WO2017157323A1

WO2017157323A1 - Lipopolysaccharide binding protein polypeptide and pharmaceutical use thereof

Info

Publication number: WO2017157323A1
Application number: PCT/CN2017/076969
Authority: WO
Inventors: 武艺; 谢展利; 阳艾珍
Original assignee: 苏州大学
Priority date: 2016-03-16
Filing date: 2017-03-16
Publication date: 2017-09-21
Also published as: CN107200783B; CN107200783A

Abstract

Provided are a lipopolysaccharide (LPS) binding protein polypeptide and the pharmaceutical use thereof, wherein the amino acid sequence of the lipopolysaccharide binding protein polypeptide is DHGHKHKHGHGHGKH, and the lipopolysaccharide binding protein polypeptide is derived from a high molecular weight kininogen (HK) protein in human plasma itself. The lipopolysaccharide binding protein polypeptide is injected into a mouse through intraperitoneal injection, and can reduce the mortality of endotoxemia caused by LPS in mice and inhibit the production of inflammatory factors.

Description

Title: A Lipopolysaccharide Binding Protein Polypeptide and Its Pharmaceutical Use

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a 15 amino acid lipopolysaccharide binding protein polypeptide and use thereof for preparing a medicament for preventing and/or treating sepsis caused by Gram-negative bacteria. .

Background technique

[0002] Sepsis is an acute systemic infection caused by pathogenic bacteria or conditional pathogens invading the blood circulation, producing toxins and other metabolites, clinically with chills, fever, rash, joint pain and hepatosplenomegaly. To be characterized, some may have septic shock and migratory lesions. There are two reasons for causing sepsis: First, human factors: When the skin and mucous membranes are damaged or purulent inflammation, bacteria are easy to invade the body; the human immune response can be divided into non-specific immune response and specific immune response, It can be divided into two aspects: cellular immunity and humoral immunity. When the body's immune function declines, it can not fully exert its phagocytosis and kill bacteria, which will cause sepsis; the iatrogenic infection caused by conditional pathogens is gradually increasing. . Second, bacterial factors: mainly related to the virulence and quantity of pathogenic bacteria. Pathogenic bacteria with strong virulence or a large number enter the body, which is more likely to cause sepsis.

[0003] Gram-negative bacterial infection is an important cause of sepsis, and systemic inflammation and microcirculatory disorders induced by lipopolysaccharide (LPS), a cell wall component, play a key role in multiple organ dysfunction and septic shock in sepsis. . Lipopolysaccharide (LPS) is one of the components of the cell wall of Gram-negative bacilli. It is usually located at the outermost layer of the cell wall and has a thickness of about 8-10 nm. It has a strong anti-inflammatory effect.

[0004] Although the current treatment of sepsis has taken comprehensive measures such as anti-infection, immune regulation and supportive treatment, the morbidity and mortality of sepsis remain high. The reason is mainly because the understanding of the pathogenesis of sepsis is far from enough. There is no free form of LPS in the blood. Organisms are known to have LPS clearance mechanisms, including LBP, sCD14, CTBP, and other lipoproteins, which bind to LPS and present them to the liver's three cells (including Kupffer cells, hepatocytes, and stellate cells). The intracellular degradation is discharged into the bile and detoxified. However, in the event of sepsis, the concentration of LPS in the blood remains high, indicating that LPS escapes the clearance mechanism by virtue of binding to the host protein, but so far, it is still unclear whether LPS is hijacked. Which host proteins.

technical problem

Problem solution

Technical solution

[0005] In view of the above situation, the present invention finds for the first time that: plasma high molecular weight kininogen (HK) can maintain its high affinity binding to LPS through its interaction with sugar chains in LPS, and can be used as A novel lipopolysaccharide binding protein. The present invention further found that a highly conserved amino acid sequence in HK (DHGHKHKHGHGHGKH) is a site for which specific binding to LPS occurs. The peptide sequence is synthesized and injected into mice by intraperitoneal injection, which can significantly improve the mortality of endotoxemia caused by LPS and inhibit the production of inflammatory factors.

In one aspect, the invention provides a lipopolysaccharide binding protein polypeptide designated DHG15, the amino acid sequence of which is set forth in SEQ ID NO: 1.

