KR102405350B1 - Composition for reverse control of cellular senescence comprising pdk1 inhibitor - Google Patents
Composition for reverse control of cellular senescence comprising pdk1 inhibitor Download PDFInfo
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- KR102405350B1 KR102405350B1 KR1020210037557A KR20210037557A KR102405350B1 KR 102405350 B1 KR102405350 B1 KR 102405350B1 KR 1020210037557 A KR1020210037557 A KR 1020210037557A KR 20210037557 A KR20210037557 A KR 20210037557A KR 102405350 B1 KR102405350 B1 KR 102405350B1
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
본 발명은 특정 분자 타겟을 이용하여 노화된 세포를 젊은 세포 상태로 되돌리는 역노화 유도 방법에 관한 것으로, 구체적으로는 PDK1(Phosphoinositide-dependent kinase-1) 저해제를 포함하는 역노화 유도용 조성물 또는 PDK1 저해제를 처리하는 단계를 포함하는 역노화 유도 방법에 관한 것이다. 본 발명에 따른 PDK1은 세포노화에 관여하는 주요 신호전달 단백질 중 MTOR과 NF-кB를 동시에 조절하는 단백질로서 세포의 성장, 분열 및 노화 관련 분비표현형을 결정하는 핵심 단백질이며, PDK1을 일시적으로 억제 시 노화된 세포 중 일부가 효과적으로 정상 젊은 세포로 가역화되며, 이러한 세포들에서는 더 이상 노화된 세포의 표현형이 관찰되지 않는 것을 확인하였다. 따라서, PDK1은 노화된 세포를 사멸 유도하는 전략에 치중되어 있는 현재의 노화치료전략에 새로운 가능성을 제시하고, 항노화 물질 및 노화 관련 질환 치료제 개발에 유용하게 사용될 수 있다.The present invention relates to a method for inducing reverse aging by using a specific molecular target to return senescent cells to a young cell state, and specifically, a composition for inducing reverse aging comprising a PDK1 (Phosphoinositide-dependent kinase-1) inhibitor or PDK1 It relates to a method for inducing reverse aging comprising the step of treating an inhibitor. PDK1 according to the present invention is a protein that simultaneously regulates MTOR and NF-κB among the major signaling proteins involved in cellular aging. It was confirmed that some of the aged cells were effectively reversible into normal young cells, and the phenotype of the aged cells was no longer observed in these cells. Therefore, PDK1 presents new possibilities for the current aging treatment strategy, which is focused on the strategy of inducing apoptosis of senescent cells, and can be usefully used for the development of anti-aging substances and therapeutic agents for aging-related diseases.
Description
본 발명은 특정 분자 타겟을 이용하여 노화된 세포를 젊은 세포 상태로 되돌리는 역노화 유도 방법에 관한 것으로, 구체적으로는 PDK1(Phosphoinositide-dependent kinase-1) 저해제를 포함하는 역노화 유도용 조성물 또는 PDK1 저해제를 처리하는 단계를 포함하는 역노화 유도 방법에 관한 것이다.The present invention relates to a method for inducing reverse aging by using a specific molecular target to return senescent cells to a young cell state, and specifically, a composition for inducing reverse aging comprising a PDK1 (Phosphoinositide-dependent kinase-1) inhibitor or PDK1 It relates to a method for inducing reverse aging comprising the step of treating an inhibitor.
세포 노화는 환경적 스트레스로 인해 손상을 입은 세포가 암화되기 전에 영구히 세포 주기를 멈추는 비가역적 생명 현상으로 알려져 있다. 세포 노화는 영구적인 세포분열의 중단과 더불어, 노화관련 분비표현형(Senescence Associated Secretory Phenotype, SASP)을 갖는다고 알려져 있다. 최근 노화된 세포가 분비하는 물질들로 인하여 주변 세포의 노화의 가속화뿐 아니라 암을 포함한 여러 질병이 발생할 수 있음이 잇따라 발표되면서 노화된 세포를 조절하려는 시도들 또한 함께 이뤄지고 있다. 하지만 대부분의 시도들은 노화된 세포들을 특징적으로 사멸시키는 전략에 집중되어 있으며, 현재까지 노화된 세포를 다시 정상 세포로 가역화하여 노화된 세포의 부수적 결과들을 방지할 수 있는지는 확인되지 않고 있다. 상기 연구들은 분자생물학적인 조작을 통하여 노화된 세포가 세포 노화 현상으로부터 벗어날 수 있다는 것을 간접적으로 보여준다. 그러나 이러한 연구들은 노화 현상의 메커니즘에 대한 이해의 부족으로 인해 노화된 세포를 가역화 시키는데 필요한 미세 컨트롤이 불가능하다는 한계를 지니고 있으며, 현재까지 효과적으로 노화된 세포를 정상 젊은 세포로 가역화시킬 수 있는 분자 타겟은 제시되어 있지 않다.Cellular aging is known as an irreversible life phenomenon in which cells damaged by environmental stress permanently stop the cell cycle before they become cancerous. Cellular senescence is known to have a senescence-associated secretory phenotype (SASP) along with the permanent cessation of cell division. Recently, as it has been announced that various diseases including cancer may occur as well as accelerated aging of surrounding cells due to substances secreted by aged cells, attempts to control aged cells are also being made. However, most attempts are focused on strategies to characteristically kill senescent cells, and it has not been confirmed so far whether it is possible to prevent side effects of senescent cells by reversing senescent cells back into normal cells. The above studies indirectly show that senescent cells can escape from cellular aging through molecular biological manipulation. However, these studies have limitations in that the fine control necessary to reversible senescent cells is impossible due to the lack of understanding of the mechanisms of aging. No target is given.
또한, 세포 노화는 세포 내부의 축적된 DNA 데미지의 축적과 세포 외부의 다양한 전달물질에 의해 일어나는 현상으로 현재까지 세포 노화를 일으키는 세 가지 주요 신호전달경로는 세포의 성장과 분열을 담당하는 IGF1-PI3K-mTOR 신호전달경로와, DNA damage -P53-RB 신호전달경로, 그리고 SASP를 주관하는 NF-kB 신호전달경로가 있다고 알려져 있다. 이처럼 여러 신호전달시스템이 종합적으로 관여하는 세포 노화를 이해하기 위해서는 네트워크적 접근을 필요로 한다.In addition, cellular senescence is a phenomenon that is caused by the accumulation of accumulated DNA damage inside the cell and various transmitters outside the cell. So far, the three major signaling pathways that cause cellular senescence are IGF1-PI3K, which is responsible for cell growth and division. It is known that there is a -mTOR signaling pathway, a DNA damage -P53-RB signaling pathway, and an NF-kB signaling pathway that controls SASP. A network approach is required to understand cellular aging in which multiple signaling systems are comprehensively involved.
이에 본 발명자들은 노화현상에 대한 이해를 바탕으로 시스템생물학적 접근을 통해 노화네트워크 모델을 구축하고, 최적의 노화제어 타겟을 제시하였다. 보다 구체적으로, 기존 문헌 정보를 바탕으로 세 가지 중심 신호전달경로를 종합한 후, 대규모 단백질 분석 결과를 이용하여 네트워크의 파라미터를 추정하고, 완성된 네트워크 모델을 이용하여 PDK1이 노화된 세포 상태를 젊은 세포 상태로 가역화시킬 수 있는 최적 분자 타겟임을 확인하고, PDK1을 억제하였을 때 인간 유래의 세포 노화 현상을 가역화시킬 수 있음을 확인함으로써 본 발명을 완성하였다.Accordingly, the present inventors built an aging network model through a systems biological approach based on an understanding of the aging phenomenon, and presented an optimal aging control target. More specifically, after synthesizing the three central signaling pathways based on existing literature information, the parameters of the network are estimated using the results of large-scale protein analysis, and the PDK1 senescent cell state is evaluated using the completed network model. The present invention was completed by confirming that it is an optimal molecular target that can be reversible to a cellular state, and reversible human-derived cellular senescence when PDK1 is inhibited.
