KR101935292B1 - Mitochondrial membrane protein MFN2 knockdown inhibition composition or method for controlling intracellular Mycobacterium tuberculosis - Google Patents
Mitochondrial membrane protein MFN2 knockdown inhibition composition or method for controlling intracellular Mycobacterium tuberculosis Download PDFInfo
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Abstract
본 발명은 미토콘드리아 막 단백질 MFN2(mitofusin 2)의 발현을 감소시키는 화합물을 유효성분으로 포함하는 결핵 예방 및 치료용 약학적 조성물에 관한 것이다. 본 발명의 유효성분인 MFN2의 발현을 감소시키는 화합물은 대식세포의 세포자멸사(apoptosis)를 유도함으로써 결핵균의 증식을 억제하는 효과가 있으므로, 이를 유효성분으로 포함하는 본 발명의 방법은 결핵의 예방 또는 치료에서 유용하게 사용될 수 있다The present invention relates to a pharmaceutical composition for preventing and treating tuberculosis comprising, as an active ingredient, a compound that reduces the expression of mitochondrial membrane protein MFN2 (mitofusin 2). Since the compound that reduces the expression of MFN2, an active ingredient of the present invention, has an effect of inhibiting the proliferation of Mycobacterium tuberculosis by inducing apoptosis of macrophages, the method of the present invention, which comprises it as an active ingredient, Can be useful in therapy
Description
본 발명은 전반적으로 면역학 분야에 관한 것이며, 부분적으로 대식세포에서 미토콘드리아 막 단백질 MFN2(mitofusin 2)의 발현을 조절하여 결핵균을 억제시키는 방법에 관한 것이다. The present invention relates generally to the field of immunology, and in part relates to a method of inhibiting Mycobacterium tuberculosis by controlling the expression of the mitochondrial membrane protein MFN2 (mitofusin 2) in macrophages.
결핵은 결핵균 및 다른 마이코박테리움(Mycobacterium)종의 감염에 의해 유발되는 만성 감염성 질병이다. 결핵은 매년 약 8백만명의 신규 감염자가 발생되며 3백만명의 목숨을 앗아가는 개발도상국의 주요 질병이며, 또한 선진국에서도 문제점으로 부각되고 있다. 상당한 기간 동안 감염은 무증상일 수 있으나, 결핵은 가장 공통적인 증상으로서 폐의 급성 염증을 나타내며, 그 결과 발열 및 마른 기침(nonproductive cough)을 유발시킨다. 치료하지 않으면, 일반적으로 심각한 합병증 및 사망을 초래한다. Tuberculosis is a chronic infectious disease caused by an infection of Mycobacterium species with Mycobacterium tuberculosis. Tuberculosis is a major disease in developing countries, with nearly 8 million new infections occurring each year, and 3 million deaths, and it is also a problem in developed countries. Infection may be asymptomatic for a considerable period of time, but tuberculosis is the most common symptom of acute inflammation of the lungs, resulting in fever and nonproductive cough. Untreated, usually causes serious complications and death.
결핵은 장기간에 걸친 항생제 요법을 이용하여 치료될 수 있으나, 이러한 치료법이 결핵의 확산을 막기에는 역부족이다. 감염된 개체들은 증상을 나타내지는 아니하나, 일정 기간 보균상태(contagious)일 수 있다. 게다가 치료 섭생을 엄격하게 따르더라도 환자의 행동을 통제하는 것은 어렵다. 일부 환자들은 치료과정을 끝마치지 못하는데, 이는 효험없는 치료와 약제 내성의 발달을 초래할 수 있다. 치료의 전 과정을 끝마친다 하더라도, 결핵균 감염은 감염된 개체로부터 근절되지 아니하며 여전히 재활성화될 수 있는 잠복성 감염으로 잔존하게 된다. Tuberculosis can be treated with long-term antibiotic therapy, but these therapies are not enough to prevent the spread of tuberculosis. Infected individuals do not exhibit symptoms, but may be contagious for a period of time. In addition, it is difficult to control the patient's behavior even though the treatment regimen is strictly followed. Some patients are unable to complete the treatment process, which can lead to ineffective treatment and development of drug resistance. Even if you complete the entire course of treatment, TB infection remains a latent infection that can not be eradicated from the infected individual and still be reactivated.
