WO2021006707A1 - Use of sgk1 inhibitor as therapeutic agent for inflammatory neurological diseases - Google Patents

Use of sgk1 inhibitor as therapeutic agent for inflammatory neurological diseases Download PDF

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WO2021006707A1
WO2021006707A1 PCT/KR2020/009141 KR2020009141W WO2021006707A1 WO 2021006707 A1 WO2021006707 A1 WO 2021006707A1 KR 2020009141 W KR2020009141 W KR 2020009141W WO 2021006707 A1 WO2021006707 A1 WO 2021006707A1
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sgk1
pyrrolo
pyridin
phenyl
disease
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이상훈
송재진
권오찬
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한양대학교 산학협력단
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Abstract

The present invention pertains to: a pharmaceutical composition for preventing or treating inflammatory neurological diseases, the pharmaceutical composition containing an Sgk1 inhibitor as an active ingredient; and a method for treating inflammatory neurological diseases using same. The effects of reducing neuronal aging, reducing neuroinflammation, increasing glutamate absorption, enhancing neuronal function and viability, and preventing α-synuclein aggregation can be achieved by inhibiting the expression of Sgk1, and thus the Sgk1 inhibitor can be advantageously used as a therapeutic agent for inflammatory neurological diseases.

Description

염증성 신경 질환의 치료제로서 Sgk1 저해제의 용도 Use of Sgk1 inhibitors as therapeutic agents for inflammatory neurological diseases
본 발명은 Sgk1 저해제의 신경세포 보호 및 신경염증 감소를 통한 염증성 신경 질환의 치료 용도에 관한 것이다.The present invention relates to the use of Sgk1 inhibitors in the treatment of inflammatory neurological diseases through neuronal protection and neuroinflammation reduction.
신경 퇴행성 질환을 비롯한 거의 대부분의 신경 질환은 기본적으로 염증에 의해 유발되거나 만성 염증을 동반한다. 이러한 신경 질환으로는 알쯔하이머병, 파킨슨병, 다발성 전신 위축, 근 위축성 측삭 경화증(ALS), 뇌경색, 척추 손상 등의 뇌신경 퇴행성 질환을 비롯하여 신경 염증(neuroinflammation)을 병인으로 하는 신경병증 통증(neuropathic pain), 복합통증증후군(complex region pain syndrome; CRPS) 등이 포함된다. Almost all neurological diseases, including neurodegenerative diseases, are basically caused by or accompanied by chronic inflammation. These neurological diseases include neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple systemic atrophy, amyotrophic lateral sclerosis (ALS), cerebral infarction and spinal injury, as well as neuropathic pain caused by neuroinflammation. , Complex region pain syndrome (CRPS), etc.
그 중에서도, 파킨슨병(Parkinson's disease)은 뇌의 염증성 환경에서 병의 진행이 가속화되며, 병변 부위의 직접적인 신약 개발 프로젝트 또한 계속적으로 실패하고 있다. 이러한 신경세포의 손실로 인해 유발되는 신경 퇴행성 질환에서 신경교세포의 기능 강화는 난치성 뇌 질환을 치료할 수 있는 잠재적 치료법으로 여겨지고 있다.Among them, Parkinson's disease accelerates the progression of the disease in the inflammatory environment of the brain, and the project of direct new drug development at the lesion site has also continued to fail. Reinforcing the function of glial cells in neurodegenerative diseases caused by the loss of these neurons is considered as a potential treatment that can treat intractable brain diseases.
신경 조직에서 교질세포는 성상세포(astrocyte)와 미세아교세포(microglia)를 포함하며, 이는 신경세포의 기능과 생존을 도와주는 보조세포이다. 그러나, 병적 상황(pathologic condition)에서는 교질세포가 염증 반응을 유발하여 오히려 신경세포를 손상하는 환경을 조성하는 방향으로 활성화(M1 activation)된다. 한편, 병적 환경을 조성하는 M1형 교질세포를 신경세포의 생존 및 재생을 도모하는 치료적 환경(therapeutic environment)을 조성하는 M2형 교질세포로 전환시킬 수 있음이 알려져 있다. 최근 이러한 신경교세포를 이로운 신경교세포로 전환시키는 방법은 중추신경계 질환의 치료에 있어서 새로운 패러다임으로 떠오르고 있다. In neural tissue, glial cells include astrocytes and microglia, which are auxiliary cells that help the function and survival of nerve cells. However, in a pathologic condition, the glial cells induce an inflammatory response and are rather activated (M1 activation) in the direction of creating an environment that damages nerve cells. Meanwhile, it is known that M1 type glial cells that create a pathological environment can be converted into M2 type glial cells that create a therapeutic environment that promotes survival and regeneration of neurons. Recently, the method of converting these glial cells into beneficial glial cells has emerged as a new paradigm in the treatment of central nervous system diseases.
이전 연구(한국 공개특허 제10-2017-0101813호(2017.09.06. 공개))에서 본 발명자들은 전사 인자인 Nurr1과 Foxa2의 공동 효과가 신경교세포의 염증 반응을 억제하는 것을 밝힌바 있으나, Nurr1과 Foxa2의 공동 작용에 대한 염증 억제 효과가 후속적으로 어떠한 방식에 의해 이루어지는지에 대해서는 언급되어 있지 않다. 이에, 본 발명은 이전 연구 결과를 토대로, Nurr1과 Foxa2의 과발현이 신경교세포에서 어떠한 작용기전을 통해 신경염증을 감소시키는지 확인하기 위함이다.In a previous study (Korean Patent Laid-Open No. 10-2017-0101813 (published on September 6, 2017)), the present inventors revealed that the joint effect of the transcription factors Nurr1 and Foxa2 inhibits the inflammatory response of glial cells, but Nurr1 and It is not stated how the inhibitory effect of the inflammation on the synergistic action of Foxa2 is subsequently achieved. Accordingly, the present invention is to determine whether overexpression of Nurr1 and Foxa2 reduces neuroinflammation through a mechanism of action in glial cells based on the results of previous studies.
한편, Sgk1(serum and glucocorticoid-regulated kinase 1)은 래트 유방 종양 세포에서 혈청과 글루코코르티코이드(GR)에 의해 상향 조절되는 유전자로 처음 발견되었다(Firestone, G. et al., Biochemistry, Stimulus-dependent regulation of serum and glucocorticoid inducible protein kinase (SGK) transcription, subcellular localization and enzymatic activity, 2003, 13(1), 1-12.). Sgk1의 발현은 뇌에서도 검출되었으며, 다양한 자극 및 Rett 증후군, 알쯔하이머병과 같은 병리학적 증상에 의해 증가된다. 특히 Sgk1은 여러 세포에서 발현되고 있으며, 뉴런의 세포 생존에 중요한 역할을 하고 NF-κB 신호를 조절하는 데 영향을 준다고 알려져 있다.Meanwhile, Sgk1 (serum and glucocorticoid-regulated kinase 1) was first discovered as a gene that is upregulated by serum and glucocorticoid (GR) in rat breast tumor cells (Firestone, G. et al., Biochemistry, Stimulus-dependent regulation). of serum and glucocorticoid inducible protein kinase (SGK) transcription, subcellular localization and enzymatic activity, 2003, 13(1), 1-12.). The expression of Sgk1 was also detected in the brain and is increased by various stimuli and pathological symptoms such as Rett syndrome and Alzheimer's disease. In particular, Sgk1 is expressed in several cells and is known to play an important role in neuronal cell survival and to regulate NF-κB signaling.
본 발명의 목적은 염증성 신경 질환을 예방, 개선 또는 치료하기 위한 Sgk1 저해제의 신규 용도를 제공하는데 있다.It is an object of the present invention to provide a novel use of an Sgk1 inhibitor for preventing, improving or treating inflammatory neurological diseases.
본 발명자들은 이전 연구를 통해 Nurr1 및 Foxa2가 과발현된 신경교세포에서 신경염증이 감소되는 결과를 확인하였으며, Nurr1 및 Foxa2가 과발현되면 Sgk1의 발현이 감소되는 것을 확인하였다. 이에, 본 발명자들은 Sgk1의 발현 저해를 통해 신경세포가 보호되고 신경염증이 억제되는 효과로부터 Sgk1 저해제가 염증성 신경 질환의 치료에 기여할 수 있음을 확인하고 본 발명을 완성하였다.The present inventors have confirmed that neuroinflammation is reduced in glial cells overexpressing Nurr1 and Foxa2 through previous studies, and it has been confirmed that the expression of Sgk1 is reduced when Nurr1 and Foxa2 are overexpressed. Accordingly, the present inventors confirmed that the Sgk1 inhibitor can contribute to the treatment of inflammatory neurological diseases from the effect of protecting nerve cells and inhibiting neuroinflammation through inhibition of the expression of Sgk1, and completed the present invention.
따라서, 본 발명은 신경염증의 감소를 위한 Sgk1의 신규한 작용기전에 관한 것이다.Accordingly, the present invention relates to a novel mechanism of action of Sgk1 for reducing neuroinflammation.
이하, 본 발명의 구성을 구체적으로 설명한다.Hereinafter, the configuration of the present invention will be described in detail.
본 발명은 Sgk1 저해제를 유효성분으로 포함하는 염증성 신경 질환의 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating inflammatory neurological diseases comprising an Sgk1 inhibitor as an active ingredient.
본 발명에서, “혈청 및 글루코코르티코이드 의존성 키나아제 1(serum and glucocorticoid-regulated kinase 1, Sgk1)”은 포유동물, 바람직하게는 인간, 생쥐, 집쥐, 토끼, 오랑우탄, 원숭이, 햄스터, 고양이, 돌고래, 고릴라 등에서 기원하는 단백질을 의미한다. 상기 Sgk1 유전자 및 단백질 서열은 공지되어 있다. 예컨대, 상기 Sgk1은 인간(Homo sapiens) 유래의 GenBank accession no. NP_001137148; 생쥐(Mus musculus) 유래의 Genbank accession no. NP_001155317 등의 아미노산 서열을 갖는 Sgk1 단백질일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, "serum and glucocorticoid-regulated kinase 1 (Sgk1)" is a mammal, preferably human, mouse, house mouse, rabbit, orangutan, monkey, hamster, cat, dolphin, gorilla It means a protein originating from the back. The Sgk1 gene and protein sequences are known. For example, the Sgk1 is human (Homo sapiens) derived from GenBank accession no. NP_001137148; Genbank accession no. from mouse (Mus musculus). It may be an Sgk1 protein having an amino acid sequence such as NP_001155317, but is not limited thereto.
상기 “Sgk1 저해제”는 염증성 신경 질환의 환자에서 신경염증의 감소 효과를 나타내는 약물을 의미한다. 예컨대, 상기 Sgk1 저해제는 Sgk1 유전자의 mRNA 발현을 넉다운(knock-down)시키거나, Sgk1 단백질의 기능 또는 활성을 감소시키는 모든 제제를 포함할 수 있다. 본 발명에 있어서, 상기 Sgk1 저해제는 Sgk1 유전자에 상보적인 서열을 포함하는 안티센스-올리고뉴클레오타이드, siRNA, shRNA, miRNA 또는 이를 포함하는 벡터; Sgk1 단백질에 특이적인 항체; 또는 화학식 1 또는 화학식 2로 표현되는 Sgk1 효소에 대한 저해 활성을 갖는 화합물; 중 어느 하나일 수 있다:The “Sgk1 inhibitor” refers to a drug exhibiting an effect of reducing neuroinflammation in patients with inflammatory neuropathy. For example, the Sgk1 inhibitor may include any agent that knocks down the mRNA expression of the Sgk1 gene or reduces the function or activity of the Sgk1 protein. In the present invention, the Sgk1 inhibitor is an antisense-oligonucleotide comprising a sequence complementary to the Sgk1 gene, siRNA, shRNA, miRNA, or a vector comprising the same; Antibodies specific for the Sgk1 protein; Or a compound having inhibitory activity against the Sgk1 enzyme represented by Formula 1 or Formula 2; It can be either:
[화학식 1][Formula 1]
Figure PCTKR2020009141-appb-img-000001
Figure PCTKR2020009141-appb-img-000001
[화학식 2][Formula 2]
Figure PCTKR2020009141-appb-img-000002
Figure PCTKR2020009141-appb-img-000002
한 구체예에서, 상기 Sgk1 저해제는 상기 화학식 1로 표현되는 화합물(GSK-650394) 및 화학식 2로 표현되는 화합물(EMD-638683) 중 하나 이상의 화합물일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment, the Sgk1 inhibitor may be one or more of the compound represented by Formula 1 (GSK-650394) and the compound represented by Formula 2 (EMD-638683), but is not limited thereto.