본 발명의 목적은 PDK1(Phosphoinositide-dependent kinase-1) 저해제를 포함하는 역노화 유도용 조성물을 제공하는 것이다.SUMMARY OF THE INVENTION It is an object of the present invention to provide a composition for inducing reverse aging comprising a phosphoinositide-dependent kinase-1 (PDK1) inhibitor.
본 발명의 또다른 목적은 PDK1 저해제를 포함하는 세포 노화 또는 노화 관련 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cellular senescence or senescence-related diseases comprising a PDK1 inhibitor.
본 발명의 또 다른 목적은 생체 외(In vitro)에서 PDK1 저해제를 처리하는 단계를 포함하는 역노화 유도 방법을 제공하는 것이다.Another object of the present invention is to provide a method for inducing reverse aging comprising treating a PDK1 inhibitor in vitro .
상기 목적을 달성하기 위하여, 본 발명은 PDK1(Phosphoinositide-dependent kinase-1) 저해제를 포함하는 역노화 유도용 조성물을 제공한다.In order to achieve the above object, the present invention provides a composition for inducing reverse aging comprising a PDK1 (Phosphoinositide-dependent kinase-1) inhibitor.
또한, 본 발명은 PDK1 저해제를 포함하는 세포 노화 또는 노화 관련 질환의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating cellular senescence or senescence-related diseases comprising a PDK1 inhibitor.
또한, 본 발명은 PDK1 저해제를 포함하는 세포 노화 또는 노화 관련 질환의 예방 또는 개선용 건강기능식품을 제공한다.In addition, the present invention provides a health functional food for preventing or improving cellular aging or aging-related diseases comprising a PDK1 inhibitor.
또한, 본 발명은 생체 외(In vitro)에서 PDK1 저해제를 처리하는 단계를 포함하는 역노화 유도 방법을 제공한다.In addition, the present invention provides a method for inducing reverse aging comprising the step of treating a PDK1 inhibitor in vitro .
또한, 본 발명은 노화 관련 질환의 예방 또는 치료 제제의 스크리닝 방법을 제공한다.In addition, the present invention provides a screening method for a prophylactic or therapeutic agent for a senescence-related disease.
본 발명에 따른 PDK1은 세포노화에 관여하는 주요 신호전달 단백질 중 mTOR과 NF-кB를 동시에 조절하는 단백질로서 세포의 성장, 분열 및 노화 관련 분비표현형을 결정하는 핵심 단백질이며, PDK1을 일시적으로 억제 시 노화된 세포 중 일부가 효과적으로 정상 젊은 세포로 가역화되며, 이러한 세포들에서는 더 이상 노화된 세포의 표현형이 관찰되지 않는 것을 확인하였다. 따라서, PDK1은 노화된 세포를 사멸 유도하는 전략에 치중되어 있는 현재의 노화치료전략에 새로운 가능성을 제시하고, 항노화 물질 및 노화 관련 질환 치료제 개발에 유용하게 사용될 수 있다.PDK1 according to the present invention is a protein that simultaneously regulates mTOR and NF-κB among the major signaling proteins involved in cellular aging. It is a key protein that determines the cell growth, division and senescence-related secretion phenotype. It was confirmed that some of the aged cells were effectively reversible into normal young cells, and the phenotype of the aged cells was no longer observed in these cells. Therefore, PDK1 presents new possibilities for the current aging treatment strategy, which is focused on the strategy of inducing apoptosis of senescent cells, and can be usefully used for the development of anti-aging substances and therapeutic agents for aging-related diseases.
도 1은 기존 문헌 정보를 기반으로 구축한 41개의 노드로 구성된 노화 네트워크 모델을 나타낸 도이다. 초록색 화살표는 활성화 링크, 빨간색 T자 화살표는 저해 링크를 의미한다. 노드의 종류는 총 3가지로, 보라색은 입력 노드(총 3개), 초록색 타원은 출력 노드(총 6개) 및 파란색 사각형은 내부노드(총 32개)를 각각 의미한다.
도 2는 섬유아세포에 대하여 대규모 단백질 분석 실험을 수행한 결과를 히트맵으로 나타낸 도이다. 히트맵의 각 열은 세포주기 상태(일시적 중단 상태, 영구적 중단 상태 또는 분열 상태)를 나타내고, 각 행은 유전자 심볼을 나타낸다. 도 2의 A는 각 유전자에 대응하는 시계열 RPPA(Reverse Phase Protein Assay) 데이터의 커브의 아래 면적을 나타낸 것이고, 도 2의 B는 커브 아래 면적 값을 0과 1로 이분화한 결과를 나타낸 것이다.
도 3은 진화 알고리즘을 통하여 2000개의 네트워크 모델들을 학습시키고, 이를 본 발명에서 구축한 노화 네트워크 모델에 적용하여 대규모 시뮬레이션한 결과를 나타낸 도이다. y축은 저해 타겟의 유전자 심볼을, x축은 2000개의 모델 중 저해 시뮬레이션 결과를 나타낸 모델의 개수를 나타낸다.
도 4는 PDK1 저해제를 노화된 섬유아세포에 처리한 세포 실험 결과를 나타낸 도이다. 도 4의 A는 Beta-galactosidase assay 결과, 도 4의 B는 Wound healing assay 결과를 나타내며, 도 4의 C는 형광염색을 수행한 결과로 왼쪽에서부터 순서대로 정상 젊은 세포, 노화 세포, mTOR 저해제를 처리한 노화세포 및 PDK1 저해제를 처리한 노화 세포를 나타낸다.1 is a diagram showing an aging network model composed of 41 nodes constructed based on existing literature information. Green arrows indicate activation links, and red T-shaped arrows indicate inhibitory links. There are a total of 3 types of nodes: purple indicates input nodes (total of 3), green ovals indicate output nodes (total of 6), and blue rectangles indicate internal nodes (total of 32).
2 is a diagram showing the results of performing a large-scale protein analysis experiment on fibroblasts as a heat map. Each column of the heatmap represents a cell cycle state (temporarily stopped, permanently stopped, or divided), and each row represents a gene symbol. 2A shows the area under the curve of the time series Reverse Phase Protein Assay (RPPA) data corresponding to each gene, and FIG. 2B shows the result of bisecting the value of the area under the curve into 0 and 1.
3 is a diagram showing the results of large-scale simulation by learning 2000 network models through an evolutionary algorithm and applying them to the aging network model constructed in the present invention. The y-axis represents the gene symbol of the inhibition target, and the x-axis represents the number of models showing the inhibition simulation result among 2000 models.
4 is a diagram showing the results of cell experiments in which the PDK1 inhibitor was treated on senescent fibroblasts. 4A is a beta-galactosidase assay result, FIG. 4B is a wound healing assay result, and FIG. 4C is a result of fluorescence staining. In order from the left, normal young cells, senescent cells, and mTOR inhibitor were treated. One senescent cell and a senescent cell treated with a PDK1 inhibitor are shown.
이하, 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 PDK1(Phosphoinositide-dependent kinase-1) 저해제를 포함하는 역노화 유도용 조성물을 제공한다.The present invention provides a composition for inducing reverse aging comprising a phosphoinositide-dependent kinase-1 (PDK1) inhibitor.
본 발명에서, “역노화”란 노화를 예방 및 치료하는 개념의 항노화와 달리 노화를 유도하는 유전인자의 발현을 원천적으로 조절하여 노화 상태를 이전으로 되돌리는 것을 의미한다.In the present invention, "reverse aging" refers to returning the aging state to the previous one by fundamentally regulating the expression of a genetic factor that induces aging, unlike the anti-aging concept of preventing and treating aging.
*항노화 및 노화 관련 질병 연구가 활발히 이루어지고 있음에도, 현재 항노화 및 노화 관련 질병 치료전략은 안 좋은 화학물질을 분비하여 주변 세포들에게 나쁜 영향을 주는 노화 세포를 사멸시키는 것에 집중되어 있어 실질적으로 거의 모든 항노화 요법들이 임상시험에서 확고한 효과와 안정성을 입증하지 못하고 있는 실정이다. 이에, 본 발명자들은 무분별한 세포 성장상태를 배제시키면서 노화된 세포를 일시적으로 세포주기 중단 상태로 되돌릴 수 있는지 여부를 확인하고자 하였다.*Even though anti-aging and aging-related disease research is being actively conducted, current anti-aging and aging-related disease treatment strategies are focused on killing senescent cells that have a bad effect on surrounding cells by secreting bad chemicals. Almost all anti-aging therapies have not proven their efficacy and safety in clinical trials. Accordingly, the present inventors tried to determine whether it is possible to temporarily return the aged cells to the cell cycle interruption state while excluding the reckless cell growth state.
