-6, binding to gp 130.

Siltuximab (currently the only FDA approved interleukin-6 mab in the united states of america) and both monoclonal antibodies, sirukumab and Clazakizumab, which are still in clinical trials, bind to site I of interleukin-6, and such monoclonal antibodies can effectively inhibit downstream signaling by inhibiting interleukin-6 binding to its receptor and thus blocking its binding to the membrane protein gp 130. However, three signaling pathways are currently known for interleukin-6, the classical signaling pathway (trans-signaling) and trans-presentation (trans-presentation), respectively. Since transfer presentation is where one cell binds interleukin-6 with interleukin-6 receptor while being presented to bind gp130 on an adjacent cell membrane, this pathway is not interfered with by interleukin-6 site I binding antibodies. These three signaling pathways can be inhibited simultaneously for interleukin-6 site IIIa (Olokizumab), or for interleukin-6 receptor (Tocilizumab, sarilumab and Vobarilizumab.) or for gp130 (Olamkizept). By 2022, antibodies that bind to interleukin-6 site IIa have not been reported.

Since interleukin-6 is involved in numerous immune responses, each drug directed against inhibiting interleukin-6 signaling pathway has one or more corresponding therapeutic conditions. For example, the earliest interleukin-6 receptor inhibiting drug, tocilizumab, has passed clinical trials as a symptomatic: rheumatoid arthritis, juvenile idiopathic arthritis, multicenter Castleman's disease, giant cell arteritis, cytokine release syndrome and high-whisker arteritis, adult stills syndrome, graves' eye disease, recurrent polychondritis and ankylosing spondylitis are in clinical stage two to three.

Among them, the monoclonal antibody Siltuximab bound to interleukin-6 has passed the FDA certification, and is a multicenter postleman disease, which is being developed for the treatment of multiple myeloma and amyloid light chain amyloidosis in clinical secondary trials, and in addition, in clinical primary to secondary trials for solid tumors, prostate cancer, metastatic renal cell carcinoma and metastatic renal cancer. Sirukumab, olokizumab and Clazakizumab are in the third, third and second phases of the rheumatoid arthritis clinical trial, respectively. Monoclonal antibodies to gp130, olamkicept, are currently in clinical second-phase trials for symptomatic inflammatory bowel disease.

Disclosure of Invention

The technical object of the present application is to provide a D-protein inhibitor against interleukin-6 (IL-6).

It is another technical object of the present application to provide a pharmaceutical composition comprising the D-protein inhibitor of the present application.

Another technical object of the present application is to provide the use of said D-protein inhibitors in the preparation of a medicament.

In one aspect, the present application provides a D-protein inhibitor against interleukin-6, the amino acid sequence of which is selected from the following cases (1) to (4):

(1) An amino acid sequence shown in SEQ ID No. 1;

(2) An amino acid sequence with the same function, wherein any 1-10 amino acids in amino acid sequences shown in SEQ ID No. 1 are replaced by the same kind of amino acids respectively, and the amino acid sequences are selected from 35, 36, 38, 39, 41, 42, 43, 45, 46, 47, 49, 50, 51, 53, 56, 59, 60, 63, 64 and 65;

(3) An amino acid sequence having the same function, which is obtained by substituting any one or more amino acids other than amino acids at positions 35, 36, 38, 39, 41, 42, 43, 45, 46, 47, 49, 50, 51, 53, 56, 59, 60, 63, 64, 65 in the amino acid sequence shown in SEQ ID No. 1 with the same kind of amino acids; and

(4) The amino acid sequence shown in SEQ ID No. 1 has the same function as the amino acid sequence obtained by substitution in any combination of the substitution in the above case (2) and the substitution in the above case (3),

wherein the amino acids of the same class are classified into the same class by amino acid side chain groups, and

the same function means that the amino acid sequence obtained by substitution has the function of binding with interleukin-6 as the unsubstituted amino acid sequence.

In the amino acid sequence shown in SEQ ID No. 1, the 35, 36, 38, 39, 41, 42, 43, 45, 46, 47, 49, 50, 51, 53, 56, 59, 60, 63, 64, 65 amino acids are positioned on the binding surface of the D-protein inhibitor and the interleukin-6.

In some embodiments, the amino acid sequence of the D-protein inhibitor against interleukin-6 of the present application is the amino acid sequence having the same function, which is obtained by substituting 1 to 5, such as 1,2,3,4,5 amino acids in amino acids 1 to 5, such as 1,2,3,4,5 amino acids, of amino acids 1, 36, 38, 39, 41, 42, 43, 45, 46, 47, 49, 50, 51, 53, 56, 59, 60, 63, 64, 65 of the amino acid sequence shown in SEQ ID No. 1.