결핵의 확산을 통제하기 위해서는, 상기 질병에 대한 효과적인 예방접종(vaccination) 및 정확한 초기 진단이 가장 중요하다. 현재까지는 생균(live bacteria)을 이용한 예방접종이 보호 면역을 유도하기 위한 가장 효율적인 방법이다. 상기 목적을 위해 이용되는 가장 일반적인 마이코박테리움은 바실러스 칼메트-게링(BacillusCalmette-Guerin, BCG)인데, M. bovis의 비병원성 종이다. 그러나, BCG의 안정성 및 효능은 논쟁의 대상이 되고 있으며, 미국과 같은 일부 국가에서는 상기 제제를 사용한 일반 대중의 예방접종을 실시하지 않고 있다. 결핵의 진단은 일반적으로 피부 검정법을 이용하여 수행되는데, 상기 검정법은 투베르쿨린 PPD(proteinpurified derivative)에 대한 피내 노출을 포함한다. 항원-특이 T 세포 반응은 주입후 48-72시간 까지 주입 부위에서의 측정가능한 정도 경화(induration)를 초래하는데, 이는 마이코박테리움 항원에 대한 노출을 의미한다. 그러나 상기 검정법은 민감도 및 특이성에서 문제가 있으며, BCG로 예방접종된 개체들은 감염된 개체들과 구별될 수 없다.In order to control the spread of tuberculosis, effective vaccination and precise initial diagnosis of the disease are most important. Until now, live vaccine vaccination is the most effective way to induce protective immunity. The most common Mycobacterium used for this purpose is Bacillus Calmette-Guerin (BCG), a non-pathogenic species of M. bovis . However, the safety and efficacy of BCG is controversial, and in some countries, such as the United States, the general public is not vaccinated with the formulation. Diagnosis of tuberculosis is generally performed using a skin test method, which involves intradermal exposure to tuberculin protein precursor derivative (PPD). The antigen-specific T cell response results in a measurable degree of induration at the site of injection up to 48-72 hours post-injection, which means exposure to the Mycobacterium antigens. However, the assay is problematic in sensitivity and specificity, and individuals vaccinated with BCG can not be distinguished from infected individuals.
결핵균은 세포내 기생하는 세균으로 주로 대식세포에 감염된다. 결핵균에 감염된 대식세포는 세포자멸사(Apoptosis)를 통하여 결핵균을 제거한다고 알려져 있고, 특히 미토콘드리아는 세포자멸사와 밀접한 상관관계가 있다. 자극을 받은 미토콘드리아는 여러 작은 조각으로 나뉘게 되는 분열이 일어나는데, 이러한 분열의 증가는 세포자멸사를 유도한다. 미토콘드리아 막 단백질 MFN2의 감소는 분열을 일으키는 원인 중 하나 알려져있다.Mycobacterium tuberculosis is an intracellular parasitic bacterium that mainly infects macrophages. Macrophages infected with Mycobacterium tuberculosis are known to remove Mycobacterium tuberculosis through apoptosis, and mitochondria are closely related to apoptosis. The stimulated mitochondria undergo division, which is divided into several smaller fragments. This increase in cleavage leads to apoptosis. Reduction of mitochondrial membrane protein MFN2 is known to be one of the causes of cleavage.
이러한 배경하에, 본 발명자들은 따라서, 결핵균 감염시 대식세포 미토콘드리아 막 단백질 MFN2의 발현 조절은 결핵균 생존 억제에 매우 효과적인 기전으로 작용함을 확인함으로써 본 발명을 완성하였다. Based on this background, the present inventors have completed the present invention by confirming that the expression of the macrophage mitochondrial membrane protein MFN2 in Mycobacterium tuberculosis infection acts as a very effective mechanism for inhibiting the survival of M. tuberculosis.
