阅读说明：本技术 一种治疗2型长qt综合征的药物组合物及其应用 (Pharmaceutical composition for treating type 2 long QT syndrome and application thereof ) 是由 廉姜芳 杨曦 郑泽群 黄晓燕 宋勇飞 袁园 于 2021-08-10 设计创作，主要内容包括：本发明属于医药领域,涉及一种治疗2型长QT综合征的药物组合物及其应用。本发明利用免疫印迹检测发现鲁玛卡托和HSF1A二者联合应用对hERG突变蛋白的成熟转运具有明显的促进作用,在转染hERG及其突变质粒的HEK-293细胞模型中应用鲁玛卡托、HSF1A,利用全细胞膜片钳检测hERG通道产生的I-(kr)电流,发现二者联合应用电流密度显著增加,且使突变通道的激活门控动力学向野生型转变,可见本发明利用鲁玛卡托和HSF1A二者联合通过上调关键的转运分子伴侣Hsp70和Hsp90来实现hERG突变蛋白的转运,通过促进hERG突变蛋白的成熟转运达到治疗2型长QT综合征的目的。(The invention belongs to the field of medicines, and relates to a pharmaceutical composition for treating type 2 long QT syndrome and application thereof. The invention discovers that the combined application of the Lumaka and the HSF1A has obvious promotion effect on the maturation transport of hERG mutant protein by utilizing the immunoblotting detection, applies the Lumaka and the HSF1A in an HEK-293 cell model for transfecting hERG and mutant plasmids thereof, and utilizes the whole-cell patch clamp to detect I generated by an hERG channel kr The current density is obviously increased by combining the two, and the activation gating dynamics of a mutation channel is changed to the wild type, so that the invention realizes the transport of the hERG mutant protein by combining the Lumakato and the HSF1A and up-regulating key transport molecular chaperones Hsp70 and Hsp90, and achieves the aim of treating the type 2 long QT syndrome by promoting the mature transport of the hERG mutant protein.)

1. A pharmaceutical composition for the treatment of long QT syndrome type 2, said composition comprising rummaca and HSF 1A.

2. The pharmaceutical composition for the treatment of long QT syndrome type 2 according to claim 1, characterized in that the molar ratio of lumacatto to HSF1A is (1.0-2.0): 1.

3. The pharmaceutical composition for treating type 2 long QT syndrome according to claim 1, wherein the pharmaceutical composition is one or more of an injectable formulation, an oral formulation or an external formulation.

4. The pharmaceutical composition for treating type 2 long QT syndrome according to claim 1, characterized in that the pharmaceutical composition is one or more of tablets, capsules, powders, pills, granules, injections or emulsions.

5. Use of a pharmaceutical composition according to claim 1 for the preparation of a medicament for the treatment of long QT syndrome type 2.

6. The use as claimed in claim 5 wherein type 2 long QT syndrome is hERG gene mutation leading to hERG transport disorders.

7. The use of claim 5, wherein the heat shock factors Hsp70 and Hsp90 are upregulated by Lumaca and HSF 1A.

8. Use according to claim 5, wherein HSF1 is down-regulated by Lumaca and HSF 1A.

Technical Field

The invention belongs to the field of medicines, and relates to a pharmaceutical composition for treating type 2 long QT syndrome and application thereof.

Background

Congenital long QT syndrome (LQTS) is a hereditary cardiac ion channel disease, caused by mutations in dominant heredity-related genes on 15 autosomes, and clinically divided into 17 subtypes, characterized by prolongation of QT interval and arrhythmia associated with mood, stress, etc. Type 2 Long QT syndrome (LQT2) is the most common subtype in China, and rapid activation of 3-phase repolarization of cardiac action potential caused by KCNH2 (also called hERG) mutation delays rectification potassium current I_krThe result is reduced.

Intracellular maturation of the hERG channel proteins requires the assistance of numerous molecular chaperones, particularly the heat shock proteins Hsp70 and Hsp90 in the cytoplasm, and associated stress proteins in the endoplasmic reticulum. Functional studies also indicate that membrane channel reduction caused by impairment of mature transport of intracellular proteins due to hERG mutations is a major mechanism in the pathogenesis of LQT 2. Due to the complex pathogenesis, the treatment of LQT2 remains a serious challenge. Although treatments such as the application of beta receptor antagonists and the implantation of cardioverter-defibrillators (ICDs) have a certain effect on preventing fatal arrhythmia in high-risk patients, none of these treatments can fundamentally correct the transport of hERG to produce a radical effect.