In a specific embodiment, the amino acid sequence of the D-protein inhibitor is SEQ ID No. 1.

In specific embodiments, the secondary structure of the D-protein inhibitor is as follows: the novel double-helix composite material consists of 4 alpha-helices, wherein the first alpha-helix sequence is SEQ ID No. 2, the second alpha-helix sequence is SEQ ID No. 3, the third alpha-helix sequence is SEQ ID No. 4, and the fourth alpha-helix sequence is SEQ ID No. 5.

In a specific embodiment, the secondary structural sequence of the D-protein inhibitor is LHHHHHHHHHHHHLLLHHHHHHHHHHHHHHHHHLLLHHHHHHHHHH HHHHHLLHHHHHHHHHHHL.

In another aspect, the application provides a pharmaceutical composition comprising a therapeutically effective amount of the above-described D-protein inhibitor and a pharmaceutically acceptable carrier.

In another aspect, the present application provides the use of the above D-protein inhibitor and the above pharmaceutical composition for the preparation of a medicament for the treatment of a disease.

In particular embodiments, the disease is an interleukin-6 mediated related disease.

In specific embodiments, the disease is selected from the group consisting of rheumatoid arthritis, juvenile idiopathic arthritis, multicenter Castleman's disease, giant cell arteritis, cytokine release syndrome, high whisker arteritis, adult stills syndrome, graves' eye disease, recurrent polychondritis, ankylosing spondylitis, and novel coronavirus infection.

In yet another aspect, the present application provides the use of the above-described D-protein inhibitor and the above-described pharmaceutical composition for the preparation of an interleukin-6 in vivo tracer.

Advantageous effects

1: the D-protein inhibitor solves the problem that L-protein is easy to hydrolyze by in vivo protease.

2 the D-protein inhibitor has better thermal stability, and is beneficial to the transportation and the preservation of medicines.

3. The D-protein inhibitor provided by the application has good inhibiting activity on interleukin-6 and has the potential of developing therapeutic drugs for interleukin-6 immune related diseases.

Drawings

Fig. 1: the chemical synthesis of D-25367, A is RP-HPLC elution curve and B is D-25367 mass spectrum, which shows that the measured molecular weight is 7329.67 and the calculated molecular weight is 7330.48.

Fig. 2: d-25367 thermal temperature change CD spectrogram.

Fig. 3: d-25367 and interleukin-6 on molecular sieve Superdex 200Increase 10/300column co-migration curve (A), and SDS-PAGE gel (B), in the figure, on: d-25367 alone; in (a): interleukin-6 alone; the following steps: d-25367 and interleukin-6 in a 1.1:1 molar ratio.

Fig. 4: the binding constants of D-25367 and interleukin-6 were determined by using a biofilm interference experiment.

Fig. 5: the effect of the point mutation (A) designed at the binding interface of D-25367 and the point mutation (B) designed at the binding interface of interleukin-6 on the interaction between proteins was studied by using a biological membrane interference experiment.

Fig. 6: the effect of D-25367 on interleukin-6 mediated downstream signaling pathway expression was verified using HEK-293T cells. The column height of each histogram in the graph is the average of every three sets of experiments, and the variance of every three sets of data is shown with thin lines protruding from the column height.

Fig. 7: SDS-PAGE gel of L-25367 and D-25367.

Detailed Description

Terminology

In the present application, the term "D-protein inhibitor" means that all amino acids constituting the protein are in the D configuration except glycine.

In the present application, in the secondary structural sequence, the letter "L" represents an irregular region, and the letter "H" represents an α -helix.

In the present application, the term "pharmaceutically acceptable carrier" refers to any kind of solid, semi-solid or inert fluid excipient, filler, encapsulating or formulation auxiliary material known to the person skilled in the art.

In the present application, the term "therapeutically effective amount" means an amount of the D-protein inhibitor of the present application contained in a pharmaceutical composition sufficient to achieve the intended purpose.

The present application is described in detail below by way of specific examples to better understand the present application by those skilled in the art, however, these examples are not intended to limit the scope of the present application.

Preparation example 1

The preparation of the interleukin-6-directed D-protein inhibitor (hereinafter referred to as D-25367) of the present application is described in detail by means of specific examples.

D-25367 was prepared by standard Fmoc solid phase peptide synthesis (Fmoc SPPS) and synthesized automatically by a Liberty blue microwave polypeptide synthesizer (CEM Corporation). The amino acid derivatives used for the experiments were purchased from Jiangsu Shen Lang Biotechnology Co., ltd (south China), N-Dimethylformamide (DMF), triisopropylsilane (TIPS), trifluoroacetic acid (TFA) and thioanisole were purchased from J & K Scientific Ltd. (Beijing.). N, N-Diisopropylcarbodiimide (DIC) and ethyl 2-oxime cyanoacetate (Oxyma) were purchased from Shanghai Tech Co., ltd., 1, 2-Ethanedithiol (EDT) was purchased from TCI (Shangghai, china) Development Co., ltd., diethyl ether was purchased from modern Development Co., ltd., ethyl ether was purchased from Oriental Chemicals, and acetonitrile was purchased from Mallinckrodt Baker, inc..