In another aspect, the present invention provides a pharmaceutical composition comprising the above-described lipopolysaccharide-binding protein polypeptide and one or more pharmaceutically acceptable excipients, the pharmaceutically acceptable excipients including (but not Limited to) p H regulators, osmotic pressure regulators, solubilizers, solubilizers, emulsifiers, stabilizers, preservatives, excipients, fillers, disintegrants, binders, flavoring agents, mediators, and Lubricant. The pharmaceutical composition can be administered to a patient by various methods such as injection administration, nasal administration, pulmonary administration, transdermal administration, oral administration, and the like.

In a final aspect, the present invention provides the use of the above lipopolysaccharide-binding protein polypeptide for the preparation of a medicament for preventing and/or treating sepsis caused by Gram-negative bacilli. Among the above drugs, the lipopolysaccharide-binding protein polypeptide of the present invention can be used as a single pharmaceutically active substance or in combination with other anti-sepsis drugs. The anti-sepsis drug includes, but is not limited to, a cephalosporin antibiotic and a quinolone antibiotic; wherein: the cephalosporin antibiotic is selected from any one of cefotaxime, ceftriaxone, ceftazidime, cefoperazone; the quinolone The antibiotic is selected from any one of the group consisting of ofloxacin, ciprofloxacin, and fleroxacin.

Advantageous effects of the invention

Beneficial effect Compared with the prior art, the present invention adopting the above technical solution has the following beneficial effects:

[0010] (1) The lipopolysaccharide-binding protein polypeptide DHG15 of the present invention has good solubility, and the sequence is derived from the human body's own HK protein, and is non-cytotoxic;

[0011] (2) After intraperitoneal injection of DHG15, the mortality of endotoxemia caused by LPS in mice can be reduced

And inhibiting the production of inflammatory factors;

[0012] (3) DHG15 binds to the polysaccharide chain of LPS, blocking the binding of LPS to HK protein (to dissociate or release it); at the same time, the lipid A portion of LPS is still exposed, LBP sCD14, CTBP and other lipoproteins can still be combined with it and presented to the liver for detoxification, thus providing dual protection.

Brief description of the drawing

DRAWINGS

1 is a diagram showing the results of Western Blotting of heavy chain (HC) and light chain (LC) protein fragments in plasma HK protein.

2 is a graph showing experimental results of binding of plasma HK protein to LPS.

[0015] FIG. 3 is a graph showing the affinity results of the binding of HK and LC to LPS.

4 is a diagram showing the results of Western Blotting identification of D4, D5 and D6 protein fragments in LC and experimental results in combination with LPS.

Figure 5 is a graph showing the distribution of eight polypeptide fragments on D5 and the results of experiments with LPS.

Figure 6 is a graph showing the results of comparison between the DHG15 polypeptide and the mutant polypeptide in inhibiting the binding of the HK light chain to the LPS.

7 is a graph showing experimental results of studying the binding of LPS to plasma HK protein.

Figure 8 is a graph showing the results of DHG15 polypeptide improving mortality in mice caused by LPS.

[0021] Figure 9 is a graphical representation of the effect of DHG15 monomer on the levels of major inflammatory factors in plasma.

Embodiments of the invention

[0022] The present invention will be further described below in conjunction with the drawings and specific embodiments. It should be noted that the following examples are only for explaining the technical solutions of the present invention, and are not intended to limit the scope of protection thereof. In addition, unless otherwise stated, the various reagents, drugs, and instruments used in the following examples are commercially available. Example 1: Screening of the functional polypeptide DHG15 from plasma HK protein and detecting its binding properties.

[0024] There are six domains in the HK protein in plasma, wherein the first to third domains (D1~D3) constitute the heavy chain of HK (Heavy)

Chain, HC), the fourth to sixth domains (D4~D6) constitute the light chain (LC) of HK. The heavy and light chain proteins of HK were prepared using the Bac-to-Bac insect cell expression system, and the purified HC (55KD) and LC (35KD) proteins were identified by Wester n blotting. The results are shown in Figure 1.