다른 구체예에서, 상기 Sgk1 저해제는 상기 화학식 1 또는 화학식 2로 표현되는 화합물 외에 Sgk1에 대해 저해 활성을 갖는 공지의 화합물로 대체하여 적용할 수 있다. 예컨대, 상기 화학식 1 또는 화학식 2로 표현되는 화합물을 대체할 수 있는 화합물은 US 2009//0233955 A1, US 2012/10238588 A1 또는 EP 2242738 B1에 개시된 화합물일 수 있다. 보다 구체적으로, 상기 화합물은 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; {3-[5-(2-naphthyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzyl}amine; 4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; {4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; 3-{4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; {3-[5-(2-naphthyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}methanol; 4-{5-[3-(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 3-(4-{5-[3-(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)propanoic acid; 3-{3-[4-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}benzonitrile; 4-{5-[3-(aminocarbonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 4-[5-(3-cyanophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-{5-[6-(methyloxy)-3-pyridinyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; m) 3-{3-[3-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}benzonitrile; 4-[5-(1-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; o) 2-fluoro-4-[5-(1-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 3-amino-5-[5-(1-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 3-{4-[5-(1-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 3-(4-{5-[3-(aminocarbonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)propanoic acid; 3-{4-[5-(3-cyanophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 3-(4-{5-[6-(methyloxy)-3-pyridinyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)propanoic acid; {4-[5-(1-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; (4-{5-[3-(aminocarbonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid; {4-[5-(3-cyanophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; (4-{5-[6-(methyloxy)-3-pyridinyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid; 3-{4-[5-(3-Methanesulfonylamino-phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}-propionic acid; {4-[5-(3-Methanesulfonylamino-phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}-acetic acid; 3-{4-[5-(3-Methanesulfonyl-phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}-propionic acid; {4-[5-(3-Methanesulfonyl-phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}-acetic acid; 3-[3-fluoro-4-(methyloxy)phenyl]-5-phenyl-1H-pyrrolo[2,3-b]pyridine; 5-phenyl-3-pyridin-4-yl-1H-pyrrolo[2,3-b]pyridine; 3-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; N-[3-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenylamine; 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenol; 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzylamine; 3-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenol; 5-(3,4-dimethoxyphenyl)-3-pyridin-4-yl-1H-pyrrolo[2,3-b]pyridine; 4-[5-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-yl]-phenol; 4-[5-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-yl]-phenylamine; 4-[5-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-benzoic acid; 4-[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-benzoic acid; N-[4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; 3,5-bis-(4-hydroxyphenyl)-1H-pyrrolo[2,3-b]pyridine; 3,5-bis-(4-carboxyphenyl)-1H-pyrrolo[2,3-b]pyridine; 4-[5-(4-aminophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzylamine; 4-[5-(4-aminophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; 4-[5-(2-fluoro-biphen-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; N-[3-(5-thiophen-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; 4-(5-thiophen-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; 4-(5-thiophen-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenol; 4-(5-thiophen-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzamide; N-[3-(5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; 4-(5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; 4-(5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenol; 4-(5-thiophen-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzylamine; 3-(1H-indol-5-yl)-5-thiophen-3-yl-1H-pyrrolo[2,3-b]pyridine; N-[4-(5-thiophen-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; 5-(3-pyridinyl)-3-(4-pyridinyl)-1H-indole; 4-(5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzamide; 4-[3-(2-fluorobiphenyl-4-yl-1H-pyrrolo[2,3-b]pyridin-5-yl]-benzylamine; 4-(5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenylamine; {3-[5-(4-methanesulfonylphenyl-1H-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}-acetic acid; N-[3-(3-thiophen-3-yl-1H-pyrrolo[2,3-b]pyridin-5-yl)-phenyl]-acetamide; N-{3-[3-(3-pyridinyl)-1H-pyrrolo[2,3-b]pyridin-5-yl]phenyl}acetamide; 4-[5-(3-acetylaminophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-benzoic acid; N-{3-[3-(2,3-difluorophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl]-phenyl}-acetamide; N-{3-[3-(4-hydroxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl]-phenyl}-acetamide; N-{3-[3-(4-aminomethylphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl]-phenyl}-acetamide; N-{3-[3-(4-aminophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl]-phenyl}-acetamide; N-{3-[3-(1H-indol-5-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl]phenyl}-acetamide; 4-[3-(2-fluorobiphenyl-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl]-benzoic acid; N-{3-[3-(4-pyridinyl)-1H-pyrrolo[2,3-b]pyridin-5-yl]phenyl}acetamide; N-{3-[5-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}-acetamide; 4-[5-(3-acetylaminophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-benzamide; 4-[5-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-benzoic acid; 4-[5-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-phenylamine; N-{4-[5-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}-acetamide; 4-[5-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-benzamide; ca) 2-chloro-N-[4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-benzamide; 2-phenyl-N-[4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; 2-chloro-N-[3-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzyl]-benzamide; 2-phenyl-N-[3-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzyl]-acetamide; (4-{5-[3-(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid; 3-[4-(5-{4-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl]propanoic acid; [4-(5-{4-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl]acetic acid; (4-{5-[3,4,5-tris(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid; 3-(4-{5-[3,4,5-tris(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)propanoic acid; {4-[5-(6-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; 3-{4-[5-(6-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; {4-[5-(3-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; {4-[5-(5-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; 3-{4-[5-(5-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 3-(4-{5-[6-(methyloxy)-2-naphthalenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)propanoic acid; 3-{4-[5-(3,4-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; {4-[5-(3,4-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; {4-[5-(2,3-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; 3-{4-[5-(2,3-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; {4-[5-(2,3-dichlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; 3-{4-[5-(2,3-dichlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; {4-[5-(1-benzothien-3-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; [(3-{5-[3-(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)methyl]amine; 7-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-1,2,3,4-tetrahydroisoquinoline; 2-fluoro-4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 2-fluoro-4-{5-[3-(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 2-methyl-4-{5-[3-(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 5-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-thiophenecarbaldehyde; 5-{5-[3-(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}-2-thiophenecarbaldehyde; 2-methyl-4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; (4-{5-[6-(methyloxy)-2-naphthalenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid; 2-fluoro-4-{5-[6-(methyloxy)-2-naphthalenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 2-fluoro-4-[5-(5-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(5-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; di) 2-methyl-4-[5-(5-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(3,4-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(3,4-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; 4-[5-(3,4-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; 4-[5-(2,3-dichlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(2,3-dichlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; 4-[5-(1-benzothien-3-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(1-benzothien-3-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; 4-[5-(1-benzothien-3-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; 6-{3-[4-(ethylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}quinoline; 4-(5-{3-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 3-[4-(butyloxy)phenyl]-5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridine; N-(3-{3-[4-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}phenyl)methane sulfonamide; N-(3-{3-[3-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}phenyl)methane sulfonamide; 3-amino-5-(5-{3-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 2-fluoro-4-(5-{3-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 3-amino-5-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 2-fluoro-4-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; [(4-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)methyl]amine; [(3-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)methyl]amine; 5-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}-2-thiophenecarbaldehyde; 4-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; N-(4-{3-[4-(butyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}phenyl)methanesulfonamide; 1,1-dimethylethyl[2-(3-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)ethyl]carbamate; 3-amino-5-(5-{4-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 2-fluoro-4-(5-{4-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 4-(5-{4-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; N-(4-{3-[4-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}phenyl)methane sulfonamide; N-(4-{3-[3-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}phenyl)methane sulfonamide; N-{4-[3-(5-formyl-2-thienyl)-1H-pyrrolo[2,3-b]pyridin-5-yl]phenyl}methanesulfonamide; 7-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}-1,2,3,4-tetrahydroisoquinoline; N-{3-[3-(1,2,3,4-tetrahydro-7-isoquinolinyl)-1H-pyrrolo[2,3-b]pyridin-5-yl]phenyl}methanesulfonamide; N-{4-[3-(1,2,3,4-tetrahydro-7-isoquinolinyl)-1H-pyrrolo[2,3-b]pyridin-5-yl]phenyl}methanesulfonamide; 2-methyl-4-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 5-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}-2-thiophenecarboxylic acid; 3-{3-[3-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}benzonitrile; 2-fluoro-4-{5-[6-(methyloxy)-3-pyridinyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 3-[3-(1,2,3,4-tetrahydro-7-isoquinolinyl)-1H-pyrrolo[2,3-b]pyridin-5-yl]benzonitrile; 7-{5-[3,4,5-tris(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}-1,2,3,4-tetrahydroisoquinoline; 4-{5-[3,4,5-tris(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 2-fluoro-4-{5-[3,4,5-tris(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 2-amino-4-{5-[3,4,5-tris(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 4-[5-(6-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(3-cyanophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; 4-[5-(2,3-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(2,3-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; 2-methyl-4-[5-(6-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 2-methyl-4-[5-(1-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(3-cyanophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; 2-methyl-4-{5-[6-(methyloxy)-3-pyridinyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 2-methyl-4-{5-[3,4,5-tris(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 2-(1-methylethyl)-4-[5-(1-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(3-cyanophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-(1-methylethyl)benzoic acid; 2-(1-methylethyl)-4-[5-(6-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(2,3-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; 2-chloro-4-{5-[3-(methylsulfonyl)phenyl]-1-H-pyrrolo-[2,3-b]pyridin-3-yl}benzoic acid; 4-{5-[3-(methylsulfonyl)phenyl]-1-H-pyrrolo-[2,3-b]pyridin-3-yl}-2,6-bis(trifluoromethyl)benzoic acid; methyl 2-(azidomethyl)-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoate; 2-ethyl-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 2-(methylamino)-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 2-(dimethylamino)-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 2-cyclopentyl-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-propylbenzoic acid; 2,6-difluoro-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 2,6-dimethyl-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 2-(2-propyl)-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 6-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-indazole; 2-(2-methylpropyl)-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 