First, rink Amide AM resin was freed from Fmoc (9-fluorenylmethoxycarbonyl) protecting group with DMF solution containing 10% piperidine and 0.1M Oxyma at 90℃for 1 min. Then, the resin was washed 3 times with DMF. The resin (0.25 mmol), 4-fold equivalents of Fmoc protected amino acid (0.2 mM,5ml in DMF), 4-fold equivalents of Oxyma (1 mM,1ml in DMF), and 4-fold equivalents of DIC (0.5 mM,2ml in DMF) were mixed and coupled under microwave heating at 90℃for 2 minutes. At the end of the procedure, the peptide was cleaved from the resin with cleavage liquid (TFA/TIPS/thioanisole/water/EDT in a volume ratio of 82.5:5:5:2.5, vol/vol/vol/vol) for 3 hours. The solution was then concentrated under nitrogen stirring, precipitated with cold diethyl ether, then centrifuged and the supernatant was decanted, and repeated 3 times. The resulting precipitate was a crude peptide, which was then further purified by HPLC (high Performance liquid chromatography) to give D-25367 (amino acid sequence: GEEEVKEYLTRKFKDDPELVRLLREAIEVLLKAGEDPELVLSLIESLIHITGDPRAAVKLAKEFG (SEQ ID No: 1)). Reverse phase HPLC was performed on Shimadzu ProminenceHPLC. The a pump mobile phase was acetonitrile (0.1% tfa) and the B pump mobile phase was deionized water (0.1% tfa). The crude polypeptide was dissolved in water containing 50% acetonitrile and 0.1% TFA, filtered through a 0.22 μm filter, and the purified solution was lyophilized to give a pure polypeptide powder designated as D-25367. HPLC and mass spectrometry detection results of D-25367 are shown in FIG. 1.

Test example 1: structural identification of D-25367

The secondary structure of D-25367 was measured by a circular dichroism spectrometer (Chirascan V100/applied photophysics). The CD spectrum tests three CD curves at 25 deg.C, at 95 deg.C and at 25 deg.C back down, at 2 deg.C/min, respectively, in the interval of 260 to 180 nm. The D-25367 test concentration was 0.2mg/ml dissolved in PBS buffer (pH 7.4).

The results are shown in FIG. 2. The results show that negative peaks at 208 and 222 nm are exhibited at 25 ℃,95 ℃ and back to 25 ℃, typical α -helical secondary structure. Through further analysis, D-25367 consists of 4 alpha-helices, the first alpha-helix sequence is SEQ ID No. 2 (EEEVKEYLTRKF), the second alpha-helix sequence is SEQ ID No. 3 (PELVRLLREAIEVLLKA), the third alpha-helix sequence is SEQ ID No. 4 (PELVLSLIESLIHIT), and the fourth alpha-helix sequence is SEQ ID No. 5 (PRAAVKLAKEF), so that its secondary structural sequence is LHHHHHHHHHHHHLLLHHHHHHHHHHHHHHHHHLLLHHHHHHHHHHHHHHHLLHHHHHHHHHHHL.

The experimental result also proves that the D-25367 has good thermal stability.

Test example 2: binding assays for D-25367 and Interleukin-6

D-25367 and interleukin binding analysis respectively use analytical molecular sieve chromatographic column co-migration experiment and biological membrane interference technology.

Analytical molecular sieve chromatographic column method

The D-25367 and interleukin-6 alone or in a 1.1:1 mixture were passed through a column (Superdex 200 increasing 10/300 column) with the same amount of substance, and then eluted samples were subjected to SDS-PAGE running to verify that D-25367 co-migrates with interleukin-6 as a target protein.

The results are shown in FIG. 3. As can be seen from FIG. 3, when interleukin-6 is mixed with D-25367, the peak position of the complex is higher than the peak position of each monomer, which means that the complex has a larger volume. And from the gel map can correspond to complexes where the peaks at the complexes are both.

Biological film interference technique (Octet RED96 e/ForteBio)

Biofilm interference techniques use an Octet instrument, using SA (streptavidin) probes that specifically bind biotin to bind biotinylated L-or D-interleukin-6. For D-interleukin-6, a biotin molecule is attached to the amino terminus of the protein during synthesis. Whereas the biotinylation of L-interleukin-6 is based on an Avi tag at the amino terminus, biotin is linked to L-interleukin-6 by biotin ligase.