[0025] In order to study the binding sites of HK protein and LPS, respectively, 200 ng

HK:, HC, LC protein was incubated with LPS or PBS (4 ° C, 30 min). Draw 30

Mix the Polymyxin-B (Sigma) into a 1.5 ml centrifuge tube, centrifuge to remove the supernatant, and wash twice with 1 ml PBS (7000 rpm, 2 min). Various protein samples were incubated with the treated Polymyxin-B (4 ° C, 5 min), the supernatant was centrifuged, and washed twice with PBS. The Polymyxin-B precipitate was resuspended in 60 μΐ protein loading buffer, boiled at 100 ° C for 10 min, and 10 μΐ was taken for Western blotting, and HK antibody (Baiqi Bio) was incubated.

As shown in FIG. 2, HK and LC were able to bind to LPS and thus coprecipitated by polymyxin-B microbeads, suggesting that H K may bind to LPS via LC.

[0027] To further verify the above conclusions, the affinity of HK and LC combined with LPS was examined using a Biacore analysis method. HK samples were diluted with a running buffer of pH = 7.4 and diluted to a concentration of 0 nM, 6.4 nM, 19.3 nM, 57.7 nM, 173 nM, 520 nM. The HK protein was coupled to the HPA chip according to the protocol of Biacore XI 00 control soft with a coupling level of 3000 RU. Set the injection time to 180 s, the dissociation time to 500 s, the regenerant to 0.1 M HC1, and the regeneration time to 120 s. Perform the test on the Biacore X100 control soft protocol.

[0028] As shown in FIG. 3, there is a physical direct combination of HK, LC and LPS, and the Kd values are 1.713*10 - ⁷ M and 1.524*10 -9 M, respectively. The above results indicate that LC binds to LPS more strongly than HK, further suggesting that LC contains a site where HK binds directly to LPS.

[0029] To further investigate which domain in the LC binds to LPS, the fourth domain (D4) and the fifth domain in LC were prepared using the Bac-to-Ba c insect cell expression system ( D5) and sixth domain (D6) proteins were identified by Western blotting. After incubation with D4, D5 or D6 protein using polymyxin-B microbeads, the binding to LPS was detected. Only D5 can be shared by polymyxin-B. Precipitating, suggesting that D5 is the binding site of HK and LPS, the results are shown in Figure 4.

[0030] Subsequently, eight polypeptide fragments on D5 (Nanjing Kingsray Biotech Co., Ltd.) were synthesized, each containing 30 amino acids. Eight polypeptide fragments were incubated with LPS and then interacted with LC, and then the binding assay was used to detect binding. Only peptide 6 and peptide 7 inhibited the binding of LPS to LC. The results are shown in Fig. 5. Comparing the amino acid sequences of polypeptide fragments 6 and 7, it is known that the consensus sequence 'J is DHGHKHKHGHGHGKH (designated as DHG15).

According to reports in the literature, the histidine-rich polypeptide can neutralize the host inflammatory response caused by LPS, while the DHG15 polypeptide of the present invention contains a large amount of histidine, so it is speculated that histidine is a combination of HK and LPS. The main amino acid. Subsequently, the histidine (H) in the DHG15 polypeptide is mutated to the same positively charged lysine.

(K), which is DKGKKKKKGKGKGKK:. The DHG15 polypeptide and the mutant polypeptide were pre-incubated with LPS, and then incubated with the above mixture using polymyxin-B microbeads, and the binding to LPS was detected by Western blotting.

As shown in FIG. 6, the DHG15 polypeptide can inhibit the binding of the HK light chain to LPS, and after the histidine (H) is mutated to lysine (K), the mutant polypeptide cannot inhibit the HK light chain and LPS. Combine. At the same time, the use of unrelated peptides from other fragments in D 5 as a negative control suggests that histidine is the key to the binding of HK to LPS.

Example 2: LPS is bound to HK via a sugar chain.