2-methyl-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 4-[5-(3-hydroxyphenyl)-1-H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 3-amino-5-[5-(3-hydroxyphenyl)1H-pyrrolo[2,3-b]pyridin-3-yl]-benzoic acid; {4-[5-hydroxyphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}acetic acid; 4-[5-(3-aminophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 3-{4-[5-(3-hydroxyphenyl)-1-H-pyrrolo[2,3-b]pyridine-3-yl]-phenyl}-propionic acid; 3-{4-[5-(3-aminophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; {4-[5-(3-aminophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; 4-{5-[3-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; (4-{5-[3-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid;) 4-[5-(3-hydroxyphenyl)-1-H-pyrrolo-[2,3-b]pyridin-3-yl]benzoic acid; 2-fluoro-4-[5-(3-hydroxyphenyl)-1-H-pyrrolo-[2,3-b]pyridin-3-yl]benzoic acid; 5-[5-(3-hydroxyphenyl)-1-H-pyrrolo-[2,3-b]pyridin-3-yl]thiophene-2-carbaldehyde; 3-{3-[3-(2-aminoethyl)phenyl]-1-H-pyrrolo-[2,3-b]pyridin-5-yl}phenol; 3-[3-(1,2,3,4-tetrahydroisoquinolin-7-yl)-1-H-pyrrolo-[2,3-b]pyridin-5-yl]phenol; 3-amino-5-[5-(3-aminophenyl)-1-H-pyrrolo-[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(3-aminophenyl)-1-H-pyrrolo-[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; 2-fluoro-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)phenol; 2,6-difluoro-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)phenol; 5-phenyl-3-[4-(1H-tetrazol-5-yl)phenyl]-1H-pyrrolo[2,3-b]pyridine; 5-(2-naphthalenyl)-3-[4-(1H-tetrazol-5-yl)phenyl]-1H-pyrrolo[2,3-b]pyridine; 3-[3-fluoro-4-(1H-tetrazol-5-yl)phenyl]-5-phenyl-1H-pyrrolo[2,3-b]pyridine; 2-methyl-2-(4-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)propanoic acid; 2-{4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 2-(4-{5-[3-(aminocarbonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)-2-methylpropanoic acid; 2-{4-[5-(3-cyanophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}-2-methylpropanoic acid; 2-methyl-2-{4-[5-(6-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 2-methyl-2-{4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 2-methyl-2-[4-(5-{3-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl]propanoic acid; 2-methyl-2-[4-(5-{4-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl]propanoic acid; 2-methyl-2-(4-{5-[6-(methyloxy)-2-naphthalenyl]-1H-pyrrolo[2,3-b]pyridin-3- yl}phenyl)propanoic acid; 2-methyl-2-{4-[5-(5-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 2-methyl-2-{4-[5-(3-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 2-{4-[5-(6-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 2-{4-[5-(5-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 2-{4-[5-(3-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 2-(4-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)propanoic acid; 2-(4-{5-[6-(methyloxy)-2-naphthalenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)propanoic acid; 2-methyl-2-[4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl]propanoic acid; 2-methyl-2-{4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 4-[5-(6-amino-3-pyridinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-{5-[6-(-alanylamino)-3-pyridinyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 4-(5-(6-indolyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; [2-(3-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)ethyl]amine; N-(3-{3-[3-(2-aminoethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}phenyl)methanesulfonamide; N-(4-{3-[3-(2-aminoethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}phenyl)methanesulfonamide; 4-{5-[3-(2-aminoethyl)phenyl]-1-H-pyrrolo-[2,3-b]pyridin-3-yl}benzoic acid; 4-{5-[3-(2-aminoethyl)phenyl]-1-H-pyrrolo-[2,3-b]pyridin-3-yl}-2-methylbenzoic acid; 4-{5-[3-({[2-(dimethylamino)ethyl]amino}carbonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 4-(5-(3-[4-(1,1-dimethylethyloxycarbonyl)aminobutanoyl]amino)phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 4-(5-(3-[3-(1,1-dimethylethyloxycarbonyl)aminopropanoyl]amino)phenyl-1H-pyrrolo[2,3-b]pyridin-3- yl)benzoic acid; 2-(2-propyl)-4-[5-[3-[3-(1,1-dimethylethyloxycarbonyl)aminopropanoyl]amino)phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-{5-[3-(-alanylamino)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 4-(5-{3-[(4-aminobutanoyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 4-{5-[3-(beta-alanylamino)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}-2-methylbenzoic acid; 4-{5-[3-(beta-alanylamino)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}-2-(1-methylethyl)benzoic acid; 4-[5-(3-{[(2-aminoethyl)amino]carbonyl}phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(3-{[(3-aminopropyl)amino]carbonyl}phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 3-amino-5-[5-(3-aminophenyl)-1-H-pyrrolo-[2,3-b]pyridin-3-yl]benzoic acid; 3-amino-5-{5-[3-(aminomethyl)phenyl]-1-H-pyrrolo-[2,3-b]pyridin-3-yl}benzoic acid; 4-{5-[3-(aminomethyl)phenyl]-1-H-pyrrolo-[2,3-b]pyridin-3-yl}-2-fluorobenzoic acid; 2-(aminomethyl)-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 3-{3-[3-(2-aminoethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}benzonitrile; 4-[5-(3-amino-1-isoquinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; 또는 이들의 염일 수 있으나, 이에 제한되는 것은 아니다.In another embodiment, the Sgk1 inhibitor may be applied by substituting a known compound having inhibitory activity against Sgk1 in addition to the compound represented by Formula 1 or Formula 2. For example, the compound capable of replacing the compound represented by Formula 1 or Formula 2 may be a compound disclosed in US 2009//0233955 A1, US 2012/10238588 A1 or EP 2242738 B1. More specifically, the compound is 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; {3-[5-(2-naphthyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzyl}amine; 4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; {4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; 3-{4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; {3-[5-(2-naphthyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}methanol; 4-{5-[3-(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 3-(4-{5-[3-(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)propanoic acid; 3-{3-[4-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}benzonitrile; 4-{5-[3-(aminocarbonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 4-[5-(3-cyanophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-{5-[6-(methyloxy)-3-pyridinyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; m) 3-{3-[3-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}benzonitrile; 4-[5-(1-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; o) 2-fluoro-4-[5-(1-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 3-amino-5-[5-(1-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 3-{4-[5-(1-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 3-(4-{5-[3-(aminocarbonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)propanoic acid; 3-{4-[5-(3-cyanophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 3-(4-{5-[6-(methyloxy)-3-pyridinyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)propanoic acid; {4-[5-(1-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; (4-{5-[3-(aminocarbonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid; {4-[5-(3-cyanophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; (4-{5-[6-(methyloxy)-3-pyridinyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid; 3-{4-[5-(3-Methanesulfonylamino-phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}-propionic acid; {4-[5-(3-Methanesulfonylamino-phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}-acetic acid; 3-{4-[5-(3-Methanesulfonyl-phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}-propionic acid; {4-[5-(3-Methanesulfonyl-phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}-acetic acid; 3-[3-fluoro-4-(methyloxy)phenyl]-5-phenyl-1H-pyrrolo[2,3-b]pyridine; 5-phenyl-3-pyridin-4-yl-1H-pyrrolo[2,3-b]pyridine; 3-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; N-[3-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenylamine; 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenol; 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzylamine; 3-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenol; 5-(3,4-dimethoxyphenyl)-3-pyridin-4-yl-1H-pyrrolo[2,3-b]pyridine; 4-[5-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-yl]-phenol; 4-[5-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-yl]-phenylamine; 4-[5-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-benzoic acid; 4-[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-benzoic acid; N-[4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; 3,5-bis-(4-hydroxyphenyl)-1H-pyrrolo[2,3-b]pyridine; 3,5-bis-(4-carboxyphenyl)-1H-pyrrolo[2,3-b]pyridine; 4-[5-(4-aminophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzylamine; 4-[5-(4-aminophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; 4-[5-(2-fluoro-biphen-4-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; N-[3-(5-thiophen-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; 4-(5-thiophen-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; 4-(5-thiophen-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenol; 4-(5-thiophen-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzamide; N-[3-(5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; 4-(5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; 4-(5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenol; 4-(5-thiophen-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzylamine; 3-(1H-indol-5-yl)-5-thiophen-3-yl-1H-pyrrolo[2,3-b]pyridine; N-[4-(5-thiophen-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; 5-(3-pyridinyl)-3-(4-pyridinyl)-1H-indole; 4-(5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzamide; 4-[3-(2-fluorobiphenyl-4-yl-1H-pyrrolo[2,3-b]pyridin-5-yl]-benzylamine; 4-(5-pyridin-3-yl-1H-pyrrolo[2, 3-b]pyridin-3-yl)-phenylamine; {3-[5-(4-methanesulfonylphenyl-1H-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}-acetic acid; N-[ 3-(3-thiophen-3-yl-1H-pyrrolo[2,3-b]pyridin-5-yl)-phenyl]-acetamide; N-{3-[3-(3-pyridinyl)-1H-pyrrolo [2,3-b]pyridin-5-yl]phenyl}acetamide; 4-[5-(3-acetylaminophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-benzoic acid; N- {3-[3-(2,3-difluorophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl]-phenyl}-acetamide; N-{3-[3-(4-hydroxyphenyl)- 1H-pyrrolo[2,3-b]pyridin-5-yl]-phenyl}-acetamide; N-{3-[3-(4-aminomethylphenyl)-1H-pyrrolo[2,3-b]pyridin-5- yl]-phenyl}-acetamide; N-{3-[3-(4-aminophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl]-phenyl}-acetamide; N-{3-[ 3-(1H-indol-5-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl]phenyl}-acetamide; 4-[3-(2-fluorobiphenyl-4-yl)-1H- pyrrolo[2,3-b]pyridin-5-yl]-benzoic acid; N-{3-[3-(4-pyridinyl)-1H-pyrrolo[2,3-b]pyridin-5-yl]phenyl} acetamide; N-{3-[5-(3-fluorophenyl)-1H-pyrrolo[2,3-b]p yridin-3-yl]-phenyl}-acetamide; 4-[5-(3-acetylaminophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-benzamide; 4-[5-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-benzoic acid; 4-[5-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-phenylamine; N-{4-[5-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}-acetamide; 4-[5-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-benzamide; ca) 2-chloro-N-[4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-benzamide; 2-phenyl-N-[4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; 2-chloro-N-[3-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzyl]-benzamide; 2-phenyl-N-[3-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-benzyl]-acetamide; (4-{5-[3-(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid; 3-[4-(5-{4-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl]propanoic acid; [4-(5-{4-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl]acetic acid; (4-{5-[3,4,5-tris(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid; 3-(4-{5-[3,4,5-tris(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)propanoic acid; {4-[5-(6-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; 3-{4-[5-(6-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; {4-[5-(3-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; {4-[5-(5-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; 3-{4-[5-(5-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 3-(4-{5-[6-(methyloxy)-2-naphthalenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)propanoic acid; 3-{4-[5-(3,4-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; {4-[5-(3,4-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; {4-[5-(2,3-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; 3-{4-[5-(2,3-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; {4-[5-(2,3-dichlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; 3-{4-[5-(2,3-dichlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; {4-[5-(1-benzothien-3-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; [(3-{5-[3-(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)methyl]amine; 7-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-1,2,3,4-tetrahydroisoquinoline; 2-fluoro-4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 2-fluoro-4-{5-[3-(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 2-methyl-4-{5-[3-(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 5-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-thiophenecarbaldehyde; 5-{5-[3-(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}-2-thiophenecarbaldehyde; 2-methyl-4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; (4-{5-[6-(methyloxy)-2-naphthalenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid; 