본 발명은 미토콘드리아 막 단백질 MFN2(mitofusin 2)의 발현을 억제하고 이를 통한 대식세포의 세포자멸사 기전을 활성화시킴으로, 기존 약제와 작용기전이 다르고 부작용이 상대적으로 적은 결핵 예방 및 효율적인 치료 방법을 제공하고자 한다. The present invention provides a preventive and effective treatment method for tuberculosis that inhibits mitochondrial membrane protein MFN2 (mitofusin 2) expression and thereby activates macrophage apoptosis mechanism, and thus has relatively few side effects with conventional drugs and side effects .
전술한 목적을 달성하기 위하여 본 발명은 미토콘드리아 막 단백질 MFN2(mitofusin 2)의 발현을 감소시키는 화합물을 유효성분으로 포함하는 결핵 예방 및 치료용 약학적 조성물을 제공하고자 한다. In order to achieve the above-mentioned object, the present invention provides a pharmaceutical composition for preventing and treating tuberculosis comprising, as an active ingredient, a compound that reduces the expression of mitofondrin membrane protein MFN2 (mitofusin 2).
상기 미토콘드리아 막 단백질 MFN2의 발현을 감소시키는 화합물은 Negative siRNA, MFN2의 siRNA 및 리포펙타민(Lipofectamine)을 포함하며 그 양은 Negative siRNA 및 MFN2의 siRNA는 80~120nM이며 리포펙타민은 1~2ul임을 특징으로 한다. The compounds that reduce the expression of the mitochondrial membrane protein MFN2 include Negative siRNA, siRNA of MFN2 and Lipofectamine. The amount of Negative siRNA and MFN2 siRNA is 80-120 nM and 1 to 2 ul of lipofectamine .
이렇게 제공된 화합물을 통하여 MFN2의 발현은 감소되며 이는 결핵균에 감염된 대식세포의 세포자멸사(apoptosis)를 유도함으로써 결핵균의 증식을 억제하게 된다. The expression of MFN2 is decreased through the compounds provided in this way, which inhibits the proliferation of Mycobacterium tuberculosis by inducing apoptosis of macrophages infected with Mycobacterium tuberculosis.
또한 미토콘드리아 막 단백질 MFN2의 발현을 감소시키는 화합물에 항결핵 약제를 병용하여 사용하는 것을 포함할 수 있다. It may also include the use of anti-tuberculosis agents in combination with a compound that reduces the expression of the mitochondrial membrane protein MFN2.
여기에서 사용되는 항결핵 약제는 리팜피신(Rifampicin), 아이소니아지드(Isoniazid), 에탐부톨(Ethambutol), 피라진아미드(Pyrazinamide)으로 이루어진 군으로부터 선택된 적어도 하나일 수 있다. The antituberculous agent used herein may be at least one selected from the group consisting of Rifampicin, Isoniazid, Ethambutol, and Pyrazinamide.
또한 상기 결핵은 안결핵, 피부 결핵, 부신 결핵, 신장결핵, 부고환 결핵, 림프선 결핵, 후두 결핵, 중이 결핵, 장결핵, 다제내성 결핵, 폐결핵, 담결핵, 골결핵, 인후결핵, 임파선 결핵, 폐허증, 유방 결핵 및 척추 결핵으로 이루어진 군으로부터 선택된 적어도 하나일 수 있다. In addition, the above-mentioned tuberculosis can be diagnosed as tuberculosis, skin tuberculosis, adrenal tuberculosis, renal tuberculosis, epidemic tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, , Breast tuberculosis, and spinal tuberculosis.