먼저, 본 발명의 일 구현예에서 노화된 세포 상태를 젊은 세포 상태로 가역화시키는 최적 분자 타겟을 규명하기 위해 노화 네트워크 모델을 구축하였다. 그 후, 섬유아세포의 대규모 단백질 분석 실험을 수행하고, 진화 알고리즘을 통하여 학습된 2000개의 네트워크 모델들을 본 발명에 따른 노화 네트워크 모델에 적용하여 PDK1이 최적의 분자 타겟임을 확인하였다. 마지막으로, 상기 PDK1 저해제를 노화된 섬유아세포에 처리하였을 때 세포의 노화 상태가 줄어들고, 정상 조건의 배지 처리 시 세포들이 다시 성장 및 분열하는 것을 확인하였다. 이를 통해, 본 발명자들은 본 발명의 PDK1 저해제가 역노화 유도용 조성물로 유용하게 이용될 수 있음을 확인하였다.First, in one embodiment of the present invention, an aging network model was constructed to identify an optimal molecular target for reversing the aged cell state to the young cell state. Thereafter, a large-scale protein analysis experiment of fibroblasts was performed, and 2000 network models learned through an evolutionary algorithm were applied to the aging network model according to the present invention to confirm that PDK1 was an optimal molecular target. Finally, it was confirmed that when the PDK1 inhibitor was treated with senescent fibroblasts, the senescent state of the cells was reduced, and cells grew and divided again when treated with a medium under normal conditions. Through this, the present inventors confirmed that the PDK1 inhibitor of the present invention can be usefully used as a composition for inducing reverse aging.
본 발명에 있어서, 상기 PDK1 저해제는 PDK1 유전자의 안티센스 올리고뉴클레오티드, siRNA, shRNA, miRNA 및 PDK1 단백질에 특이적인 항체로 구성된 군에서 선택되는 것을 특징으로 할 수 있다. 또한, 상기 PDK1 저해제는 PDK1 단백질에 직접/간접적으로 결합하여 이의 활성을 억제하는 화합물을 포함할 수 있다. 상기 화합물로는 BX-795, BX-912, PHT-427, GSK2334470, OSU-03012 등이 있을 수 있으나 이에 제한되는 것은 아니며, 본 발명의 일 실시예에서는 PDK1 활성을 억제하는 화합물로 PDK1 저해제인 BX-795를 사용하였다.In the present invention, the PDK1 inhibitor may be selected from the group consisting of an antisense oligonucleotide of the PDK1 gene, siRNA, shRNA, miRNA, and an antibody specific to PDK1 protein. In addition, the PDK1 inhibitor may include a compound that directly/indirectly binds to the PDK1 protein and inhibits its activity. The compound may include, but is not limited to, BX-795, BX-912, PHT-427, GSK2334470, OSU-03012, and the like. -795 was used.
본 발명에서 "안티센스 올리고뉴클레오타이드”란 특정 mRNA의 서열에 상보적인 핵산 서열을 함유하고 있는 DNA 또는 RNA 또는 이들의 유도체를 의미하고, mRNA 내의 상보적인 서열에 결합하여 mRNA의 단백질로의 번역을 저해하는 작용을 한다. 상기 안티센스 서열은 상기 유전자의 mRNA에 상보적이고 상기 mRNA에 결합할 수 있는 DNA 또는 RNA 서열을 의미하며, 상기 mRNA의 번역, 세포질내로의 전위(translocation), 성숙(maturation) 또는 다른 모든 전체적인 생물학적 기능에 대한 필수적인 활성을 저해할 수 있다.In the present invention, "antisense oligonucleotide" means DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a specific mRNA sequence, and inhibits translation of mRNA into protein by binding to a complementary sequence in mRNA. The antisense sequence refers to a DNA or RNA sequence that is complementary to the mRNA of the gene and capable of binding to the mRNA, and is used for translation of the mRNA, translocation into the cytoplasm, maturation, or any other It can inhibit essential activity for overall biological function.
또한, 상기 안티센스 핵산은 효능을 증진시키기 위하여 하나 이상의 염기, 당 또는 골격(backbone)의 위치에서 변형될 수 있다. 핵산 골격은 포스포로티오에이트, 포스포트리에스테르, 메틸 포스포네이트, 단쇄 알킬, 시클로알킬, 단쇄 헤테로아토믹, 헤테로시클릭 당간 결합 등으로 변형될 수 있다. 또한, 안티센스 핵산은 하나 이상의 치환된 당 모이어티(sugar moiety)를 포함할 수 있다. 안티센스 핵산은 변형된 염기를 포함할 수 있다. 변형된 염기에는 하이포크잔틴, 6-메틸아데닌, 5-메틸피리미딘(특히 5-메틸시토신), 5-하이드록시메틸시토신(HMC), 글리코실 HMC, 젠토비오실 HMC, 2-아미노아데닌, 2-티오우라실, 2-티오티민, 5-브로모우라실, 5-하이드록시메틸우라실, 8-아자구아닌, 7-데아자구아닌, N6(6-아미노헥실)아데닌, 2,6-디아미노퓨린 등이 있다. 또한, 상기 안티센스 핵산은 상기 안티센스 핵산의 활성 및 세포 흡착성을 향상시키는 하나 이상의 모이어티(moiety) 또는 컨쥬게이트(conjugate)와 화학적으로 결합될 수 있다. 콜레스테롤 모이어티, 콜레스테릴 모이어티, 콜릭산, 티오에테르, 티오콜레스테롤, 지방성 사슬, 인지질, 폴리아민, 폴리에틸렌 글리콜 사슬, 아다맨탄 아세트산, 팔미틸 모이어티, 옥타데실아민, 헥실아미노카르보닐-옥시콜에스테롤 모이어티 등의 지용성 모이어티 등이 있으며 이에 제한되지 않는다. 상기 안티센스 올리고뉴클레오타이드는 통상의 방법으로 시험관에서 합성되어 생체 내로 투여하거나 생체 내에서 안티센스 올리고뉴클레오타이드가 합성되도록 할 수 있다.In addition, the antisense nucleic acid may be modified at the position of one or more bases, sugars or backbones to enhance efficacy. The nucleic acid backbone can be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyls, cycloalkyls, short chain heteroatomics, heterocyclic intersugar linkages, and the like. Antisense nucleic acids may also include one or more substituted sugar moieties. Antisense nucleic acids may include modified bases. Modified bases include hypoxanthine, 6-methyladenine, 5-methylpyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosyl HMC, 2-aminoadenine, 2 -Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, 2,6-diaminopurine, etc. There is this. In addition, the antisense nucleic acid may be chemically bound to one or more moieties or conjugates that enhance the activity and cell adsorption of the antisense nucleic acid. Cholesterol moiety, cholesteryl moiety, cholic acid, thioether, thiocholesterol, fatty chain, phospholipid, polyamine, polyethylene glycol chain, adamantane acetic acid, palmityl moiety, octadecylamine, hexylaminocarbonyl-oxychol fat soluble moieties such as esterol moieties, and the like. The antisense oligonucleotide may be synthesized in vitro by a conventional method and administered in vivo, or the antisense oligonucleotide may be synthesized in vivo.
본 발명에서 "siRNA”란 RNA 방해 또는 유전자 사일런싱을 매개할 수 있는 핵산 분자를 의미한다. siRNA는 표적 유전자의 발현을 억제할 수 있기 때문에 효율적인 유전자 넉다운(knockdown) 방법 또는 유전자치료 방법으로 제공된다.In the present invention, "siRNA" refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing. Since siRNA can inhibit the expression of a target gene, it is provided as an efficient gene knockdown method or gene therapy method .