2-fluoro-4-{5-[6-(methyloxy)-2-naphthalenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 2-fluoro-4-[5-(5-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(5-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; di) 2-methyl-4-[5-(5-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(3,4-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(3,4-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; 4-[5-(3,4-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; 4-[5-(2,3-dichlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(2,3-dichlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; 4-[5-(1-benzothien-3-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(1-benzothien-3-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; 4-[5-(1-benzothien-3-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; 6-{3-[4-(ethylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}quinoline; 4-(5-{3-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 3-[4-(butyloxy)phenyl]-5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridine; N-(3-{3-[4-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}phenyl)methane sulfonamide; N-(3-{3-[3-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}phenyl)methane sulfonamide; 3-amino-5-(5-{3-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 2-fluoro-4-(5-{3-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 3-amino-5-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 2-fluoro-4-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; [(4-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)methyl]amine; [(3-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)methyl]amine; 5-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}-2-thiophenecarbaldehyde; 4-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; N-(4-{3-[4-(butyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}phenyl)methanesulfonamide; 1,1-dimethylethyl[2-(3-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)ethyl]carbamate; 3-amino-5-(5-{4-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 2-fluoro-4-(5-{4-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 4-(5-{4-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; N-(4-{3-[4-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}phenyl)methane sulfonamide; N-(4-{3-[3-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}phenyl)methane sulfonamide; N-{4-[3-(5-formyl-2-thienyl)-1H-pyrrolo[2,3-b]pyridin-5-yl]phenyl}methanesulfonamide; 7-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}-1,2,3,4-tetrahydroisoquinoline; N-{3-[3-(1,2,3,4-tetrahydro-7-isoquinolinyl)-1H-pyrrolo[2,3-b]pyridin-5-yl]phenyl}methanesulfonamide; N-{4-[3-(1,2,3,4-tetrahydro-7-isoquinolinyl)-1H-pyrrolo[2,3-b]pyridin-5-yl]phenyl}methanesulfonamide; 2-methyl-4-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 5-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}-2-thiophenecarboxylic acid; 3-{3-[3-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}benzonitrile; 2-fluoro-4-{5-[6-(methyloxy)-3-pyridinyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 3-[3-(1,2,3,4-tetrahydro-7-isoquinolinyl)-1H-pyrrolo[2,3-b]pyridin-5-yl]benzonitrile; 7-{5-[3,4,5-tris(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}-1,2,3,4-tetrahydroisoquinoline; 4-{5-[3,4,5-tris(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 2-fluoro-4-{5-[3,4,5-tris(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 2-amino-4-{5-[3,4,5-tris(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 4-[5-(6-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(3-cyanophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; 4-[5-(2,3-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(2,3-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; 2-methyl-4-[5-(6-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 2-methyl-4-[5-(1-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(3-cyanophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; 2-methyl-4-{5-[6-(methyloxy)-3-pyridinyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 2-methyl-4-{5-[3,4,5-tris(methyloxy)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 2-(1-methylethyl)-4-[5-(1-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(3-cyanophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-(1-methylethyl)benzoic acid; 2-(1-methylethyl)-4-[5-(6-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(2,3-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; 2-chloro-4-{5-[3-(methylsulfonyl)phenyl]-1-H-pyrrolo-[2,3-b]pyridin-3-yl}benzoic acid; 4-{5-[3-(methylsulfonyl)phenyl]-1-H-pyrrolo-[2,3-b]pyridin-3-yl}-2,6-bis(trifluoromethyl)benzoic acid; methyl 2-(azidomethyl)-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoate; 2-ethyl-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 2-(methylamino)-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 2-(dimethylamino)-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 2-cyclopentyl-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-propylbenzoic acid; 2,6-difluoro-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 2,6-dimethyl-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 2-(2-propyl)-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 6-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-indazole; 2-(2-methylpropyl)-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 2-methyl-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 4-[5-(3-hydroxyphenyl)-1-H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 3-amino-5-[5-(3-hydroxyphenyl)1H-pyrrolo[2,3-b]pyridin-3-yl]-benzoic acid; {4-[5-hydroxyphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}acetic acid; 4-[5-(3-aminophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 3-{4-[5-(3-hydroxyphenyl)-1-H-pyrrolo[2,3-b]pyridine-3-yl]-phenyl}-propionic acid; 3-{4-[5-(3-aminophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; {4-[5-(3-aminophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; 4-{5-[3-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; (4-{5-[3-(aminomethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid;) 4-[5-(3-hydroxyphenyl)-1- H-pyrrolo-[2,3-b]pyridin-3-yl]benzoic acid; 2-fluoro-4-[5-(3-hydroxyphenyl)-1-H-pyrrolo-[2,3-b]pyridin-3-yl]benzoic acid; 5-[5-(3-hydroxyphenyl)-1-H-pyrrolo-[2,3-b]pyridin-3-yl]thiophene-2-carbaldehyde; 3-{3-[3-(2-aminoethyl)phenyl]-1-H-pyrrolo-[2,3-b]pyridin-5-yl}phenol; 3-[3-(1,2,3,4-tetrahydroisoquinolin-7-yl)-1-H-pyrrolo-[2,3-b]pyridin-5-yl]phenol; 3-amino-5-[5-(3-aminophenyl)-1-H-pyrrolo-[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(3-aminophenyl)-1-H-pyrrolo-[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; 2-fluoro-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)phenol; 2,6-difluoro-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)phenol; 5-phenyl-3-[4-(1H-tetrazol-5-yl)phenyl]-1H-pyrrolo[2,3-b]pyridine; 5-(2-naphthalenyl)-3-[4-(1H-tetrazol-5-yl)phenyl]-1H-pyrrolo[2,3-b]pyridine; 3-[3-fluoro-4-(1H-tetrazol-5-yl)phenyl]-5-phenyl-1H-pyrrolo[2,3-b]pyridine; 2-methyl-2-(4-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)propanoic acid; 2-{4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 2-(4-{5-[3-(aminocarbonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)-2-methylpropanoic acid; 2-{4-[5-(3-cyanophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}-2-methylpropanoic acid; 2-methyl-2-{4-[5-(6-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 2-methyl-2-{4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 2-methyl-2-[4-(5-{3-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl]propanoic acid; 2-methyl-2-[4-(5-{4-[(methylsulfonyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl]propanoic acid; 2-methyl-2-(4-{5-[6-(methyloxy)-2-naphthalenyl]-1H-pyrrolo[2,3-b]pyridin-3- yl}phenyl)propanoic acid; 2-methyl-2-{4-[5-(5-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 2-methyl-2-{4-[5-(3-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 2-{4-[5-(6-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 2-{4-[5-(5-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 2-{4-[5-(3-quinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 2-(4-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)propanoic acid; 2-(4-{5-[6-(methyloxy)-2-naphthalenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)propanoic acid; 2-methyl-2-[4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl]propanoic acid; 2-methyl-2-{4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; 4-[5-(6-amino-3-pyridinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-{5-[6-(-alanylamino)-3-pyridinyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 4-(5-(6-indolyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; [2-(3-{5-[3-(methylsulfonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}phenyl)ethyl]amine; N-(3-{3-[3-(2-aminoethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}phenyl)methanesulfonamide; N-(4-{3-[3-(2-aminoethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}phenyl)methanesulfonamide; 4-{5-[3-(2-aminoethyl)phenyl]-1-H-pyrrolo-[2,3-b]pyridin-3-yl}benzoic acid; 4-{5-[3-(2-aminoethyl)phenyl]-1-H-pyrrolo-[2,3-b]pyridin-3-yl}-2-methylbenzoic acid; 4-{5-[3-({[2-(dimethylamino)ethyl]amino}carbonyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 4-(5-(3-[4-(1,1-dimethylethyloxycarbonyl)aminobutanoyl]amino)phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 4-(5-(3-[3-(1,1-dimethylethyloxycarbonyl)aminopropanoyl]amino)phenyl-1H-pyrrolo[2,3-b]pyridin-3- yl)benzoic acid; 2-(2-propyl)-4-[5-[3-[3-(1,1-dimethylethyloxycarbonyl)aminopropanoyl]amino)phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid ; 4-{5-[3-(-alanylamino)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; 4-(5-{3-[(4-aminobutanoyl)amino]phenyl}-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 4-{5-[3-(beta-alanylamino)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}-2-methylbenzoic acid; 4-{5-[3-(beta-alanylamino)phenyl]-1H-pyrrolo[2,3-b]pyridin-3-yl}-2-(1-methylethyl)benzoic acid; 4-[5-(3-{[(2-aminoethyl)amino]carbonyl}phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 4-[5-(3-{[(3-aminopropyl)amino]carbonyl}phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 3-amino-5-[5-(3-aminophenyl)-1-H-pyrrolo-[2,3-b]pyridin-3-yl]benzoic acid; 3-amino-5-{5-[3-(aminomethyl)phenyl]-1-H-pyrrolo-[2,3-b]pyridin-3-yl}benzoic acid; 4-{5-[3-(aminomethyl)phenyl]-1-H-pyrrolo-[2,3-b]pyridin-3-yl}-2-fluorobenzoic acid; 2-(aminomethyl)-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; 3-{3-[3-(2-aminoethyl)phenyl]-1H-pyrrolo[2,3-b]pyridin-5-yl}benzonitrile; 4-[5-(3-amino-1-isoquinolinyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; Or it may be a salt thereof, but is not limited thereto.
본 명세서에서, “siRNA(small interfering RNA)”는 표적 유전자의 mRNA의 절단을 통해 RNA 간섭현상을 유도하는 이중사슬 RNA를 의미하며, 표적 유전자의 mRNA와 같은 서열을 가지는 센스서열의 RNA 가닥과 이와 상보적인 서열을 가지는 안티센스 서열의 RNA 가닥으로 구성된다. In the present specification, “siRNA (small interfering RNA)” refers to a double-stranded RNA that induces RNA interference by cleaving the mRNA of the target gene, and the RNA strand of the sense sequence having the same sequence as the mRNA of the target gene and It consists of an RNA strand of an antisense sequence having a complementary sequence.
상기 siRNA는 인간 및/또는 생쥐 상의 siRNA 공통 핵산서열 부위를 표적으로 하여 통상의 방법에 따라 제작된 것을 사용할 수 있다. The siRNA may be prepared according to a conventional method by targeting the siRNA common nucleic acid sequence site on human and/or mouse.
본 발명에서, 상기 siRNA는 ThermoFisher Scientific사의 Assay ID Mm00441380_m1, catalog #4331182인 것을 사용할 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the siRNA may be ThermoFisher Scientific's Assay ID Mm00441380_m1, catalog #4331182, but is not limited thereto.
상기 siRNA는 시험관 내에서 합성한 siRNA 자체 또는 siRNA를 코딩하는 핵산서열을 발현 벡터에 삽입하여 발현되는 형태를 포함할 수 있다.The siRNA may include a siRNA synthesized in vitro or a form expressed by inserting a nucleic acid sequence encoding the siRNA into an expression vector.
본 발명에 있어서, 상기 “벡터”는 폴리펩타이드를 코딩하는 게놈 내로 삽입된 외부 DNA를 포함하는 유전자 작제물을 말한다.In the present invention, the "vector" refers to a genetic construct including an external DNA inserted into a genome encoding a polypeptide.
본 발명과 관련된 벡터는 상기 유전자를 저해하는 핵산 서열이 게놈 내로 삽입된 벡터로서, 이들 벡터는 DNA 벡터, 플라스미드 벡터, 코즈미드 벡터, 박테리오파아지 벡터, 효모 벡터 또는 바이러스 벡터를 예로 들 수 있다.The vector related to the present invention is a vector in which a nucleic acid sequence that inhibits the gene is inserted into the genome, and these vectors include DNA vectors, plasmid vectors, cosmid vectors, bacteriophage vectors, yeast vectors, or viral vectors.
본 발명에 있어서, 적합한 발현 벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서 같은 발현 조절 엘리먼트 외에도 막 표적화 또는 분비를 위한 시그널 서열 또는 리더 서열을 포함하며, 목적에 따라 다양하게 제조될 수 있다. 벡터의 프로모터는 구성적 또는 유도성일 수 있다. 또한, 발현 벡터는 벡터를 함유하는 숙주세포를 선택하기 위한 선택 마커를 포함하고, 복제가능한 발현벡터인 경우 복제 기원을 포함한다. In the present invention, suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, start codons, stop codons, polyadenylation signals and enhancers, and variously according to the purpose. Can be manufactured. The promoter of the vector can be constitutive or inducible. In addition, the expression vector includes a selection marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, it includes an origin of replication.
또한, 상기 안티센스는 Sgk1 유전자 또는 그의 단편으로부터 전사되는 mRNA 서열 전체 또는 일부와 상보적인 서열을 지니고, 상기 mRNA와 결합하여 상기 Sgk1 유전자 또는 단편의 발현을 억제할 수 있다.In addition, the antisense has a sequence complementary to all or part of the mRNA sequence transcribed from the Sgk1 gene or fragment thereof, and may bind to the mRNA to inhibit the expression of the Sgk1 gene or fragment.