이상과 같이 본 발명에 의하면 종래 전혀 알려져 있지 않던 미토콘드리아 막 단백질 MFN2의 녹다운에 의한 대식세포의 세포자멸사 유도를 통해 결핵균을 억제하는 효과를 가지고 있다. 또한 본 발명은 결핵균 감염시 대식세포의 미토콘드리아 막 단백질 MFN2의 발현을 감소시킬 수 있는 화합물 자극 요법을 사용한다면 다약제내성이 있는 결핵균에도 더욱 효과적이었다. As described above, the present invention has an effect of suppressing Mycobacterium tuberculosis through induction of macrophage apoptosis by knockdown of mitochondrial membrane protein MFN2, which has not been known at all. In addition, the present invention is more effective for drug-resistant Mycobacterium tuberculosis by using compound stimulation therapy that can reduce the expression of macrophage mitochondrial membrane protein MFN2 upon infection with M. tuberculosis.
도 1은 마우스 유래 큰포식세포주 Raw264.7 세포에서 MFN2 특이적인 siRNA를 형질전환하여 MFN2 단백질의 발현 감소를 보여주는 Western blot을 통한 전기영동사진이다.
도 2는 MFN2에 특이적인 siRNA를 형질전환시킨 큰포식세포주 Raw264.7 세포에 결핵균을 24시간 동안 감염시킨 후, 세포내 결핵균의 생존을 비교한 그래프이다. FIG. 1 is a Western blot electrophoresis image showing reduction of expression of MFN2 protein by transforming MFN2-specific siRNA in mouse-derived macrophage cell line Raw264.7 cells.
Fig. 2 is a graph comparing the survival of intracellular Mycobacterium tuberculosis after infecting M. tuberculosis bacterium with Raw264.7 cells expressing MFN2-specific siRNA in a large predominant cell line for 24 hours.
이하 첨부된 도면을 참조하여 본 발명을 보다 상세히 설명한다. 그러나 첨부된 도면은 본 발명의 기술적 사상의 내용과 범위를 쉽게 설명하기 위한 예시일 뿐, 이에 의해 본 발명의 기술적 범위가 한정되거나 변경되는 것은 아니다. 또한 이러한 예시에 기초하여 본 발명의 기술적 사상의 범위 안에서 다양한 변형과 변경이 가능함은 당업자에게는 당연할 것이다. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail with reference to the accompanying drawings. It should be understood, however, that the appended drawings illustrate only the contents and scope of technology of the present invention, and the technical scope of the present invention is not limited thereto. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical idea of the present invention based on these examples.
실시예Example 1 : One : 큰포식세포Large predominant cell 미토콘드리아 막 단백질 Mitochondrial membrane protein MFN2의Of MFN2 발현을 조절하는 단계 The step of regulating expression
큰포식세포주 Raw264.7 세포에서 미토콘드리아 막 단백질 MFN2의 발현을 억제하기 위하여 각각 100nM의 Negative siRNA와 MFN2의 siRNA를 lipofectamine(1.5ul)을 이용하여 세포내 형질전환 해주었다. 도 1은 형질전환 18시간 후, 세포내 MFN2 단백질 발현정도를 Western blotting 방법으로 확인한 결과, 큰포식세포 내 MFN2 단백질 발현의 감소를 확인할 수 있었다.In order to inhibit mitochondrial membrane protein MFN2 expression in Raw264.7 cells, 100 nM of negative siRNA and MFN2 siRNA were transfected intracellularly with lipofectamine (1.5 ul). FIG. 1 shows that the expression of MFN2 protein in the cells after 18 hours of transformation was confirmed by Western blotting, and the expression of MFN2 protein in the large phagocytic cells was decreased.