본 발명의 siRNA 분자는, 센스 가닥(본 발명의 일 구현예에 따르면, 타겟 유전자인 PDK1 유전자의 mRNA 서열에 상응하는 서열)과 안티센스 가닥(상기 mRNA 서열에 상보적인 서열)이 서로 반대쪽에 위치하여 이중쇄를 이루는 구조를 가질 수 있으며, 본 발명의 siRNA 분자는 자기-상보성(self-complementary) 센스 및 안티센스 가닥을 가지는 단일쇄 구조를 가질 수 있다. 나아가 siRNA는 RNA끼리 짝을 이루는 이중사슬 RNA 부분이 완전히 쌍을 이루는 것에 한정되지 않고 미스매치(대응하는 염기가 상보적이지 않음), 벌지(일방의 사슬에 대응하는 염기가 없음) 등에 의하여 쌍을 이루지 않는 부분이 포함될 수 있다. 또한, siRNA 말단 구조는 타겟 유전자의 발현을 RNAi 효과에 의하여 억제할 수 있는 것이면 평활(blunt) 말단 혹은 점착(cohesive) 말단 모두 가능하고, 점착 말단 구조는 3'-말단 돌출 구조와 5'-말단 돌출 구조 모두 가능하다. siRNA를 제조하는 방법은 시험관에서 siRNA를 직접 합성한 뒤, 형질전환 과정을 거쳐 세포 안으로 도입시키는 방법과 siRNA가 세포 안에서 발현되도록 제조된 siRNA 발현 벡터 또는 PCR-유래의 siRNA 발현 카세트 등을 세포 안으로 형질전환 또는 감염(infection)시키는 방법이 있다.In the siRNA molecule of the present invention, the sense strand (according to one embodiment of the present invention, a sequence corresponding to the mRNA sequence of the target gene, PDK1 gene) and the antisense strand (sequence complementary to the mRNA sequence) are located opposite to each other. It may have a double-stranded structure, and the siRNA molecule of the present invention may have a single-stranded structure having self-complementary sense and antisense strands. Furthermore, siRNA is not limited to the complete pairing of double-stranded RNA parts that are paired with each other, but pairs are formed by mismatch (corresponding bases are not complementary), bulges (there is no base corresponding to one chain), etc. There may be parts that are not achieved. In addition, the siRNA end structure can have either a blunt end or a cohesive end as long as it can suppress the expression of a target gene by the RNAi effect, and the cohesive end structure includes a 3'-end protrusion structure and a 5'-end structure. Both protrusion structures are possible. Methods for producing siRNA include direct synthesizing siRNA in a test tube and introducing it into cells through transformation, and transfecting an siRNA expression vector or PCR-derived siRNA expression cassette prepared so that siRNA is expressed in cells into cells. There is a method of converting or infecting.
본 발명에서 “shRNA”란 small hairpin RNA 혹은 short hairpin RNA으로 불리며, RNA 간섭으로 유전자를 침묵시킬 수 있는 용도로 사용되는 것이다. 보통 벡터를 이용하여 목적 세포로 도입한다. 이러한 shRNA 헤어핀 구조는 세포 내 다른 물질에 의하여 절단되어 siRNA가 되기도 한다.In the present invention, “shRNA” is called small hairpin RNA or short hairpin RNA, and is used for the purpose of silencing genes by RNA interference. Usually, a vector is used to introduce into the target cell. This shRNA hairpin structure is cleaved by other substances in the cell to become siRNA.
본 발명에서 “항체”란 생체의 면역계에서 혈액이나 림프를 순환하면서 외부 물질인 항원이 침입한 경우 이에 반응하는 물질을 말하며, 림프조직에서 형성되는 글로불린계 단백질로 면역글로불린이라고도 불린다. 항체는 B 세포가 생산해서 체액으로 흘려보내는 단백질로 항원과 특이적으로 결합하며, 하나의 항체 분자에는 두 개의 중사슬 (heavy chain)과 두 개의 경사슬 (light chain)이 있으며, 각각의 중사슬과 경사슬은 그의 N-터미널 말단에 가변영역을 갖고 있다. 각각의 가변영역은 3 개의 상보성 결정부위 (Complementarity determining region: CDR)와 4 개의 구조 형성부위 (framework regions: FRs)로 구성되는데, 상보성 결정 부위들은 항체의 항원 결합 특이성을 결정하고, 가변영역의 구조형성 부위들로 유지되는 비교적 짧은 펩티드 서열로 존재한다. 본 발명의 목적상 상기 항체는 PDK1과 결합하여 PDK1의 활성을 억제할 수 있는 항체일 수 있다.In the present invention, the term “antibody” refers to a substance that reacts when an antigen, which is a foreign substance, invades while circulating blood or lymph in the immune system of a living body. Antibodies are proteins produced by B cells and released into body fluids and specifically bind to antigens. One antibody molecule has two heavy chains and two light chains, and each heavy chain and light chains have a variable region at their N-terminal end. Each variable region consists of three complementarity determining regions (CDRs) and four framework regions (FRs). The complementarity determining regions determine the antigen-binding specificity of an antibody, and the structure of the variable region It exists as a relatively short peptide sequence that is maintained by forming sites. For the purposes of the present invention, the antibody may be an antibody capable of inhibiting the activity of PDK1 by binding to PDK1.
또한, 본 발명에 있어서, 상기 PDK1 저해제는 mTOR(mammalian Target Of Rapamycin) 및 NF-кB(Nuclear Factor Kappa B)의 전사활성을 저해하는 것을 특징으로 할 수 있다. 본 발명에 따른 PDK1 저해제는 따라서 mTOR 및 NF-Кb를 동시에 조절하는 것을 특징으로 할 수 있다.In addition, in the present invention, the PDK1 inhibitor may be characterized in that it inhibits the transcriptional activity of mTOR (mammalian target of rapamycin) and NF-кB (nuclear factor Kappa B). The PDK1 inhibitor according to the present invention can thus be characterized as simultaneously modulating mTOR and NF-Кb.
본 발명에 따른 역분화 유도용 조성물은 생체 내(in vivo) 및 생체 외(in vitro) 처리가 모두 가능하며, 예를 들어 약학적 조성물, 건강기능식품 조성물, 화장료 조성물, 배지 첨가용 조성물, 시약 조성물 등의 다양한 형태로 이용될 수 있다.The composition for inducing reverse differentiation according to the present invention can be treated both in vivo and in vitro , for example, pharmaceutical compositions, health functional food compositions, cosmetic compositions, media addition compositions, reagents It can be used in various forms, such as a composition.
또한, 본 발명은 PDK1 저해제를 포함하는 세포 노화 또는 노화 관련 질환의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating cellular senescence or senescence-related diseases comprising a PDK1 inhibitor.
본 발명에서 “노화”란 시간이 지남에 따라 일어나는 신체의 모든 생리적 변화를 통칭하는 것으로 개체에 따라 수많은 요인에 의해 매우 다양하게 일어나는 생명 현상을 의미한다. 노화 현상을 구체적으로 살펴보면 각 구성 기관 및 조직의 기능 변화가 일어나는 것으로, 개체의 노화는 결국 그 개체를 구성하는 세포들의 노화에 기인한다.In the present invention, “aging” refers to all physiological changes of the body that occur over time, and refers to a life phenomenon that occurs in a very diverse manner due to numerous factors depending on the individual. If we look at the aging phenomenon in detail, the functional changes of each constituent organ and tissue occur, and the aging of an individual is ultimately due to the aging of the cells constituting the individual.