Lumakato is clinically proven to have a promoting effect on the transport of hERG mutant protein as a drug for treating cystic fibrosis, and the phenotype of LQT2 is corrected. However, the intracellular action process and action target of the medicine for promoting the hERG mutant protein transport are not characterized, so that the study on the action target of macadam in the cell aiming at the mature transport mechanism of the hERG has important significance in providing ideas for efficiently promoting the hERG protein maturation and new treatment strategies.

Disclosure of Invention

The invention aims to solve the problems and provides a pharmaceutical composition for treating type 2 long QT syndrome, which promotes the mature transport of hERG mutant protein, increases the expression level of mature hERG and corrects the electrophysiological properties of the mature hERG mutant protein.

The purpose of the invention can be realized by the following technical scheme: a pharmaceutical composition for the treatment of long QT syndrome type 2, said composition comprising rummaca and HSF 1A.

In the pharmaceutical composition for treating type 2 long QT syndrome, the molar ratio of the Rumaca to the HSF1A is (1.0-2.0): 1.

In the above pharmaceutical composition for treating type 2 long QT syndrome, the pharmaceutical composition is one or more of an injection preparation, an oral preparation or an external preparation.

In the pharmaceutical composition for treating type 2 long QT syndrome, the pharmaceutical composition is one or more of tablets, capsules, powders, pills, granules, injections or emulsions.

The invention also provides application of the pharmaceutical composition in preparing a medicament for treating type 2 long QT syndrome.

In the application of the pharmaceutical composition in preparing the medicine for treating the type 2 long QT syndrome, the type 2 long QT syndrome is hERG transport disorder caused by hERG gene mutation.

In the application of the pharmaceutical composition in preparing the medicine for treating the type 2 long QT syndrome, the heat shock factors Hsp70 and Hsp90 are up-regulated by the Lumaca and HSF 1A. Hsp70 and Hsp90 are heat shock proteins, have chaperone activities and promote misfolded and mature proteins, and a single dose of Lumakato can increase hERG protein maturation. HSF1A is an activator of Hsp70 and Hsp90, and a single administration can activate Hsp70 and Hsp 90. Compared with single medicine, the invention combines the Lumakato and the HSF1A to up-regulate key transport molecular chaperone Hsp70 and Hsp90 to realize the transport of hERG mutant protein, increase the expression level of mature hERG and correct the electrophysiological properties of the mature hERG, and the purpose of treating type 2 long QT syndrome is achieved by promoting the mature transport of the hERG mutant protein.

In the application of the pharmaceutical composition in preparing the medicine for treating type 2 long QT syndrome, HSF1 is down-regulated through Rumaca and HSF 1A. Promotion of hERG mutein transport is achieved by upregulation of the key transport chaperones Hsp70 and Hsp90, whose upregulation feedback inhibits HSF1 expression.

Compared with the prior art, the invention has the following beneficial effects:

the invention discovers that the combined application of the Lumaka and the HSF1A has obvious promotion effect on the maturation transport of hERG mutant protein by utilizing the immunoblotting detection, applies the Lumaka and the HSF1A in an HEK-293 cell model for transfecting hERG and mutant plasmids thereof, and utilizes the whole-cell patch clamp to detect I generated by an hERG channel_krThe current density is obviously increased by combining the two, and the activation gating dynamics of a mutation channel is changed to the wild type, so that the invention realizes the transport of the hERG mutant protein by combining the Lumakato and the HSF1A and up-regulating key transport molecular chaperones Hsp70 and Hsp90, and achieves the aim of treating the type 2 long QT syndrome by promoting the mature transport of the hERG mutant protein.

Drawings

FIG. 1 is a graph showing the expression results of Hsp70, Hsp90 and HSF 1.

FIG. 2 is a graph showing the results of SDS polyacrylamide gel electrophoresis of hERG, Hsp70, Hsp90, and β -tubulin.

FIG. 3 is a graph of the enhancement of the hERG channel current by Lumaca (LUM) and/or HSF 1A.

Detailed Description

The following are specific examples of the present invention and further describe the technical solutions of the present invention, but the present invention is not limited to these examples.

Example 1:

cell culture and construction of hERG transport disorder model: the pcDNA3-Mock, pcDNA3-HERG, pcDNA3-HERG/pcDNA3-HERG-A561V, pcDNA3-HERG-A561V plasmids were transfected with transient transfection into HEK293 cell lines producing control, wild, heterozygous and mutant hERG. The above cells were cultured in DMEM (high glucose) containing 10% fetal bovine serum and maintained in an environment of a 37 ℃ incubator containing 5% carbon dioxide. Cells were harvested from the culture dish, resuspended to single cells and cultured in fresh MEM dishes for at least 4 hours for use.