실시예Example 2 : 2 : MFN2MFN2 단백질의 발현 감소가 결핵균 억제에 영향을 미치는 것을 확인하는 단계 Identifying that the decreased expression of the protein affects mycobacteria inhibition
도 2는 도 1에서 확인한 MFN2 단백질의 발현을 감소시킨 큰포식세포에 결핵균 H37Rv를 Multiplicity of infection (MOI)=1로 감염시킨 후, 세포내 결핵균 생존비율을 비교한 그래프이다. 결핵균 감염 24시간 이후, 3차 증류수를 사용하여 세포에서 결핵균을 용출시키고 7H10 배지에서 14일간 배양하여 세포내 결핵균 생존율을 확인하였다. 결핵균 감염 시 MFN2 단백질의 발현이 감소된 큰포식세포에서 세포내 결핵균의 생존이 억제되는 것을 확인할 수 있었다.FIG. 2 is a graph comparing survival rates of Mycobacterium tuberculosis bacteria with the multiplicity of infection (MOI) = 1 of Mycobacterium tuberculosis H37Rv in a large predominant cell with reduced expression of MFN2 protein identified in FIG. After 24 hours of Mycobacterium tuberculosis infection, Mycobacterium tuberculosis was eluted from the cells using third distilled water and cultured in 7H10 medium for 14 days to confirm the survival rate of intracellular Mycobacterium tuberculosis. It was confirmed that the survival of intracellular Mycobacterium tuberculosis was inhibited in the large phagocytic cells in which the expression of MFN2 protein was decreased upon infection with M. tuberculosis.
상기 실시예에서는 큰포식세포에서 MFN2 단백질의 감소가 결핵균의 생존 억제에 효과적인 기전임을 나타낸다. 본 발명은 결핵균뿐만 아니라 다양한 세포내 기생세균의 감염에 관련한 큰포식세포의 병원균 생존 억제 연구에 적용가능하며, MFN2조절 화합물을 통한 치료제 개발에도 광범위하게 활용이 가능할 것으로 사료된다. The above example shows that reduction of MFN2 protein in macrophages is an effective mechanism for inhibiting the survival of Mycobacterium tuberculosis. The present invention can be applied to inhibition of pathogen viability of large phagocytic cells associated with infection of various intracellular parasitic bacteria as well as to tubercular bacillus, and may be widely applicable to the development of therapeutic agents through MFN2 modulating compounds.
Claims (7)
상기 MFN2의 siRNA는 80~120nm이며 리포펙타민은 1~2ul임을 특징으로 하는 결핵 예방 및 치료용 약학적 조성물. The method according to claim 1,
Wherein the MFN2 siRNA is 80-120 nm and the lipofectamine is 1-2 ul.
상기 MFN2의 발현의 감소는 결핵균에 감염된 대식세포의 세포자멸사(apoptosis)를 유도함으로써 결핵균의 증식을 억제하는 것을 특징으로 하는 결핵 예방 및 치료용 약학적 조성물. The method according to claim 1,
Wherein the decrease in expression of MFN2 induces apoptosis of macrophages infected with Mycobacterium tuberculosis, thereby inhibiting proliferation of Mycobacterium tuberculosis.
상기 미토콘드리아 막 단백질 MFN2의 발현을 감소시키는 화합물에 항결핵 약제를 병용하여 사용하는 결핵 예방 및 치료용 약학적 조성물. The method according to claim 1,
A pharmaceutical composition for the prevention and treatment of tuberculosis, which is used by using an anti-tuberculosis drug in combination with a compound that reduces the expression of the mitochondrial membrane protein MFN2.
상기 항결핵 약제는 리팜피신(Rifampicin), 아이소니아지드(Isoniazid), 에탐부톨(Ethambutol) 또는 피라진아미드(Pyrazinamide)인 결핵 예방 및 치료용 약학적 조성물.
5. The method of claim 4,
The pharmaceutical composition for the prevention and treatment of tuberculosis, wherein the anti-tuberculosis drug is Rifampicin, Isoniazid, Ethambutol or Pyrazinamide.
상기 결핵은 안결핵, 피부 결핵, 부신 결핵, 신장결핵, 부고환 결핵, 림프선 결핵, 후두 결핵, 중이 결핵, 장결핵, 다약제내성 결핵, 폐결핵, 담결핵, 골결핵, 인후결핵, 임파선 결핵, 패혈증, 유방 결핵 또는 척추 결핵인 결핵 예방 및 치료용 약학적 조성물.6. The method according to any one of claims 1 to 5,
The above-mentioned tuberculosis can be classified as tuberculosis, skin tuberculosis, adrenal tuberculosis, renal tuberculosis, epidemic tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, A pharmaceutical composition for the prevention and treatment of tuberculosis which is breast tuberculosis or spinal tuberculosis.
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