본 발명에 있어서, 상기 노화 관련 질환은 탈모, 주름, 곱사등, 골다공증, 류마티스 관절염(Rheumatoid Arthritis), 천식(Asthma), 피부염(Dermititis), 건선(Psoriasis), 낭섬유증(Cystic Fibrosis), 고형장기 이식 후기 및 만성 거부증(Post transplantation late and chronic solid organ rejection), 다발성 경화증(Multiple Sclerosis), 전신성 홍반성 루푸스(systemic lupus erythematosus), 쇼그렌 증후군(Sjogren syndrome), 하시모토 갑상선(Hashimoto thyroiditis), 다발성근염(polymyositis), 경피증(scleroderma), 아디슨병(Addison disease), 백반증(vitiligo), 악성빈혈(pernicious anemia), 사구체신염(glomerulonephritis), 폐섬유증(pulmonary fibrosis), 염증성장질환(Inflammatory Bowel Dieseses), 자가면역성 당뇨(Autoimmune Diabetes), 당뇨 망막증(Diabetic retinopathy), 비염(Rhinitis), 혀혈-재관류 손상(Ischemia-reperfusion injury), 혈관성형술후 재협착(Post-angioplasty restenosis), 만성 폐색성 심장 질환(Chronic obstructive pulmonary diseases; COPD), 그레이브병(Graves disease), 위장관 알러지(Gastrointestinal allergies), 결막염(Conjunctivitis), 죽상경화증(Atherosclerosis), 관상동맥질환(Coronary artery disease), 협심증(Angina), 암(Cancer), 소동맥 질환 및 이식편대숙주질환(graft-versus-host disease)으로 이루어진 군으로부터 선택되는 1종 이상일 수 있고, 바람직하게는 류마티스 관절염, 피부염, 낭섬유증, 폐섬유증, 염증성장질환, 죽상경화증, 관상동맥질환협심증 및 암으로 이루어진 군으로부터 선택되는 것일 수 있으나, 이에 제한되지는 않는다.In the present invention, the aging-related diseases are hair loss, wrinkles, humpbacks, osteoporosis, rheumatoid arthritis, asthma, dermatitis, psoriasis, cystic fibrosis, solid organ transplantation Post transplantation late and chronic solid organ rejection, Multiple Sclerosis, systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis ), scleroderma, Addison disease, vitiligo, pernicious anemia, glomerulonephritis, pulmonary fibrosis, Inflammatory Bowel Dieseses, autoimmunity Autoimmune Diabetes, Diabetic retinopathy, Rhinitis, Ischemia-reperfusion injury, Post-angioplasty restenosis, Chronic obstructive pulmonary disease diseases; COPD, Graves disease, Gastrointestinal allergies, Conjunctivitis, Atherosclerosis, Coronary artery disease, Angina, Cancer, Arterioles It may be at least one selected from the group consisting of diseases and graft-versus-host disease, and , Preferably, it may be selected from the group consisting of rheumatoid arthritis, dermatitis, cystic fibrosis, pulmonary fibrosis, inflammatory bowel disease, atherosclerosis, coronary artery disease angina, and cancer, but is not limited thereto.
본 발명에 있어서 상기 약학적 조성물은 캡슐, 정제, 과립, 주사제, 연고제, 분말 또는 음료 형태임을 특징으로 할 수 있으며, 상기 약학적 조성물은 인간을 대상으로 하는 것을 특징으로 할 수 있다. 상기 약학적 조성물은 이들로 한정되는 것은 아니지만, 각각 통상의 방법에 따라 산제, 과립제, 캡슐, 정제, 수성 현탁액 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. In the present invention, the pharmaceutical composition may be characterized in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be characterized in that it is targeted to humans. The pharmaceutical composition is not limited thereto, but may be formulated in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. .
본 발명에 따른 약학적 조성물은 약제학적으로 허용가능한 담체를 포함할 수 있다. 약제학적으로 허용되는 담체는 경구투여시에는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등을 사용할 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등을 혼합하여 사용할 수 있으며, 국소투여용의 경우에는 기제, 부형제, 윤활제, 보존제 등을 사용할 수 있다. 본 발명에 따른 약학적 조성물의 제형은 상술한 바와 같은 약제학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구투여 시에는 정제, 트로키, 캡슐, 엘릭서(elixir), 서스펜션, 시럽, 웨이퍼 등의 형태로 제조할 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수회 투약 형태로 제조할 수 있다. The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, fragrances, etc. for oral administration, and in the case of injections, buffers, preservatives, pain-free agents A topical agent, solubilizer, isotonic agent, stabilizer, etc. can be mixed and used, and in the case of topical administration, a base, excipient, lubricant, preservative, etc. can be used. The dosage form of the pharmaceutical composition according to the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in the form of unit dose ampoules or multiple doses. have.
한편, 제제화에 적합한 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말디톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유 등이 사용될 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다.Meanwhile, examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used. In addition, it may further include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.
본 발명에 따른 약학적 조성물의 투여 경로는 이들로 한정되는 것은 아니지만 구강, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장이 포함된다. 상기 "비경구"는 피하, 피내, 정맥내, 근육내, 관절내, 활액낭내, 흉골내, 경막내, 병소내 및 두개골 내 주사 또는 주입기술을 포함한다. 본 발명에 따른 약학적 조성물은 또한 직장 투여를 위한 좌제의 형태로 투여될 수 있다.The route of administration of the pharmaceutical composition according to the present invention is not limited thereto, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical , sublingual or rectal. The term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition according to the present invention may also be administered in the form of a suppository for rectal administration.
본 발명에 따른 약학적 조성물은 사용된 특정 유효성분의 활성, 연령, 체중, 일반적인 건강, 성별, 정식, 투여시간, 투여경로, 배출율, 약물 배합 및 예방 또는 치료될 특정 질환의 중증을 포함한 여러 요인에 따라 다양하게 변할 수 있고, 상기 약학적 조성물의 투여량은 환자의 상태, 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만 당업자에 의해 적절하게 선택될 수 있다. 바람직하게는 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여할 수 있으며, 더욱 바람직하게는 1~10000㎍/체중kg/day, 더욱더 바람직하게는 10~1000㎎/체중kg/day의 유효용량으로 하루에 수회 반복 투여될 수 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The pharmaceutical composition according to the present invention contains several factors including the activity of the specific active ingredient used, age, weight, general health, sex, formula, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, disease severity, drug form, administration route and period, but may be appropriately selected by those skilled in the art. Preferably, taking all of the above factors into consideration, it is possible to administer an amount that can obtain the maximum effect with a minimum amount without side effects, more preferably 1 to 10000 μg/weight kg/day, even more preferably 10 to 1000 mg It can be administered repeatedly several times a day at an effective dose of /weight kg/day. The above dosage does not limit the scope of the present invention in any way.
본 발명에 따른 세포 노화는 섬유아세포, 혈관내피세포, 근섬유육모세포, 조골세포, 연골세포, 심근세포, 조혈모세포, 간세포, 췌장세포, 생식세포 및 각질세포로 이루어진 군으로부터 선택된 1종 이상의 노화일 수 있으며, 바람직하게는 섬유아세포 또는 혈관내피세포의 노화일 수 있으나, 이에 제한되지는 않는다.Cellular aging according to the present invention is at least one selected from the group consisting of fibroblasts, vascular endothelial cells, myofibroblastic cells, osteoblasts, chondrocytes, cardiomyocytes, hematopoietic stem cells, hepatocytes, pancreatic cells, germ cells, and keratinocytes. It may be, and preferably may be aging of fibroblasts or endothelial cells, but is not limited thereto.
본 발명에 따른 세포 노화 또는 노화 관련 질환의 예방 또는 치료용 약학적 조성물은 추가적으로 다른 노화 관련 질환의 약제와 병용투여할 수 있다. 또한, 단독으로 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The pharmaceutical composition for preventing or treating cellular aging or aging-related diseases according to the present invention may be additionally administered in combination with other agents for aging-related diseases. In addition, it can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.
또한, 본 발명은 PDK1 저해제를 포함하는 세포 노화 또는 노화 관련 질환의 예방 또는 개선용 건강기능식품을 제공한다.In addition, the present invention provides a health functional food for preventing or improving cellular aging or aging-related diseases comprising a PDK1 inhibitor.
본 발명에 따른 건강기능식품은 세포 노화 또는 노화 관련 질환의 예방 및 개선을 목적으로, 정제, 캅셀, 분말, 과립, 액상, 환 등의 형태로 제조 및 가공할 수 있다.The health functional food according to the present invention may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing and improving cellular aging or aging-related diseases.