Quantitative RT-PCR: cells before and after drug treatment are cracked by using TransZol Up (all-gold) and total RNA is extracted, after reverse transcription is carried out, 1 mu g is used as a template, a 20 mu l reaction system is prepared according to a 2 XT 5 Fast qPCR Mix (engine biology) scheme to detect target gene expression, SYBR Green I is used as an expression signal, GAPDH is used as a housekeeping gene in the detection process, and related primers are shown in Table 1.

RNA extraction conditions:

1. collecting 1X 10⁶Adding 1ml of TransZol Up reagent to the cells to lyse the cells;

2. adding 200ul chloroform solution, shaking vigorously, and standing for 10 min;

3. centrifuging at 12000rpm for 20min, transferring the upper aqueous phase layer into a new centrifuge tube, adding 500ul isopropanol, mixing, and standing for 10 min;

4. centrifuging at 12000rpm for 10min, removing the upper layer, adding 1.5ml 75% ethanol, and blowing to suspend the precipitate;

5. centrifuging at 12000rpm for 5min, removing supernatant, and drying for 10 min;

6. 50ul DEPC water was added, dissolved on ice for 10min, RNA concentration was determined using the Nanodrop instrument, and all sample RNA concentrations were diluted to 500 ng/ul.

Reverse transcription reaction conditions:

preparing a system: RNA 2ul, oligo (dt) Primer (0.5ug/ul)1ul,2XES reaction MIX 10ul, easy script RT/RI enzyme MIX 1ul, gDNA remover 1ul, RNase free water 5 ul.

Reaction conditions are as follows: in a PCR apparatus (brand: eppendorf; model: master), the program: 15min at 42 ℃; 5s at 85 ℃; the sample was taken out, 180ul of DEPC water was added and mixed well, and stored in a refrigerator at-20 ℃ for later use.

Table 1: fluorescent quantitative PCR reaction conditions and system configuration:

reaction conditions are as follows: at 95 ℃ for 2 min; 95 ℃ for 10 s; 60 ℃, 15s (signal collected) (35 cycles); melting curve: signals are collected at 60-95 ℃ and 2 ℃/s.

Table 2: HSF1, Hsp70, Hsp90 and GAPDH primers

Drug and reagent preparation: lammaca (brand: Selleck cat # S1565) and HSF1A (brand: Selleck cat # S0294) were stored in 10mmol in DMSO, diluted to final concentrations of 12.5. mu. mol and 10. mu. mol, respectively, using DMSO, and an equal amount of DMSO was added to the unaddressed group for comparison. The five groups are respectively as follows:

wild type: hERG-WT; heterozygous group: hERG-WT + hERG-A561V; heterozygous garumakato treatment group: hERG-WT + hERG-A561V + LUM; heterozygous plus HSF1A treatment group: hERG-WT + hERG-A561V + HSF 1A; hybrid garumaki and HSF1A treatment groups: hERG-WT + hERG-A561V + LUM + HSF 1A.

Protein immunoblotting: after 24 hours incubation of the drug, different groups of proteins were extracted with RIPA lysate. Equal amounts of protein (20. mu.g) were separated on a 7.5% SDS polyacrylamide electrophoresis gel for the different proteins of interest. After membrane transfer and blocking, a primary antibody incubation strip of specific target protein is selected for overnight. Wherein the hERG antibody (brand: Alomone/Alomone Labs, cat # APC-062), the Hsp70 antibody (brand: Abcam, cat # ab2787), the Hsp90 antibody (brand: Abcam, cat # ab282108) and the beta-tubulin antibody (brand: transgene, cat # HC101-01) are diluted sequentially in a ratio of 1:400, a ratio of 1:1000, a ratio of 1: 10000. 1: 3000). Using corresponding secondary antibodiesGoat Anti-Rabbit IgG (H + L), HRP Conjugate andthe bands were visualized using ImageQuant LAS 500 after 1 hour incubation with Goat Anti-Mouse IgG (H + L), HRP Conjugate (brand: transgene Cat: HS101-01 and HS201-01) (1:3000), beta-tubulin as the normalized internal reference protein.

Patch clamp electrophysiological analysis: the cells were re-digested and separated into single cells after 24 hours of treatment with different cell models and plated in 35mm dishes for electrophysiological analysis after 5 hours. The whole-cell patch clamp system is used for leading Ikr current of an hERG channel, and specifically comprises the following steps: depolarized to-60 mV at-80 mV resting state, then depolarized to +60mV at a step current of 10mV for 3s, followed by 3s repolarization to-40 mV to draw the hERG tail current.