본 명세서에서, “shRNA(short hairpin RNA)”는 단일 가닥 50-60개로 구성된 뉴클레오타이드를 의미하며, in vivo 상에서 스템-루프(stem-loop) 구조를 이루고 있다. 즉, shRNA는 RNA 간섭을 통해 유전자 발현을 억제하기 위한 타이트한 헤어핀 구조를 만드는 RNA 서열이다. 5-10개 뉴클레오타이드의 루프 부위 양쪽으로 상보적인 15-30개의 뉴클레오타이드의 긴 RNA가 염기쌍을 이루어 이중가닥의 스템을 형성한다. shRNA는 언제나 발현되도록 하기 위해 U6 프로모터를 포함하는 벡터를 통해 세포 내로 형질도입되며 대개 딸세포로 전달되어 유전자 발현 억제가 유전되도록 한다. shRNA의 헤어핀 구조는 세포 내 기작에 의해 절단되어 siRNA가 된 후 RISC(RNA-induced silencing complex)에 결합한다. 이들 RISC는 mRNA에 결합하여 이를 절단한다. shRNA는 RNA 폴리머레이즈(polymerase)에 의해 전사된다. In the present specification, “shRNA (short hairpin RNA)” refers to a nucleotide consisting of 50-60 single strands, and constitutes a stem-loop structure in vivo. That is, shRNA is an RNA sequence that creates a tight hairpin structure to suppress gene expression through RNA interference. Long RNAs of 15-30 nucleotides complementary to both sides of the loop of 5-10 nucleotides form a base pair to form a double-stranded stem. In order to be always expressed, shRNA is transduced into cells via a vector containing the U6 promoter and is usually transferred to daughter cells, allowing gene expression inhibition to be inherited. The hairpin structure of shRNA is cleaved by an intracellular mechanism to become siRNA and then binds to the RNA-induced silencing complex (RISC). These RISCs bind to and cleave mRNA. shRNA is transcribed by RNA polymerase.
또한, 상기 shRNA(short hairpin RNA)는 인간 또는 생쥐 상의 shRNA 공통 핵산서열 부위를 표적으로 하여 통상의 방법에 따라 제작된 것을 사용할 수 있다. In addition, the shRNA (short hairpin RNA) may be prepared according to a conventional method by targeting the shRNA common nucleic acid sequence site in human or mouse.
한 구체예에서, 상기 shRNA는 SEQ ID NO: 9의 핵산서열로 이루어진 것일 수 있다:In one embodiment, the shRNA may consist of the nucleic acid sequence of SEQ ID NO: 9:
[SEQ ID NO: 9][SEQ ID NO: 9]
5'-GCACTTCGATCCCGAGTTTAC-3'5'-GCACTTCGATCCCGAGTTTAC-3'
본 명세서에서, “miRNA(micro RNA)”는 약 22개의 핵산서열로 이루어진 짧은 non-coding RNA를 의미한다. 유전자의 발현 과정에서 전사 후 조절인자(post-transcriptional regulator)로서 기능을 한다고 알려져 있다. 상보적인 핵산서열을 가진 표적 mRNA에 상보적으로 결합함으로써 표적 mRNA를 분해시키거나 단백질로 번역되는 것을 억제한다. 상기 miRNA의 존재는 프로브를 이용하여 확인할 수 있다.In the present specification, “miRNA (micro RNA)” refers to a short non-coding RNA consisting of about 22 nucleic acid sequences. It is known to function as a post-transcriptional regulator in the process of gene expression. By binding complementarily to a target mRNA having a complementary nucleic acid sequence, the target mRNA is degraded or translation into a protein is inhibited. The presence of the miRNA can be confirmed using a probe.
본 명세서에서, “프로브”는 자연의 또는 변형된 모노머 또는 연쇄(linkage)의 선형 올리고머를 의미하며, 디옥시리보뉴클레오타이드 및 리보뉴클레오타이드를 포함하고 표적 뉴클레오타이드 서열에 특이적으로 혼성화할 수 있으며, 자연적으로 존재하거나 또는 인위적으로 합성된 것일 수 있다. In the present specification, “probe” refers to a natural or modified monomer or a linear oligomer of a linkage, including deoxyribonucleotides and ribonucleotides, and can specifically hybridize to a target nucleotide sequence, and exist naturally or Or it may be artificially synthesized.
또한, 항체는 Sgk1 단백질에 대한 다클론 항체, 단클론 항체, 인간 항체 및 인간화 항체를 사용할 수 있다.In addition, as the antibody, polyclonal antibodies, monoclonal antibodies, human antibodies, and humanized antibodies against Sgk1 protein can be used.
상기 항체 단편의 예로는, Fab, Fab', F(ab') 2 및 Fv 단편; 디아바디(diabody); 선형 항체(Zapata et al. Protein Eng. 8(10):1057-1062(1995)); 단일쇄 항체 분자; 및 항체 단편으로부터 형성된 다중 특이성 항체 등이 포함된다.Examples of such antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments; Diabody; Linear antibodies (Zapata et al. Protein Eng. 8(10):1057-1062(1995)); Single chain antibody molecules; And multispecific antibodies formed from antibody fragments.
항체를 파파인(papain)으로 분해하면 2개의 동일한 항원 결합 단편, 즉 단일 항원 결합 부위가 있는 각 “Fab” 단편, 및 그 나머지인 “Fc” 단편이 생성된다. 펩신을 처리하면, 2개의 항원 결합 부위가 있으며 여전히 항원에 교차결합할 수 있는 F(ab') 2 단편이 생성된다. Fv는 완전한 항원 인식 및 결합 부위를 포함하는 최소한의 항체 단편이다. 이 부위는 하나의 중쇄 및 하나의 경쇄 가변 영역의 이합체로 구성되며 비공유결합으로 단단히 결합되어 있다.Digestion of an antibody with papain yields two identical antigen-binding fragments, each “Fab” fragment with a single antigen binding site, and the remainder, “Fc” fragment. Treatment with pepsin yields an F(ab') 2 fragment that has two antigen binding sites and is still capable of cross-linking to the antigen. Fv is a minimal antibody fragment containing a complete antigen recognition and binding site. This site consists of a dimer of one heavy chain and one light chain variable region, and is tightly bound by a non-covalent bond.
다클론 항체의 제조방법은 당업자에게 공지되어 있다. 다클론 항체는 포유 동물에 1회 이상 면역화제를 주입, 필요한 경우 면역 보강제와 함께 주입하여 제조할 수 있다. 통상, 면역화제 및/또는 면역 보강제는 포유 동물에 피하주사 또는 복강내 주사로 수회 주입된다. 면역화제는 본 발명의 단백질 또는 이의 융합 단백질일 수 있다. 면역화되는 포유동물에 면역원성이 있는 것으로 공지된 단백질과 함께 면역화제를 주사하는 것이 효과적일 수 있다.Methods for preparing polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be prepared by injecting an immunizing agent into a mammal one or more times and, if necessary, injecting with an adjuvant. Typically, the immunizing agent and/or adjuvant is injected several times by subcutaneous injection or intraperitoneal injection into the mammal. The immunizing agent may be a protein of the invention or a fusion protein thereof. It may be effective to inject an immunizing agent with a protein known to be immunogenic in the mammal being immunized.
본 발명에 따른 단클론 항체는 문헌(Kohler et al. Nature, 256:495(1975))에 기재된 하이브리도마 방법으로 제조하거나 또는 재조합 DNA 방법(예를 들어, 미국 특허 제4,816,576호 참조)으로 제조할 수 있다. 단클론 항체는 또한, 문헌(Clackson et al. Nature, 352:624-628(1991) 및 Marks et al. J. Mol. Biol., 222:581-597(1991))에 기재된 기술을 이용하여 파지 항체 라이브러리로부터 단리할 수 있다.The monoclonal antibody according to the present invention can be prepared by the hybridoma method described in Kohler et al. Nature, 256:495 (1975) or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,576). I can. Monoclonal antibodies are also phage antibody using techniques described in Clackson et al. Nature, 352:624-628 (1991) and Marks et al. J. Mol. Biol., 222:581-597 (1991). It can be isolated from the library.
본 발명에서의 단클론 항체는 구체적으로, 목적하는 활성을 발휘한다면 중쇄 및/또는 경쇄의 일부분이 특정 종으로부터 유래된 항체 또는 특정 항체 클래스 또는 서브 클래스에 속하는 항체에 상응하는 서열과 동일하거나 상동성이 있지만, 쇄(들)의 나머지는 다른 종으로부터 유래된 항체 또는 다른 항체 클래스 또는 서브 클래스에 속하는 항체 또는 그러한 항체의 단편과 동일하거나 상동성이 있는 “키메라” 항체를 포함한다(Morrison et al. Proc. Natl. Acad. Sci. USA, 81:6851-6855(1984)).In the monoclonal antibody of the present invention, specifically, if it exhibits the desired activity, a portion of the heavy chain and/or light chain is identical or homologous to the sequence corresponding to an antibody derived from a specific species or an antibody belonging to a specific antibody class or subclass. However, the remainder of the chain(s) includes antibodies derived from other species or antibodies belonging to different antibody classes or subclasses, or "chimeric" antibodies that are identical or homologous to fragments of such antibodies (Morrison et al. Proc). Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
비-인간(예컨대, 쥐과 동물) 항체의 “인간화” 형태는 비-인간 면역글로불린으로부터 유도된 최소서열을 포함하는 키메라 면역글로불린, 면역글로불린 쇄 또는 그의 단편(예를 들어, Fv, Fab, Fab', F(ab') 2 또는 항체의 다른 항원 결합 서열)이다. 대부분의 경우 인간화 항체는 수용자의 상보성 결정(CDR)의 잔기를 원하는 특이성, 친화도 및 능력을 갖는 생쥐, 쥐 또는 토끼와 같은 인간 이외의 종(공여자 항체)의 CDR 잔기로 치환시킨 인간 면역글로불린(수용자 항체)을 포함한다. 몇몇 경우에 인간 면역글로불린의 Fv 프레임워크 잔기는 상응하는 비-인간 잔기에 의해 치환된다. 또한, 인간화 항체는 수용 항체, 또는 도입되는 CDR 또는 프레임워크 서열에서 발견되지 않는 잔기를 포함할 수 있다. 인간화 항체는 하나 이상, 일반적으로 둘 이상의 가변 도메인을 실질적으로 모두 포함하며, 여기서 모든 또는 실질적으로 모든 CDR 영역은 비-인간 면역글로불린의 영역에 대응하며, 모든 또는 실질적으로 모든 FR 영역은 인간 면역글로불린 서열의 영역에 해당한다. 또한, 인간화 항체는 면역글로불린 불변 영역(Fc)의 적어도 일부, 일반적으로 인간 면역글로불린 영역의 일부를 포함한다(Presta, Curr. Op. Struct. Biol. 2:593-596(1992)).“Humanized” forms of non-human (eg, murine) antibodies include chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg, Fv, Fab, Fab′) containing minimal sequences derived from non-human immunoglobulins. , F(ab') 2 or other antigen binding sequence of the antibody). In most cases, humanized antibodies are human immunoglobulins in which residues of the recipient's complementarity determination (CDR) are replaced with CDR residues of a non-human species (donor antibody) such as mice, rats, or rabbits having the desired specificity, affinity and ability. Acceptor antibody). In some cases, the Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, the humanized antibody may comprise residues not found in the recipient antibody or in the CDR or framework sequence to be introduced. Humanized antibodies comprise substantially all of one or more, generally two or more variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are human immunoglobulins. It corresponds to a region of the sequence. In addition, humanized antibodies comprise at least a portion of an immunoglobulin constant region (Fc), generally a portion of a human immunoglobulin region (Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)).