본 발명에서 “건강기능식품”이란 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 말하며, 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다.In the present invention, “health functional food” refers to food manufactured and processed using raw materials or ingredients useful for the human body according to Health Functional Food Act No. 6727, and controls nutrients for the structure and function of the human body. It means to consume for the purpose of obtaining useful effects for health purposes, such as physiological action or the like.
본 발명에 따른 건강기능식품은 통상의 식품 첨가물을 포함할 수 있으며, 식품 첨가물로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전청에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.The health functional food according to the present invention may contain normal food additives, and, unless otherwise specified, whether it is suitable as a food additive is applied to the item according to the general rules and general test methods of food additives approved by the Food and Drug Administration. It is judged according to the relevant standards and standards.
상기 “식품 첨가물 공전”에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼슘, 니코틴산, 계피산 등의 화학적 합성물; 감색소, 감초 추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물; L-글루타민산나트륨 제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합제제류 등을 들 수 있다.Examples of the items listed in the “Food Additives Code” include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as dark pigment, licorice extract, crystalline cellulose, high pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, noodles added alkalis, preservatives, and tar dye preparations.
예를 들어, 정제 형태의 건강기능식품은 본 발명의 유효성분인 PDK1의 저해제를 부형제, 결합제, 붕해제 및 다른 첨가제와 혼합한 혼합물을 통상의 방법으로 과립화한 다음, 활택제 등을 넣어 압축성형하거나, 상기 혼합물을 직접 압축 성형할 수 있다. 또한 상기 정제 형태의 건강기능식품은 필요에 따라 교미제 등을 함유할 수도 있다.For example, for health functional food in tablet form, a mixture obtained by mixing the PDK1 inhibitor, the active ingredient of the present invention, with an excipient, binder, disintegrant and other additives is granulated in a conventional manner, and then a lubricant is added and compressed. Or, the mixture may be directly compression molded. In addition, the health functional food in the form of tablets may contain a corrosive agent and the like, if necessary.
캅셀 형태의 건강기능식품 중 경질 캅셀제는 통상의 경질 캅셀에 본 발명의 유효성분인 PDK1의 저해제를 부형제 등의 첨가제와 혼합한 혼합물을 충진하여 제조할 수 있으며, 연질 캅셀제는 유효성분인 PDK1의 저해제를 부형제 등의 첨가제와 혼합한 혼합물을 젤라틴과 같은 캅셀기제에 충진하여 제조할 수 있다. 상기 연질 캅셀제는 필요에 따라 글리세린 또는 소르비톨 등의 가소제, 착색제, 보존제 등을 함유할 수 있다.Among health functional foods in capsule form, hard capsules can be prepared by filling a conventional hard capsule with a mixture of the active ingredient, PDK1 inhibitor of the present invention, mixed with additives such as excipients, and soft capsules are active ingredient, PDK1 inhibitor It can be prepared by filling a mixture mixed with additives such as excipients in a capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.
환 형태의 건강기능식품은 본 발명의 유효성분인 PDK1의 저해제와 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 성형하여 조제할 수 있으며, 필요에 따라 백당이나 다른 제피제로 제피할 수 있으며, 또는 전분, 탈크와 같은 물질로 표면을 코팅할 수도 있다.The health functional food in the form of a ring can be prepared by molding a mixture of an inhibitor of PDK1, which is the active ingredient of the present invention, an excipient, a binder, a disintegrant, etc. by a known method. It can be peeled off, or the surface can be coated with a material such as starch or talc.
과립 형태의 건강기능식품은 본 발명의 유효성분인 PDK1의 저해제와 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 입상으로 제조할 수 있으며, 필요에 따라 착향제, 교미제 등을 함유할 수 있다.A health functional food in the form of granules can be prepared in a granular form by a conventionally known method by mixing a mixture of an inhibitor of PDK1, which is the active ingredient of the present invention, an excipient, a binder, a disintegrant, etc. and the like.
상기 건강기능식품은 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제 및 건강보조식품류 등일 수 있다.The health functional food may be beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements.
또한, 본 발명은 생체 외(In vitro)에서 PDK1 저해제를 노화된 세포에 처리하는 단계를 포함하는 역노화 유도 방법을 제공한다.In addition, the present invention provides a method for inducing reverse aging comprising the step of treating senescent cells with a PDK1 inhibitor in vitro .
또한, 본 발명은 (a) 분리된 노화된 세포에 후보물질을 처리하는 단계; (b) 상기 후보물질이 처리된 노화된 세포에서 PDK1 발현 수준을 측정하는 단계; 및 (c) 상기 (b) 단계에서 측정된 PDK1 발현 수준이 후보물질이 처리되지 않은 분리된 노화된 세포에 비해 낮은 경우, 상기 후보물질을 노화 관련 질환의 예방 또는 치료 제제로 사용할 수 있을 것으로 판정하는 단계; 를 포함하는 노화 관련 질환의 예방 또는 치료 제제의 스크리닝 방법을 제공한다.In addition, the present invention comprises the steps of (a) treating the candidate material to the isolated senescent cells; (b) measuring the PDK1 expression level in senescent cells treated with the candidate substance; And (c) when the PDK1 expression level measured in step (b) is low compared to isolated senescent cells that are not treated with the candidate substance, it is determined that the candidate substance can be used as a prophylactic or therapeutic agent for senescence-related diseases to do; It provides a screening method of a preventive or therapeutic agent for a senescence-related disease comprising a.
본 발명의 스크리닝 방법에 따르면, 먼저 분리된 노화된 세포에 분석하고자 하는 후보물질을 접촉시킬 수 있다. 상기 후보물질은 PDK1의 발현량, 단백질의 양 또는 단백질의 활성에 영향을 미치는지의 여부를 검사하기 위하여 스크리닝에서 이용되는 미지의 물질을 의미한다. 상기 시료는 화학물질, 안티센스 올리고뉴클레오티드, 안티센스-RNA, siRNA, shRNA, miRNA, 상기 단백질에 특이적인 항체 또는 천연물 추출물을 포함할 수 있으나, 이에 제한되지 않는다.According to the screening method of the present invention, a candidate substance to be analyzed can be contacted with the isolated senescent cells first. The candidate substance refers to an unknown substance used in screening to examine whether it affects the expression level of PDK1, the amount of the protein, or the activity of the protein. The sample may include a chemical substance, antisense oligonucleotide, antisense-RNA, siRNA, shRNA, miRNA, an antibody specific for the protein, or a natural product extract, but is not limited thereto.
이후, 후보물질이 처리된 세포에서 상기 유전자의 발현량, 단백질의 양 또는 단백질의 활성을 측정할 수 있으며, 측정 결과 상기 유전자의 발현량, 단백질의 양 또는 단백질의 활성이 감소되는 것이 측정되면 상기 후보물질은 노화 관련 질환을 치료 또는 예방할 수 있는 제제로 사용할 수 있을 것으로 판정할 수 있다.Thereafter, the expression level of the gene, the amount of the protein, or the activity of the protein can be measured in the cell treated with the candidate substance, and when it is measured that the expression level of the gene, the amount of the protein, or the activity of the protein is reduced as a result of the measurement, the Candidate substances can be determined to be usable as agents that can treat or prevent aging-related diseases.
본 발명에 있어서, 유전자의 발현 수준 또는 단백질의 양을 측정하는 방법은 공지의 기술을 이용하여 생물학적 시료로부터 mRNA 또는 단백질을 분리하는 공지의 공정을 포함하여 수행될 수 있다. 본 발명에서 상기 "생물학적 시료"란 상기 유전자의 발현 수준 또는 단백질의 수준이 정상 대조군과는 다른, 생체로부터 채취된 시료를 말하며, 상기 시료로는 예를 들면, 조직, 세포, 혈액, 혈청, 혈장, 타액 및 뇨 등이 포함될 수 있으나, 이에 제한되지 않는다.In the present invention, the method for measuring the expression level of a gene or the amount of protein may be performed including a known process for isolating mRNA or protein from a biological sample using a known technique. In the present invention, the "biological sample" refers to a sample collected from a living body in which the expression level of the gene or the level of the protein is different from that of the normal control, and the sample includes, for example, tissues, cells, blood, serum, plasma. , saliva and urine may be included, but is not limited thereto.