Wherein the electrode external liquid is: 4mM KCl, 137mM NaCl, 1.8mM CaCl₂10mM glucose, 1mM MgSO₄10mM HEPES, pH adjusted to 7.4 with NaOH;

the electrode internal solution is as follows: 30mM KCl, 5mM EGTA, 5mM Mg-ATP, 1mM MgCl₂10mM HEPES, adjusted to pH 7.2 with KOH.

PCR and immunoblotting were performed for at least 3 biological replicates and electrophysiological analysis mapped at least 6 different cell currents per group. Data are expressed using mean M ± standard deviation SD. Molecular biology experiments used a two-sided paired t-test, electrophysiological experiments used the Wilcoxon Mann-Whitney U-test to detect statistical differences among groups, with P <0.05 representing significant differences.

Table 3: semi-activation voltage and slope factor test results for each cell line

FIG. 1 is a graph showing the expression results of Hsp70, Hsp90 and HSF 1. From the figure, it is known that the romacatto significantly up-regulates the heat shock factors Hsp70 and Hsp90 and down-regulates the upstream factor HSF1 in hERG transport-impaired HEK-293 cells, suggesting that the promotion of the transport of the hERG mutant protein by the romacatto is probably achieved by the key transport molecular partners Hsp70 and Hsp90, and the up-regulation feedback thereof inhibits the expression of HSF 1.

FIG. 2 is a graph showing the results of SDS polyacrylamide gel electrophoresis of hERG, Hsp70, Hsp90, and β -tubulin. As can be seen from the figure, both the Rolmatoc and HSF1 activators HSF1A, alone or in combination, can promote expression of hERG mature protein (155KDa) to different degrees, and the combination of the two can make the promotion more obvious (FIG. 2-A); this protein transport-promoting effect not only plays a role in the model of hERG mutant protein, but it still exists in cells of wild-type hERG; and accordingly, protein expression levels of Hsp70 and Hsp90 were up-regulated following the action of lummaca and HSF1A (fig. 2-B).

FIG. 3 is a graph of the enhancement of the hERG channel current by Lumaca (LUM) and/or HSF 1A. FIG. 3-A shows a trace of current recordings in different cell models (wild type: hERG-WT; heterozygous group: hERG-WT + hERG-A561V; heterozygous Caragacat Tuo-treated group: hERG-WT + hERG-A561V + LUM; heterozygous Caragacat Tuo-HSF 1A-treated group: hERG-WT + hERG-A561V + HSF 1A; heterozygous Caragacat Tuo and HSF 1A-treated group: hERG-WT + hERG-A561V + LUM + HSF1A), with ordinate representing current I (pA) and abscissa representing time T (ms); FIG. 3-B shows the stimulation pulse protocol used to elicit hERG channel current; FIG. 3-C shows that Lumaka Tor (LUM) and HSF1A increase the activation current of the channel, and the increase in current (pA/pF) is more pronounced after the combination treatment; FIGS. 3-D and 3-E show a significant increase in channel tail current, more pronounced in combination, and normalized peak tail current (I/I), respectively, for the two drugs alone or in combination_max) The activation-gated kinetics of the channel after drug treatment was shown to be wild-type after fitting using the Boltzmann equation, especially when the two drugs were combined.

In summary, while both lumacatto and HSF1A act alone or in combination to increase hERG activation current and tail current to different extents, the combined effect is particularly more pronounced with increased current amplitude and more pronounced correction of the cellular phenotype. Normalized peak tail current the corresponding half-activation voltages (V) of each set were obtained using the Boltzmann fitting equation_1/2) And activation curve slope k, indicated in LuWhen maca-To and HSF1A act independently (particularly HSF1A), the hERG channel activation curve parameter can be closer to a wild-type channel, and the effect is more obvious when the maca-To and HSF1A act in a combined manner, which shows that the maca-To and HSF1A not only increase the current of the hERG mutant channel, but also correct the negative activation gating change after mutation.

The technical scope of the invention claimed by the embodiments of the present application is not exhaustive, and new technical solutions formed by equivalent replacement of single or multiple technical features in the technical solutions of the embodiments are also within the scope of the invention claimed by the present application; in all the embodiments of the present invention, which are listed or not listed, each parameter in the same embodiment only represents an example (i.e., a feasible embodiment) of the technical solution, and there is no strict matching and limiting relationship between the parameters, wherein the parameters may be replaced with each other without departing from the axiom and the requirements of the present invention, unless otherwise specified.

The technical means disclosed by the scheme of the invention are not limited to the technical means disclosed by the technical means, and the technical scheme also comprises the technical scheme formed by any combination of the technical characteristics. While the foregoing is directed to embodiments of the present invention, it will be appreciated by those skilled in the art that various changes may be made in the embodiments without departing from the principles of the invention, and that such changes and modifications are intended to be included within the scope of the invention.

The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.