상기 약학 조성물은 Sgk1 저해제를 통해 Sgk1을 넉다운(knock-down)시킴으로써, 신경세포를 보호하고 신경염증을 억제시킬 수 있으므로, 염증성 신경 질환을 예방, 개선 또는 치료할 수 있다. 하기 실시예에서는, Nurr1 및 Foxa2가 과발현된 신경교세포에서 Sgk1의 발현이 감소하는 결과를 토대로, Sgk1을 넉다운시킨 신경교세포에서의 항염증 효과를 확인하였다. 그 결과, Sgk1의 발현을 저해함에 따라 신경교세포에서 염증성 사이토카인인 IL-1ß와 TNF-α의 발현이 유의하게 감소하였으며, 세포 노화와 관련된 활성산소종(ROS)의 양이 감소하였다. 또한, 본 발명은 Sgk1의 발현 저해에 의해 염증조절 복합체(inflammasome)의 활성이 감소하고, 글루타메이트 운송 유전자인 GLAST와 GLT-1의 발현이 증가되는 결과로부터 글루타메이트의 흡수량이 증가된다는 것을 확인할 수 있었으며, Sgk1 저해제가 α-synuclein의 응집을 방지하여 염증성 신경 질환 모델의 치료제로서의 효과를 나타낼 수 있음을 동물 실험을 통해 밝혔다. The pharmaceutical composition can protect nerve cells and inhibit neuroinflammation by knocking down Sgk1 through an Sgk1 inhibitor, and thus can prevent, improve or treat inflammatory neurological diseases. In the following examples, the anti-inflammatory effect in glial cells knocked down Sgk1 was confirmed based on the result of a decrease in the expression of Sgk1 in glial cells overexpressing Nurr1 and Foxa2. As a result, inhibition of Sgk1 expression significantly reduced the expression of inflammatory cytokines IL-1ß and TNF-α in glial cells, and the amount of reactive oxygen species (ROS) associated with cellular senescence decreased. In addition, the present invention confirmed that the absorption of glutamate was increased from the result that the activity of the inflammation regulatory complex (inflammasome) was decreased by the inhibition of the expression of Sgk1, and the expression of glutamate transport genes GLAST and GLT-1 was increased. It was revealed through animal experiments that Sgk1 inhibitors can prevent the aggregation of α-synuclein and thus exhibit an effect as a therapeutic agent for an inflammatory neurological disease model.
따라서, 염증성 신경 질환을 갖는 환자의 경우, Sgk1 유전자 또는 단백질의 발현 양을 억제하거나 이의 저해제를 환자에 투여하는 경우 염증성 사이토카인의 발현을 감소시키고 α-synuclein의 응집을 억제할 수 있으며, 이로 인한 신경보호 및 신경염증 억제할 수 있으므로, Sgk1 저해제로부터 파킨슨병 및 알쯔하이머병과 같은 염증성 신경 질환 환자의 증상을 개선, 완화 또는 치료할 수 있다. Therefore, in the case of a patient with inflammatory neurological disease, when the amount of expression of the Sgk1 gene or protein is suppressed or an inhibitor thereof is administered to the patient, the expression of inflammatory cytokines can be reduced and the aggregation of α-synuclein can be suppressed. Since neuroprotection and neuroinflammation can be suppressed, symptoms of patients with inflammatory neurological diseases such as Parkinson's disease and Alzheimer's disease can be improved, alleviated or treated from Sgk1 inhibitors.
또한, 본 발명의 약학 조성물은 약제학적으로 허용가능한 담체를 더 포함할 수 있다. 상기 약제학적으로 허용가능한 담체는 의약 분야에서 통상적으로 사용되는 담체 및 비히클을 포함하며, 구체적으로 이온 교환 수지, 알루미나, 알루미늄 스테아레이트, 레시틴, 혈청 단백질(예, 사람 혈청 알부민), 완충 물질(예, 각종 인산염, 글리신, 소르브산, 칼륨 소르베이트, 포화 식물성 지방산의 부분적인 글리세라이드 혼합물), 물, 염 또는 전해질(예, 프로타민설페이트, 인산수소이나트륨, 인산수소칼륨, 염화나트륨 및 아연염), 교질성실리카, 마그네슘 트리실리케이트, 폴리비닐피롤리돈, 셀룰로오즈계 기질, 폴리에틸렌글리콜, 나트륨 카르복시메틸셀룰로오즈, 폴리아릴레이트, 왁스 또는 양모지 등을 포함하나, 이에 제한되는 것은 아니다. In addition, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes carriers and vehicles commonly used in the field of medicine, and specifically, ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer substances (eg , Various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloids Including, but not limited to, silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax or wool paper, and the like.
또한, 본 발명의 조성물은 상기 성분들 이외에 윤활제, 습윤제, 유화제, 현탁제 또는 보존제 등을 추가로 포함할 수 있다.In addition, the composition of the present invention may further include a lubricant, a wetting agent, an emulsifier, a suspending agent or a preservative in addition to the above components.
한 구체예에서, 본 발명에 따른 조성물은 비경구 투여를 위한 수용성 용액으로 제조할 수 있으며, 바람직하게는 한스 용액(Hank's solution), 링거 용액(Ringer's solution) 또는 물리적으로 완충된 염수와 같은 완충 용액을 사용할 수 있다. 수용성 주입(injection) 현탁액은 소디움카르복시메틸셀룰로오즈, 솔비톨 또는 덱스트란과 같이 현탁액의 점도를 증가시킬 수 있는 기질을 첨가할 수 있다.In one embodiment, the composition according to the present invention may be prepared as an aqueous solution for parenteral administration, preferably a buffer solution such as Hank's solution, Ringer's solution or physically buffered saline You can use Aqueous injection suspensions may be added with a substrate capable of increasing the viscosity of the suspension such as sodium carboxymethylcellulose, sorbitol or dextran.
본 발명의 조성물은 전신계 또는 국소적으로 투여될 수 있으며, 이러한 투여를 위해 공지의 기술로 적하반 제형으로 제제화될 수 있다. 예를 들어, 경구 투여시에는 불활성 희석제 또는 식용 담체와 혼합하거나, 경질 또는 연질 젤라틴 캡슐에 밀봉되거나 또는 정제로 압형하여 투여할 수 있다. 경구 투여용의 경우, 활성 화합물은 부형제와 혼합되어 섭취형 정제, 협측 정제, 트로키, 캡슐, 엘릭시르, 서스펜션, 시럽, 웨이퍼 등의 형태로 사용될 수 있다.The composition of the present invention may be administered systemically or locally, and for such administration may be formulated in a dropping plate formulation by a known technique. For example, when administered orally, it may be administered by mixing with an inert diluent or an edible carrier, sealed in a hard or soft gelatin capsule, or compressed into tablets. For oral administration, the active compound may be mixed with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
주사용, 비경구 투여용 등의 각종 제형은 당해 기술 분야 공지된 기법 또는 통용되는 기법에 따라 제조할 수 있다. 또한, 유효량의 Sgk1 저해제를 정맥 내 주입, 피하 주입, 근육 주입, 복강 주입, 경피 투여 등에 적합한 형태로 식염수 또는 완충액에 투여 직전에 용액으로 제제화하여 투여할 수도 있다.Various formulations, such as for injection and parenteral administration, can be prepared according to techniques known or commonly used in the art. In addition, an effective amount of the Sgk1 inhibitor may be formulated as a solution immediately before administration in saline or buffer in a form suitable for intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like.
본 발명에서 용어, "투여"는 어떠한 적절한 방법으로 환자에게 본 발명의 조성물을 도입하는 것을 의미하며, 본 발명의 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있다. 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여, 경구 투여, 국소 투여, 비내 투여, 폐내 투여, 직장내 투여 될 수 있으나, 이에 제한되지는 않는다.In the present invention, the term "administration" means introducing the composition of the present invention to a patient by any suitable method, and the route of administration of the composition of the present invention is through various oral or parenteral routes as long as it can reach the target tissue. Can be administered. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration may be performed, but are not limited thereto.
또한, 본 발명은 치료상 유효량의 Sgk1 저해제를 이를 필요로 하는 대상체에게 투여하는 것을 포함하는 염증성 신경 질환의 치료 방법을 제공한다.In addition, the present invention provides a method for treating inflammatory neurological diseases comprising administering a therapeutically effective amount of an Sgk1 inhibitor to a subject in need thereof.
여기에서 사용된 용어 “대상체”는 치료, 관찰 또는 실험의 대상인 포유동물을 말하며, 바람직하게는 인간을 말한다.The term "subject" as used herein refers to a mammal that is the subject of treatment, observation or experiment, and preferably refers to a human.
본 명세서에서, "치료상 유효량"은 연구자, 수의사, 의사 또는 기타 임상의에 의해 생각되는 조직계, 동물 또는 인간에서 생물학적 또는 의학적 반응을 유도하는 유효성분 또는 약학 조성물의 양을 의미하는 것으로, 이는 목적하는 치료되어야 할 특정 질환의 발병 또는 진행을 지연하거나 전적으로 중지시키는 데 필요한 양을 포함한다. 본 발명의 약학 조성물에 포함되는 Sgk1 저해제의 유효량은 파킨슨병, 알쯔하이머병, 다발성 전신 위축, 근 위축성 측삭 경화증(ALS), 뇌경색, 척추손상, 신경병증 통증(neuropathic pain) 및 복합통증증후군(complex region pain syndrome; CRPS)에서 선택되는 질환에 있어서 신경보호 및 신경염증 감소 효과를 이루는데 요구되는 양을 의미한다. 따라서, 상기 치료상 유효량은 질환의 종류, 질환의 중증도, 조성물에 함유된 다른 성분의 종류 및 함량, 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로, 치료 기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다. 본 발명의 치료 방법에 있어서, 적합한 총 1일 사용량은 올바른 의학적 판단범위 내에서 처치의에 의해 결정될 수 있다는 것은 당업자에게 자명한 일이다.In the present specification, the "therapeutically effective amount" refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human thought by a researcher, veterinarian, doctor or other clinician, which It includes the amount necessary to delay or completely stop the onset or progression of the particular disease to be treated. The effective amount of the Sgk1 inhibitor contained in the pharmaceutical composition of the present invention is Parkinson's disease, Alzheimer's disease, multiple systemic atrophy, amyotrophic lateral sclerosis (ALS), cerebral infarction, spinal injury, neuropathic pain, and complex region Pain syndrome (CRPS) refers to the amount required to achieve neuroprotective and neuroinflammation reduction effects. Accordingly, the therapeutically effective amount is the type of disease, severity of disease, type and content of other ingredients contained in the composition, and the age, weight, general health condition, sex and diet of the patient, administration time, administration route, treatment period, It can be adjusted according to a variety of factors, including drugs used simultaneously. In the treatment method of the present invention, it is obvious to those skilled in the art that a suitable total daily amount can be determined by the treating physician within the range of correct medical judgment.
본 발명의 목적상, 특정 환자에 대한 구체적인 치료적 유효량은 달성하고자 하는 반응의 종류와 정도, 경우에 따라 다른 제제가 사용되는지의 여부를 비롯한 구체적 조성물, 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료 기간, 구체적 조성물과 함께 사용되거나 동시 사용되는 약물을 비롯한 다양한 인자와 의약 분야에 잘 알려진 유사 인자에 따라 다르게 적용하는 것이 바람직하다.For the purposes of the present invention, a specific therapeutically effective amount for a specific patient is the type and extent of the reaction to be achieved, the specific composition including whether or not other agents are used in some cases, the patient's age, weight, general health status, and sex. And it is preferable to apply differently according to various factors including diet, administration time, route of administration and secretion rate of the composition, duration of treatment, drugs used with or concurrently with the specific composition, and similar factors well known in the medical field.
본 발명의 치료 방법에서 본 발명의 Sgk1 저해제를 유효성분으로 포함하는 조성물은 경구, 직장, 정맥내, 동맥내, 복강내, 근육내, 흉골내, 경피, 국소, 안구내 또는 피내 경로를 통해 통상적인 방식으로 투여할 수 있다.In the treatment method of the present invention, the composition comprising the Sgk1 inhibitor of the present invention as an active ingredient is conventionally used through oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. It can be administered in a phosphorus manner.