[0034] LPS consists mainly of a sugar chain and a lipid A. In order to detect which part of the structure LPS binds to HK, 2 lipid A, defatted LPS, LPS or BSA are added to each well of a 96-well plate. (containing 0.1 M Na ₂ C0 ₃ , pH = 9.6), coated at 4 ° C, overnight. After washing the wells with PBS, 1% gelatin was added and blocked at 37 ° C for 1 h. After washing the wells with PBS, FITC-labeled HK protein (100 nM) was added to each well and incubated at 37 ° C for 1 h. After the liquid was aspirated, the fluorescence intensity in each well was measured using a microplate reader (excitation light: 494 nm, emission: 518 nm).

[0035] As shown in FIG. 7, the binding ability of defatted LPS and HK is the strongest, and there is a significant difference compared with the binding ability of lipid A and HK, suggesting that the sugar chain on LPS is the main site of LPS binding with HK. point.

Example 3: Activity of DHG15 polypeptide.

[0037] To investigate whether DHG15 polypeptides can ameliorate mouse death by LPS, mice were randomized into two groups, administered by intraperitoneal injection, and a group of injections co-incubated with an unrelated polypeptide (200 g/mouse). LPS (control group), another group of LPS (treatment group) injected with DHG15 polypeptide (200 g/mouse), view The survival rate of the two groups of mice was examined. It was found that treatment with DHG15 polypeptide significantly improved the mortality caused by L PS in mice.

[0038] To investigate whether DHG15 affects the levels of major inflammatory factors in plasma, the plasma levels of major inflammatory factors were compared between control mice and DHG15 treated mice. As shown in Figure 9, the levels of TNF-a, IL-lb, and IL-6 in the plasma of DHG15-treated mice were significantly lower than those in the control group, indicating that DHG15 was significant in the inflammatory response caused by LPS. Inhibits the level of major inflammatory factors in plasma.

Industrial applicability

[0039] The above results indicate that the application of DHG15 polypeptide can block the binding of plasma HK protein to LPS, which is beneficial for preventing the onset of sepsis caused by Gram-negative bacilli, and is beneficial for alleviating the symptoms after onset, and taking preventive measures. And the dual effect of treatment.

Sequence table free content

[0040] <110> Suzhou University

<120> A lipopolysaccharide binding protein polypeptide and pharmaceutical use thereof

<004> 1

<170> Patentln version 3.3

<210> 1

<211> 15

<212> PRT

<213>Artificial sequence

[0048] <220>

<223> DHG15 polypeptide

<400> 1

[0051] Asp His Gly His Lys His Lys His Gly His Gly His Gly Lys His

1 5 10 15

Claims

Claim

A lipopolysaccharide binding protein polypeptide having an amino acid sequence as shown in SEQ ID NO: 1.

A pharmaceutical composition comprising the lipopolysaccharide binding protein polypeptide of claim 1 and one or more pharmaceutically acceptable excipients.

The pharmaceutical composition according to claim 2, characterized by:

The pharmaceutically acceptable excipients include pH adjusters, osmotic pressure regulators, solubilizers, solubilizers, emulsifiers, stabilizers, preservatives, excipients, fillers, disintegrants, binders, flavors Agents, mediators and lubricants.

The pharmaceutical composition according to claim 2, characterized by:

The pharmaceutical composition is administered to a patient by injection administration, nasal administration, pulmonary administration, transdermal administration or oral administration.

Use of the lipopolysaccharide binding protein polypeptide according to claim 1 for the preparation of a medicament for preventing and/or treating sepsis caused by Gram-negative bacilli.

The use according to claim 5, characterized in that:

In the medicament for preventing and/or treating sepsis caused by Gram-negative bacilli, the lipopolysaccharide-binding protein polypeptide according to claim 1 is as a single pharmacodynamically active substance.

The use according to claim 5, characterized in that:

In the medicament for preventing and/or treating sepsis caused by Gram-negative bacilli, the lipopolysaccharide-binding protein polypeptide according to claim 1 is used in combination with other anti-sepsis drugs.

The use according to claim 7, characterized in that:

The anti-sepsis drugs include cephalosporin antibiotics and quinolone antibiotics.

The use according to claim 8, characterized in that:

The cephalosporin antibiotic is selected from any one of cefotaxime, ceftriaxone, ceftazidime, and cefoperazone.

The use according to claim 8, characterized in that:

The quinolone antibiotic is selected from any one of the group consisting of ofloxacin, ciprofloxacin and fleroxacin.