상기 유전자의 발현 수준 측정은 바람직하게는 mRNA의 수준을 측정하는 것이며, mRNA의 수준을 측정하는 방법으로는 역전사 중합효소연쇄반응(RT-PCR), 실시간 역전사 중합효소연쇄반응, RNase 보호 분석법, 노던 블럿 및 DNA 칩 등이 있으나, 이에 제한되지 않는다.Measurement of the expression level of the gene is preferably to measure the level of mRNA, and as a method for measuring the level of mRNA, reverse transcription polymerase chain reaction (RT-PCR), real-time reverse transcription polymerase chain reaction, RNase protection assay, Northern blots and DNA chips, but are not limited thereto.
상기 단백질 수준의 측정은 항체를 이용할 수 있는데, 이러한 경우 생물학적 시료 내의 상기 마커 단백질과 이에 특이적인 항체는 결합물, 즉, 항원-항체 복합체를 형성하며, 항원-항체 복합체의 형성량은 검출 라벨 (detection label)의 시그널의 크기를 통해서 정량적으로 측정할 수 있다. 이러한 검출 라벨은 효소, 형광물, 리간드, 발광물, 미소입자(microparticle), 레독스 분자 및 방사선 동위원소로 이루어진 그룹 중에서 선택할 수 있으며, 이에 제한되지 않는다. 단백질 수준을 측정하기 위한 분석 방법으로는 웨스턴 블랏, ELISA, 방사선면역분석, 방사선 면역 확산법, 오우크테로니 면역 확산법, 로케트 면역전기영동, 조직면역 염색, 면역침전 분석법, 보체 고정분석법, FACS, 단백질 칩 등이 있으나, 이에 제한되지 않는다.The protein level can be measured using an antibody. In this case, the marker protein in the biological sample and an antibody specific therefor form a binding substance, that is, an antigen-antibody complex, and the amount of the antigen-antibody complex formed is determined by the detection label ( It can be quantitatively measured through the magnitude of the signal of the detection label). The detection label may be selected from the group consisting of an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, a redox molecule, and a radioisotope, but is not limited thereto. Analysis methods for measuring protein levels include Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, Oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chips, and the like, but are not limited thereto.
본 명세서에서 달리 정의되지 않은 용어들은 본 발명이 속하는 기술분야에서 통상적으로 사용되는 의미를 갖는 것이다.Terms not defined otherwise herein have the meanings commonly used in the art to which the present invention pertains.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.
실시예 1. 노화 네트워크 모델 구축Example 1. Aging network model construction
기존 문헌 정보를 기반으로 41개의 노드로 구성된 노화 네트워크 모델을 구축하고 이를 도 1에 나타내었다.An aging network model consisting of 41 nodes was constructed based on the existing literature information, and this is shown in FIG. 1 .
네트워크는 세포 노화를 일으키는 세 가지 주요 신호전달경로인 세포의 성장과 분열을 담당하는 IGF1-PI3K-mTOR 신호전달경로와, DNA damage-P53-RB 신호전달경로 및 SASP를 주관하는 NF-kB 신호전달경로를 종합하여 구축하였으며, 네트워크에 포함된 77개 링크의 기존 문헌정보는 하기 표 1과 같다.The network consists of three major signaling pathways that cause cellular senescence: the IGF1-PI3K-mTOR signaling pathway responsible for cell growth and division, the DNA damage-P53-RB signaling pathway, and the NF-kB signaling pathway responsible for SASP. The routes were synthesized and constructed, and the existing literature information of 77 links included in the network is shown in Table 1 below.
실시예 2. 대규모 단백질 분석 실험을 통한 세포주기 상태 분석Example 2. Analysis of cell cycle status through large-scale protein assays
섬유아세포에 대하여 대규모 단백질 분석 실험을 수행하여 그 결과를 히트맵으로 나타내고 이를 도 2에 나타내었다. 구체적으로, 도 2의 A는 유전자 심볼 및 세포주기 상태 별로 각 유전자에 대응하는 시계열 RPPA(Reverse Phase Protein Assay) 데이터의 커브의 아래 면적을 나타낸 것이고, 세포주기 상태는 일시적 중단 상태(Quiescence), 영구적 중단 상태(Senescence) 또는 분열 상태(Proliferation)로 구분하였다. 도 2의 B는 커브 아래 면적 값을 0과 1로 이분화한 결과를 나타낸 것으로, 이를 위해서는 가중합 불리언 로직(weighted sum Boolean logic) 모델을 사용하였다. 상기 모델은 각 링크가 실수의 값을 가지고 각 노드가 기저 활성도를 가지고 있어 이를 통하여 노드의 상태가 0 또는 1로 정해지는 모델을 의미하며, 예를 들어 하기 그림에서
따라서 네트워크에 포함된 노드의 상태(0 또는 1)는 다른 노드들의 상태(0 또는 1)에 따라서 변하게 된다. 위의 방법을 이용하여 네트워크 전체의 노드들의 상태를 N번 갱신하였을 때의 모든 노드들의 상태가 (N+1)번 갱신하였을 때의 모든 노드들의 상태와 같다면 고정 끌개에 도달하였다고 하며, N번 갱신하였을 때의 모든 노드들의 상태가 (N-M)번 갱신하였을 때의 모든 노드들의 상태와 같다면 크기가 M인 순환 끌개에 도달하였다고 표현한다(단, 1<M<N).Therefore, the state (0 or 1) of a node included in the network changes according to the state (0 or 1) of other nodes. If the state of all nodes when the state of the entire network is updated N times using the above method is the same as the state of all nodes when the state of all nodes is updated (N+1) times, it is said that a fixed attraction has been reached, If the state of all nodes at the time of updating is the same as the state of all nodes at the time of updating (N-M) times, it is expressed that a recursive attraction of size M has been reached (provided that 1<M<N).
실시예 3. 노화 네트워크 모델 시뮬레이션을 통한 최적 분자 타겟 발굴Example 3. Optimal molecular target discovery through aging network model simulation
진화 알고리즘을 통하여 2000개의 네트워크 모델들을 학습시키고, 이를 상기 실시예 1에서 구축한 노화 네트워크 모델에 적용하여 대규모 시뮬레이션하고 노화된 세포 상태를 젊은 세포 상태로 가역화시킬 수 있는 최적 분자 타겟을 발굴하였다. 이 때, 상기 진화알고리즘은 생물의 진화과정을 모방하여 개발된 최적화 기법으로 불리언 피팅과 같은 불연속적인 문제를 해결하는데 적절한 알고리즘이며, 본 실시예에서는 하기 비용 함수를 사용하였다.Through an evolutionary algorithm, 2,000 network models were trained, and by applying them to the aging network model constructed in Example 1, a large-scale simulation was performed, and an optimal molecular target capable of reversing the aged cell state to the young cell state was discovered. At this time, the evolution algorithm is an optimization technique developed by imitating the evolutionary process of living things, and is an algorithm suitable for solving discontinuous problems such as Boolean fitting. In this embodiment, the following cost function is used.
여기서, i는 각 상태(세포주기 일시적 중단 상태, 영구적 중단 상태, 분열 상태)를 의미하고 j는 각 41개의 노드 번호를 의미한다.
상기 진화 알고리즘을 통하여 학습된 2000개의 모델 네트워크에 대하여, 입력 노드 조합을 영구적 중단 상태(노화 상태)로 주어서 노화 끌개 상태로 보낸 다음, 입력 노드 조합을 유지한 상태에서 모든 노드의 단일 저해 시뮬레이션과 2개의 노드 조합의 저해 시뮬레이션을 진행하고 그 결과를 도 3에 나타내었다. 도 3에 나타낸 바와 같이, 저해 타겟의 유전자 심볼 중 PDK1이 노화된 세포 상태를 젊은 세포 상태로 가역화시킬 수 있는 최적 분자 타겟이 될 수 있음을 확인하였다.For 2000 model networks trained through the above evolutionary algorithm, the input node combination is given a permanent stop state (aging state) and sent to the aging attraction state, and then a single inhibition simulation of all nodes is performed while maintaining the input node combination. Inhibition simulation of the node combination was performed, and the results are shown in FIG. 3 . As shown in FIG. 3 , it was confirmed that PDK1 among the gene symbols of the inhibition target could be an optimal molecular target capable of reversing the aged cell state to the young cell state.