본 명세서에서, “치료”는 이롭거나 바람직한 임상적 결과를 수득하기 위한 접근을 의미한다. 본 발명의 목적을 위해서, 이롭거나 바람직한 임상적 결과는 비제한적으로, 증상의 완화, 질병 범위의 감소, 질병 상태의 안정화(즉, 악화되지 않음), 질병 진행의 지연 또는 속도의 감소, 질병 상태의 개선 또는 일시적 완화 및 경감(부분적이거나 전체적으로), 검출 가능하거나 또는 검출되지 않거나의 여부를 포함한다. 또한, “치료”는 치료를 받지 않았을 때 예상되는 생존율과 비교하여 생존율을 늘이는 것을 의미할 수도 있다. “치료”는 치료학적 치료 및 예방적 또는 예방 조치 방법 모두를 가리킨다. 상기 치료들은 예방되는 장애뿐만 아니라 이미 발생한 장애에 있어서 요구되는 치료를 포함한다. 질병을 “완화”하는 것은 치료를 하지 않은 경우와 비교하여, 질병 상태의 범위 및/또는 바람직하지 않은 임상적 징후가 감소되거나 및/또는 진행의 시간적 추이(time course)가 늦춰지거나 길어지는 것을 의미한다.As used herein, “treatment” means an approach to obtain beneficial or desirable clinical outcomes. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (i.e., not exacerbation), delay or decrease in disease progression, disease state. Amelioration or temporal mitigation and mitigation (partially or entirely) of, detectable or undetectable. In addition, “treatment” may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include the disorder to be prevented as well as the treatment required for a disorder that has already occurred. “Relieving” the disease means that the extent of the disease state and/or undesirable clinical signs are reduced and/or the time course of progression is slowed or prolonged compared to without treatment. do.
또한, 본 발명은 염증성 신경 질환의 치료를 위한 Sgk1 저해제의 용도를 제공한다.In addition, the present invention provides the use of an Sgk1 inhibitor for the treatment of inflammatory neurological diseases.
상기 염증성 신경 질환의 치료 방법에 사용되는 약학 조성물 및 투여 방법은 상기에서 설명하였는바, 이 둘 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여 그 기재를 생략한다.The pharmaceutical composition and the administration method used in the treatment method for the inflammatory neurological disease have been described above, and descriptions of the content in common between the two are omitted in order to avoid excessive complexity of the present specification.
상기 염증성 신경 질환의 예방 또는 치료용 조성물을 투여할 수 있는 개체는 인간을 제외한 모든 동물을 포함한다. 예컨대, 개, 고양이, 생쥐와 같은 동물일 수 있다.Individuals to which the composition for preventing or treating inflammatory neurological diseases can be administered include all animals except humans. For example, it may be an animal such as a dog, a cat, or a mouse.
본 발명은 Sgk1(serum and glucocorticoid-regulated kinase 1) 유전자의 mRNA 또는 이의 단백질 수준을 측정하는 제제를 포함하는 염증성 신경 질환의 진단용 조성물을 제공한다.The present invention provides a composition for diagnosing inflammatory neurological diseases comprising an agent measuring the level of mRNA or protein thereof of the Sgk1 (serum and glucocorticoid-regulated kinase 1) gene.
또한, 본 발명은 개체로부터 분리된 생물학적 시료로부터 Sgk1 유전자의 mRNA 또는 이의 단백질 수준을 측정하는 제제를 이용하여 Sgk1의 발현 수준을 측정하는 단계를 포함하는 염증성 신경 질환의 진단 방법을 제공한다.In addition, the present invention provides a method for diagnosing inflammatory neurological disorders comprising the step of measuring the expression level of Sgk1 using an agent measuring the level of mRNA or protein thereof of the Sgk1 gene from a biological sample isolated from an individual.
염증성 신경 질환의 경우, Sgk1의 발현이 증가되어 있는 상태이므로, 상기 Sgk1의 발현 수준을 확인하여 염증성 신경 질환을 진단할 수 있다. In the case of inflammatory neurological disease, since the expression of Sgk1 is increased, the inflammatory neurological disease can be diagnosed by checking the expression level of Sgk1.
본 발명에서, “진단”은 병리 상태를 확인하는 것을 의미하는 것으로서, 본 발명의 목적상 상기 진단은 염증성 신경 질환의 진단 마커의 발현 유무를 확인하여 이들 질환의 발병 여부, 발전 및 경감 여부를 확인하는 것을 의미한다. In the present invention, “diagnosis” refers to confirming a pathological state, and for the purposes of the present invention, the diagnosis is to confirm the onset, development and alleviation of these diseases by checking the expression of diagnostic markers of inflammatory neurological diseases. Means to do.
본 발명에서, “진단 마커”는 염증성 신경 질환의 세포를 정상 세포와 구분하여 진단할 수 있는 물질을 의미하며, 정상 세포에 비해 염증성 신경 세포에서 증가 또는 감소를 보이는 폴리펩타이드 또는 핵산(예컨대, mRNA 등), 지질, 당지질, 당단백질, 당(단당류, 이당류, 올리고당류 등) 등과 같은 유기 생체 분자를 포함한다. 본 발명에서 제공하는 염증성 신경 질환 진단용 마커는 정상 세포에 비해 염증성 신경 질환의 세포에서 발현양이 증가 또는 감소하는 Sgk1 유전자로부터 발현된 단백질일 수 있다. In the present invention, “diagnostic marker” refers to a substance capable of distinguishing and diagnosing cells of inflammatory neurological disease from normal cells, and a polypeptide or nucleic acid (eg, mRNA) showing an increase or decrease in inflammatory neurons compared to normal cells. Etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.), and the like. The marker for diagnosing inflammatory neurological disease provided by the present invention may be a protein expressed from the Sgk1 gene whose expression level is increased or decreased in cells of inflammatory neurological disease compared to normal cells.
본 발명의 염증성 신경 질환 진단용 조성물은 Sgk1 유전자의 mRNA 발현 수준 또는 상기 유전자로부터 발현된 단백질의 양을 측정하는 제제를 포함하고, 이러한 제제로 Sgk1 mRNA에 상보적인 서열을 갖는 올리고뉴클레오타이드, 예컨대 Sgk1 mRNA에 특이적으로 결합하는 프라이머 또는 핵산 프로브나 Sgk1 단백질에 특이적인 항체를 포함할 수 있다.The composition for diagnosing inflammatory neurological diseases of the present invention includes an agent for measuring the level of mRNA expression of the Sgk1 gene or the amount of protein expressed from the gene, and with such an agent, an oligonucleotide having a sequence complementary to Sgk1 mRNA, such as Sgk1 mRNA. It may contain a primer or nucleic acid probe that specifically binds, or an antibody specific for the Sgk1 protein.
또한, 본 발명은,In addition, the present invention,
Sgk1(serum and glucocorticoid-regulated kinase 1) 유전자를 후보물질과 인체 외에서 접촉시키고, Sgk1 (serum and glucocorticoid-regulated kinase 1) gene is brought into contact with a candidate substance outside the human body,
상기 후보물질이 상기 유전자의 발현을 촉진하는지 또는 억제하는지를 판단하는 것을 포함하는 염증성 신경 질환의 예방 또는 치료용 약물의 스크리닝 방법을 제공한다.It provides a method for screening a drug for preventing or treating inflammatory neurological diseases, comprising determining whether the candidate substance promotes or inhibits the expression of the gene.
또한, 본 발명은,In addition, the present invention,
Sgk1(serum and glucocorticoid-regulated kinase 1) 단백질을 후보물질과 인체 외에서 접촉시키고, Sgk1 (serum and glucocorticoid-regulated kinase 1) protein is brought into contact with a candidate substance outside the human body,
상기 후보물질이 상기 단백질의 기능 또는 활성을 증진하는지 또는 억제하는지를 판단하는 것을 포함하는 염증성 신경 질환의 예방 또는 치료용 약물의 스크리닝 방법을 제공한다.It provides a method for screening a drug for preventing or treating inflammatory neurological diseases, comprising determining whether the candidate substance enhances or inhibits the function or activity of the protein.
본 발명의 스크리닝 방법에 따르면, 먼저 상기 유전자 또는 단백질을 포함하는 염증성 신경 질환의 환자로부터 채취한 생물학적 시료에 분석하고자 하는 후보물질을 접촉시킬 수 있다.According to the screening method of the present invention, first, a candidate substance to be analyzed may be brought into contact with a biological sample collected from a patient of inflammatory neurological disease including the gene or protein.
본 발명에 있어서, 상기 후보물질은 통상적인 선정방식에 따라 Sgk1 유전자 핵산서열에서 mRNA, 단백질로의 전사, 번역을 촉진하거나 억제하는 물질 또는 Sgk1 단백질의 기능 또는 활성을 증진하거나 억제, 넉다운(knock-down) 시키는 의약으로서의 가능성을 지닌 것으로 추정되는 물질 또는 무작위로 선정된 개별적인 핵산, 단백질, 펩타이드, 기타 추출물 또는 천연물, 화합물 등이 될 수 있다.In the present invention, the candidate substance is a substance that promotes or inhibits transcription and translation from the Sgk1 gene nucleic acid sequence to mRNA, protein, or enhances or inhibits the function or activity of the Sgk1 protein according to a conventional selection method, knock-down (knock-down). down) It may be a substance that is assumed to have the potential as a drug or a randomly selected individual nucleic acid, protein, peptide, other extract, natural product, or compound.
이후, 후보물질이 처리된 시료에서 상기 유전자의 발현양, 단백질의 양, 단백질의 활성을 측정 또는 확인할 수 있으며, 측정 결과, 상기 유전자의 발현양, 단백질의 양 또는 단백질의 활성이 감소되는 것이 측정되면 상기 후보물질은 염증성 신경 질환을 치료할 수 있는 물질로 판단할 수 있다.Thereafter, the expression amount of the gene, the amount of protein, and the activity of the protein can be measured or confirmed in the sample treated with the candidate substance, and as a result of the measurement, it is determined that the amount of expression of the gene, the amount of protein, or the activity of the protein is decreased. If so, the candidate substance can be determined as a substance capable of treating an inflammatory neurological disease.
한 구체예에서, 후보물질이 Sgk1 유전자의 발현을 억제시키면 상기 후보물질이 염증성 신경 질환을 치료할 수 있는 물질로 판단할 수 있다. In one embodiment, when the candidate substance inhibits the expression of the Sgk1 gene, the candidate substance may be determined as a substance capable of treating an inflammatory neurological disease.
상기 Sgk1의 유전자 발현 수준 또는 단백질의 양을 측정하는 방법은 공지의 기술을 이용하여 생물학적 시료로부터 mRNA 또는 단백질을 분리하는 공지의 공정을 포함하여 수행될 수 있다. The method of measuring the gene expression level of Sgk1 or the amount of protein may be performed by using a known technique, including a known process of separating mRNA or protein from a biological sample.
상기 생물학적 시료는 염증성 신경 질환의 발생 또는 진행 정도에 따른 상기 유전자의 발현 수준 또는 단백질의 수준이 정상 대조군과는 다른, 생체로부터 채취된 시료를 말하며, 상기 시료로는 예컨대, 조직, 세포, 혈액, 혈청, 혈장, 타액 및 뇨 등이 포함될 수 있으나 이에 제한되는 것은 아니다.The biological sample refers to a sample taken from a living body whose expression level or protein level of the gene is different from that of a normal control group according to the incidence or progression of inflammatory neurological disease, and the sample includes, for example, tissue, cells, blood, Serum, plasma, saliva, urine, and the like may be included, but are not limited thereto.