The experimental procedure is as follows:

(1) Baseline 1: the SA probe was immersed in a phosphate buffer solution containing two parts per million of Tween-20 at pH7.4 (hereinafter abbreviated as PBST) for 60 seconds,

(2) Curing: biotinylated interleukin-6 was diluted in PBST to a final concentration of 10. Mu.g/ml, and SA probe was immersed in this solution for 120 seconds;

(3) Closing: (blocking solution 10. Mu.g/ml of biotin in PBST), immersing the SA probe in the blocking solution for 60 seconds,

(4) Baseline 2: immersed in the PBST for 60 seconds,

(5) Combining: immersed in a solution of D-25367 diluted in PBST at different concentrations of 250, 125, 62.5, 31.1, 15.6nM for 180 seconds (duration of single point mutation, fig. 5), or 300 seconds (duration of binding constant measurement, fig. 4),

(6) Dissociation: immersed in PBST for 180 seconds,

(7) The binding constant was measured to be 28.3.+ -. 0.3nM (see FIG. 4).

In addition, point mutations were designed at the binding interface of D-25367 or interleukin-6, respectively, to verify the effect of these point mutations on protein interaction performance.

The results are shown in FIG. 5. From FIG. 5 it can be seen that the binding interface of D-25367 with interleukin-6 is consistent with the design.

Test example 3: d-25367 cell Activity experiment

Interleukin-6 mediated JAK-STAT signaling pathway has been demonstrated to be useful in measuring human placental secreted alkaline phosphatase (SEAP) expression to quantify signaling pathways.

In this experiment, a plasmid containing PhCMV-h interleukin-6R-pA (40 ng), phCMV-hSTAT3-pA (40 ng) and PhSTAT3-SEAP-pA (20 ng) was transfected into 1X 10 ⁴ SEAP was expressed in HEK-293T cells to sense extracellular interleukin-6. Transfected cells were incubated with varying concentrations of D-25367 and human interleukin-6 in Dulbecco's Modified Eagle Medium (DMEM) medium containing 10% Fetal Bovine Serum (FBS) at 37℃in an incubator with 5% carbon dioxide for 48 hours. The cell culture supernatant was then heat-inactivated (65 ℃ C., 30 min) and 80. Mu.l of the supernatant was combined with 120. Mu.l of a substrate solution [ 100. Mu.l of a double concentration SEAP detection buffer containing 20mmol of homoarginine, 1mmol of magnesium chloride, 21% (v/v) diethanolamine pH 9.8 and 20. Mu.l of a substrate solution containing 120mmol of p-nitrophenylphosphoric acid]Mixing. Interleukin-6 signaling was assessed by measuring SEAP production in the cell culture medium. Absorbance in enzyme activity units/L was recorded at 405nmol (37 ℃) using a Synergy H1 hybrid multimode microplate reader (BioTek instruments).

The results are shown in FIG. 6, from which it can be seen that D-25367 can significantly inhibit interleukin-6 mediated signaling pathway.

Test example 4: hydrolysis experiments of D-25367 protease

In the proteolytic experiments, the protein concentration was 0.2mg/ml, incubated at 37℃in trypsin and pepsin solutions, respectively, while the chiral mirror polypeptide L-25367 of D-25367 (identical in amino acid sequence but composed of L-amino acids) was used as a control. In the experiments, the final concentrations of trypsin and pepsin were 2.2 and 0.22mg/ml, respectively, and samples were taken at 6 and 20 hours of incubation, and verified by SDS-PAGE running.

The results are shown in FIG. 7. As can be seen from FIG. 7, the D-25367 of the present application is not easily hydrolyzed by protease, showing good in vivo stability.

Claims

1. A D-protein inhibitor for interleukin-6 has the amino acid sequence of SEQ ID No. 1.

2. The D-protein inhibitor according to claim 1, wherein the D-protein inhibitor consists of 4 a-helices, the first a-helix sequence being SEQ ID No. 2, the second a-helix sequence being SEQ ID No. 3, the third a-helix sequence being SEQ ID No. 4, the fourth a-helix sequence being SEQ ID No. 5.

3. A pharmaceutical composition comprising a therapeutically effective amount of the D-protein inhibitor of claim 1 or 2 and a pharmaceutically acceptable carrier.

4. Use of a D-protein inhibitor according to claim 1 or 2 or a pharmaceutical composition according to claim 3 in the manufacture of a medicament for the treatment of a disease, wherein the disease is an interleukin-6 mediated related disease and the disease is selected from the group consisting of rheumatoid arthritis, juvenile idiopathic arthritis, multicenter Castleman's disease, giant cell arteritis, cytokine release syndrome, high-whisker arteritis, adult stills syndrome, graves' eye disease, recurrent polychondritis and ankylosing spondylitis.