실시예 4. PDK1 억제에 따른 노화된 세포에서의 세포주기 변화 확인Example 4. Confirmation of cell cycle changes in senescent cells according to PDK1 inhibition
PDK1 억제에 따라 노화된 세포가 정상 젊은 세포로 가역화되는지 여부를 확인하기 위하여, 노화된 섬유아세포에 PDK1 저해제(BX-795, Selleckchem)를 처리하고 Beta-galactosidase assay, Wound healing assay 및 형광염색실험을 수행하여 세포주기 변화 양상을 확인하였다. 구체적으로, IGF1 100ng/ml, 독소루비신(doxorubicin) 100ng/ml, 10% FBS, 글루코스(glucose) 4.5g/L을 포함하는 세포의 노화를 일으키는 배양 조건에 세포를 7일간 노출시켜 세포의 노화를 일으켰다. 이 때 대부분의 세포는 노화되며, 이후 동일한 배양 조건에서 7일간 PDK1 저해제를 처리하였다. 총 14일간의 처리가 종료되면 IGF1, 독소루비신, PDK1 저해제를 포함하지 않은 성장조건 배양액을 처리하고, 그로부터 하루가 지난 시점에 beta-galactosidase assay를 수행하고 그 결과를 도 4의 A에 나타내었다. 같은 조건으로 wound healing assay를 수행하였으며 성장 조건 배양액을 총 7일간 처리한 후 그 결과를 도 4의 B에 나타내었다. 세포의 분열 활성을 평가하기 위하여 ki-67 발현을 분석하고 그 결과를 도 4의 C에 나타내었다. In order to confirm whether senescent cells are reversible into normal young cells according to PDK1 inhibition, senescent fibroblasts are treated with a PDK1 inhibitor (BX-795, Selleckchem), and beta-galactosidase assay, Wound healing assay and fluorescence staining test was performed to confirm the cell cycle change pattern. Specifically, cell senescence was caused by exposing the cells to senescence-causing culture conditions including IGF1 100ng/ml, doxorubicin 100ng/ml, 10% FBS, and glucose 4.5g/L for 7 days. . At this time, most of the cells are aged, and then treated with a PDK1 inhibitor for 7 days under the same culture conditions. When the treatment for a total of 14 days was completed, the culture medium under growth conditions not containing IGF1, doxorubicin, and PDK1 inhibitors was treated, and a beta-galactosidase assay was performed one day after that, and the results are shown in FIG. 4A . The wound healing assay was performed under the same conditions, and the results were shown in FIG. 4B after treatment with the culture medium under growth conditions for a total of 7 days. To evaluate the cell division activity, ki-67 expression was analyzed and the results are shown in FIG. 4C .
도 4의 A 내지 C에 나타낸 바와 같이, PDK1 저해제를 노화된 섬유아세포에 처리하였을 때 세포의 노화상태가 줄어듦을 확인하였다. 나아가, ki-67 염색을 통하여 약물 처리 7일 경과 후 성장 조건의 배양액을 처리하였을 때 세포 중 일부가 분열하고 있음을 확인하였으며, 이를 통해 정상 조건의 배지 처리시 세포들이 다시 성장 및 분열함을 확인하였다.As shown in FIG. 4A to C, it was confirmed that the senescent state of the cells was reduced when the PDK1 inhibitor was treated with senescent fibroblasts. Furthermore, through ki-67 staining, it was confirmed that some of the cells were dividing when the culture medium was treated with the growth condition after 7 days of drug treatment. did
상기 결과를 통하여, 본 발명에 따른 PDK1은 세포노화에 관여하는 주요 신호전달 단백질 중 mTOR과 NF-кB를 동시에 조절하는 단백질로서 세포의 성장, 분열 및 노화 관련 분비표현형을 결정하는 핵심 단백질이며, 이를 억제 시 노화된 세포 중 일부가 효과적으로 정상 젊은 세포로 가역화되며, 이러한 세포들에서는 더 이상 노화된 세포의 표현형이 관찰되지 않는 것을 확인하였다. 따라서, PDK1은 노화된 세포를 사멸 유도하는 전략에 치중되어 있는 현재의 노화치료전략에 새로운 가능성을 제시하고, 항노화 물질 및 노화 관련 질환 치료제 개발에 유용하게 사용될 수 있어 관련 산업 분야에 큰 파급효과를 가져올 것으로 기대된다.Through the above results, PDK1 according to the present invention is a protein that simultaneously regulates mTOR and NF-κB among the major signaling proteins involved in cell aging, and is a key protein that determines the cell growth, division, and senescence-related secretory phenotype. Upon inhibition, some of the aged cells were effectively reversible into normal young cells, and it was confirmed that the phenotype of the aged cells was no longer observed in these cells. Therefore, PDK1 presents new possibilities for the current aging treatment strategy, which is focused on the strategy of inducing apoptosis of senescent cells, and can be usefully used in the development of anti-aging substances and treatments for aging-related diseases, which has a large ripple effect on related industries. is expected to bring
비록 본 발명이 상기에 언급된 바람직한 실시예로서 설명되었으나, 발명의 요지와 범위로부터 벗어남이 없이 다양한 수정이나 변형을 하는 것이 가능하다. 또한 첨부된 청구 범위는 본 발명의 요지에 속하는 이러한 수정이나 변형을 포함한다.Although the present invention has been described as the above-mentioned preferred embodiment, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also intended that the appended claims cover such modifications and variations as fall within the scope of the present invention.
Claims (10)
(b) 상기 후보물질이 처리된 노화된 세포에서 PDK1 발현 수준을 측정하는 단계;
(c) 상기 후보물질이 처리된 노화된 세포에 성장 조건을 처리하는 단계; 및
(d) 상기 (b) 단계에서 측정된 PDK1 발현 수준이 후보물질이 처리되지 않은 분리된 노화된 세포에 비해 낮고,
상기 (c) 단계의 후보물질 및 성장 조건 처리 전 세포 분열하지 않았던 노화된 세포가 후보 물질 처리 및 성장 조건 처리 후 분열하는 경우,
상기 후보물질을 분열이 중단된 노화된 세포를 분열가능 세포로 전환시킬 수 있는 물질로 판정하는 단계; 를 포함하는 분열이 중단된 노화된 세포를 성장조건 하에 분열이 가능한 세포로 전환시킬 수 있는 물질을 선별하는 스크리닝 방법.
(a) treating the candidate material to the senescent cells whose division has been stopped;
(b) measuring the PDK1 expression level in senescent cells treated with the candidate substance;
(c) treating the senescent cells treated with the candidate substance under growth conditions; and
(d) the PDK1 expression level measured in step (b) is lower than that of isolated senescent cells that are not treated with a candidate substance,
When the senescent cells that did not divide before treatment with the candidate material and growth conditions of step (c) divide after treatment with the candidate material and the growth conditions,
determining the candidate substance as a substance capable of converting senescent cells whose division is stopped into divisionable cells; A screening method for selecting substances capable of converting senescent cells whose division has been stopped into cells capable of division under growth conditions, comprising a.
The method according to claim 1, wherein the substance capable of converting the senescent cells in which division has been stopped into cells capable of dividing under growth conditions is to reinduce division and growth of the senescent cells in which division has been stopped. A screening method for selecting substances capable of converting stopped senescent cells into cells capable of dividing under growth conditions.
According to claim 1, wherein the aged cells are fibroblasts, vascular endothelial cells, myofibroblasts, osteoblasts, chondrocytes, cardiomyocytes, hematopoietic stem cells, hepatocytes, pancreatic cells, germ cells and keratinocytes 1 selected from the group consisting of A screening method for selecting a substance capable of converting senescent cells whose division is stopped into cells capable of division under growth conditions, characterized in that they are cells of a species.
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