상기 방법에서 유전자의 발현양, 단백질의 양 또는 단백질의 활성을 측정하는 방법은 당업계에 공지된 다양한 방법을 통해 수행될 수 있는데, 예컨대 역전사 중합효소 연쇄반응(reverse transcripatase-polymerase chain reaction), 실시간 중합효소 연쇄반응(real time-polymerase chain reaction), 웨스턴 블랏, 노던 블랏, 서던 블랏, EMSA(electrophoric mobility shift assay), 면역형광염색(immunofluorescence), ELISA(enzyme-linked immunosorbent assay), 방사선 면역분석(RIA; radioimmuno assay), 방사면역확산법(radioimmunodiffusion) 또는 면역침전분석(immunoprecipitation) 등을 이용하여 수행할 수 있다.In the above method, the method of measuring the amount of gene expression, the amount of protein, or the activity of the protein can be performed through various methods known in the art, such as reverse transcripatase-polymerase chain reaction, real-time Real time-polymerase chain reaction, Western blot, Northern blot, Southern blot, EMSA (electrophoric mobility shift assay), immunofluorescence, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay ( RIA (radioimmuno assay), radioimmunodiffusion, or immunoprecipitation can be used.
본 발명의 스크리닝 방법을 통해 얻은, 유전자 발현을 억제시키거나 단백질의 기능을 저하시키는 활성을 나타내는 후보물질은 염증성 신경 질환 치료제의 후보물질이 될 수 있다.Candidate substances obtained through the screening method of the present invention and exhibiting activity of inhibiting gene expression or reducing the function of proteins may be candidate substances for therapeutic agents for inflammatory neurological diseases.
이와 같은 염증성 신경 질환 치료제 후보물질은 이후의 염증성 신경 질환의 치료제 개발 과정에서 선도물질(leading compound)로서 작용하게 되며, 선도물질이 Sgk1 유전자 또는 그로부터 발현되는 단백질의 기능을 촉진 또는 억제 효과를 나타낼 수 있도록 그 구조를 변형시키고 최적화함으로써, 새로운 염증성 신경 질환 치료제를 개발할 수 있다.Such candidates for inflammatory neurological disease treatment will act as a leading compound in the development of a treatment for inflammatory neurological disease in the future, and the leading material can promote or inhibit the function of the Sgk1 gene or the protein expressed therefrom. By modifying and optimizing its structure so that it is possible to develop new therapeutic agents for inflammatory neurological diseases.
본 발명에서 유전공학적 기술과 관련된 사항은 샘브룩 등의 문헌(Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y(2001)) 및 프레드릭 등의 문헌(Prederick M. Ausubel et al. Current protocols in molecular biology volume 1, 2, 3, John Wiley & Sons, Inc. (1994))에 개시되어 있는 내용에 의해 보다 명확하게 된다.In the present invention, matters related to genetic engineering techniques are described in Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001) and Frederick et al. (Prederick M. Ausubel et al. Current protocols in molecular biology volume 1, 2, 3, John Wiley & Sons, Inc. (1994)).
본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 상세하게 후술되어 있는 실시예들을 참조하면 명확해질 것이다. 그러나 본 발명은 이하에서 개시되는 실시예들에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 것이며, 단지 본 실시예들은 본 발명의 개시가 완전하도록 하고, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이며, 본 발명은 청구항의 범주에 의해 정의될 뿐이다.Advantages and features of the present invention, and a method of achieving them will become apparent with reference to embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, only the present embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to completely inform the scope of the invention to those who have it, and the invention is only defined by the scope of the claims.
본 발명은 Sgk1의 넉다운을 통해 신경염증을 감소시키고, 신경교세포의 노화를 억제하며, 신경교세포에서의 글루타메이트 흡수를 증가시키고, 신경세포의 성숙 및 생존성을 증진시킬 수 있으므로, Sgk1 저해제로부터 염증성 신경 질환의 예방, 완화 또는 치료에 기여할 수 있다.The present invention reduces neuroinflammation through knockdown of Sgk1, inhibits senescence of glial cells, increases glutamate absorption in glial cells, and improves maturation and viability of neurons, so that inflammatory neurons from Sgk1 inhibitors It can contribute to the prevention, alleviation or treatment of disease.
도 1a는 마우스 피질에서 얻은 Nurr1과 Foxa2가 과발현된 신경교세포의 1차 배양 세포에서의 마이크로어레이 분석 결과를 나타낸 것이다. 1A shows the results of microarray analysis in primary cultured cells of glial cells overexpressing Nurr1 and Foxa2 obtained from mouse cortex.
도 1b는 마우스 중뇌에서 얻은 Nurr1과 Foxa2가 과발현된 신경교세포의 RNA-sequencing 분석 결과를 나타낸 것이다.Figure 1b shows the results of RNA-sequencing analysis of glial cells overexpressing Nurr1 and Foxa2 obtained from mouse midbrain.
도 1c는 Nurr1 및/또는 Foxa2의 발현에 따른 Sgk1의 발현 수준을 qPCR을 통해 확인한 결과이다.1C is a result of confirming the expression level of Sgk1 according to the expression of Nurr1 and/or Foxa2 through qPCR.
도 2a는 Nurr1 및/또는 Foxa2가 과발현된 성상교세포에서의 Sgk1의 발현 수준을 확인하기 위한 western blot 분석 결과를 나타낸 것이다.Figure 2a shows the results of western blot analysis to confirm the expression level of Sgk1 in astrocytes overexpressing Nurr1 and/or Foxa2.
도 2b는 TLR 리간드인 LPS를 신경교세포에 처리한 후 NFκB 활성을 확인한 결과이다.Figure 2b is a result of confirming the NFκB activity after the treatment of the TLR ligand LPS to glial cells.
도 3은 Sgk1 저해제를 처리한 신경교세포에서의 염증성 사이토카인(IL-1β 및 TNF-α)의 발현 수준을 확인한 결과이다. 3 is a result of confirming the expression level of inflammatory cytokines (IL-1β and TNF-α) in glial cells treated with an Sgk1 inhibitor.
도 4는 Sgk1 조절에 따른 세포 간 상호작용 효과를 나타낸 것으로, 도 4a는 Sgk1 발현이 감소된 성상교세포와 Sgk1이 과발현된 성상교세포의 공동 배양을 도식화한 것이다.Figure 4 shows the effect of the interaction between cells according to the regulation of Sgk1, Figure 4a is a schematic diagram of the co-culture of astrocytes with reduced Sgk1 expression and Sgk1 overexpressed astrocytes.
도 4b 및 도 4c는 공동 배양한 두 세포의 활성산소종(ROS)의 활성을 측정한 결과이다.4B and 4C are results of measuring the activity of reactive oxygen species (ROS) of two co-cultured cells.
도 5는 Sgk1 조절에 따른 각 세포 유형(미세아교세포 또는 성상교세포)의 기여도를 확인한 결과로, 도 5a는 미세아교세포와 성상교세포를 분리하여 배양한 후 Sgk1 발현 수준을 측정한 결과이다.Figure 5 is a result of confirming the contribution of each cell type (microglia or astrocyte) according to the Sgk1 regulation, Figure 5a is a result of measuring the Sgk1 expression level after separating and culturing microglia and astrocytes.
도 5b와 도 5c는 H 2O 2가 처리된 중뇌 부위의 성상교세포와 피질 부위의 성상교세포에서의 Sgk1의 발현 수준과 Sgk1 형광 발현 강도를 확인한 결과이다. 5B and 5C are the results of confirming the expression level of Sgk1 and the intensity of Sgk1 fluorescence in astrocytes in the midbrain region treated with H 2 O 2 and astrocytes in the cortical region.
도 6은 성상교세포에서의 Sgk1 저해 효과를 확인한 것으로, 중뇌 유래의 신경교세포의 Sgk1 저해제의 유무에 따른 RNA 서열분석 결과를 나타낸 것이다.6 shows the results of RNA sequencing according to the presence or absence of an Sgk1 inhibitor in midbrain-derived glial cells as confirming the Sgk1 inhibitory effect on astrocytes.
도 7은 성상교세포에서의 Sgk1 저해 효과를 확인한 것으로, 상기 도 6의 결과 중 염증/면역과 관련된 유전자 그룹을 heat-map으로 나타낸 것이다.FIG. 7 shows the Sgk1 inhibitory effect on astrocytes, and shows a heat-map of a group of genes related to inflammation/immunity among the results of FIG. 6.
도 8은 Sgk1 저해가 NRLP3 염증조절 복합체(inflammasome)의 활성에 미치는 영향을 확인한 결과로, 도 8a는 Sgk1 저해제를 처리하여 배양한 중뇌 유래의 신경교세포에 LPS와 ATP를 차례로 처리하여 inflammasome 활성을 자극한 후 caspase-1과 IL-1ß의 발현 수준을 확인한 결과이다.8 is a result of confirming the effect of Sgk1 inhibition on the activity of the NRLP3 inflammation-regulating complex (inflammasome). FIG. 8A is a result of confirming the effect of Sgk1 inhibitory treatment on the midbrain-derived glial cells cultured with LPS and ATP sequentially to stimulate inflammasome activity. This is the result of confirming the expression levels of caspase-1 and IL-1ß.
도 8b는 Sgk1 저해제가 처리된 중뇌 유래 신경교세포의 배양액 내 IL-1ß와 IL-6의 발현 수준을 확인한 그래프이다.Figure 8b is a graph confirming the expression levels of IL-1ß and IL-6 in the culture medium of midbrain-derived glial cells treated with an Sgk1 inhibitor.
도 8c는 Sgk1 저해제의 처리에 따른 신경교세포에서의 cGAS-STING 경로 활성을 측정한 결과이다.Figure 8c is a result of measuring the cGAS-STING pathway activity in glial cells according to the treatment of the Sgk1 inhibitor.
도 9는 Sgk1 저해가 NRLP3 염증조절 복합체(inflammasome)의 활성에 미치는 영향을 확인한 결과로, 도 9a는 Sgk1 저해제가 처리된 신경교세포의 활성산소종(ROS)의 활성을 측정한 결과이다. 도 9b는 Sgk1 저해제를 처리한 Mito-Sox 세포의 면역염색 및 FACS 결과를 나타낸 것이다.9 is a result of confirming the effect of Sgk1 inhibition on the activity of the NRLP3 inflammation-regulating complex (inflammasome), FIG. 9a is a result of measuring the activity of reactive oxygen species (ROS) of glial cells treated with the Sgk1 inhibitor. Figure 9b shows the results of immunostaining and FACS of Mito-Sox cells treated with Sgk1 inhibitor.
도 10의 왼쪽은 Sgk1 저해제가 처리된 신경교세포에서의 세포 응집/ECM 분자 관련 유전자의 발현을 heat-map으로 나타낸 것이고, 오른쪽은 Sgk1 저해제가 처리된 신경교세포에서의 글루타메이트 흡수 정도를 확인한 결과이다.The left side of FIG. 10 is a heat-map showing the expression of a cell aggregation/ECM molecule-related gene in glial cells treated with the Sgk1 inhibitor, and the right side is the result of confirming the degree of glutamate absorption in glial cells treated with the Sgk1 inhibitor.
도 11은 신경세포에서의 Sgk1 저해제의 처리에 따른 세포의 성숙과 생존성을 확인한 결과를 나타낸 것으로, Sgk1 저해제 농도에 따른 중뇌 유래 신경세포와 도파민 신경세포의 신경돌기 길이 및 TH 발현율을 확인한 것이다.Figure 11 shows the results of confirming the maturation and viability of cells according to the treatment of the Sgk1 inhibitor in neurons, confirming the neurite length and TH expression rate of midbrain-derived neurons and dopamine neurons according to the concentration of the Sgk1 inhibitor.
도 12는 노화된 세포에서의 Sgk1 저해제의 처리에 따른 신경세포의 생존율을 나타낸 결과이다.12 is a result showing the survival rate of neurons according to the treatment of an Sgk1 inhibitor in aged cells.
도 13은 중뇌 유래의 신경세포의 Sgk1 저해제 처리에 따른 TH 발현율을 확인한 결과이다.13 is a result of confirming the TH expression rate of midbrain-derived neurons treated with an Sgk1 inhibitor.
도 14는 도파민 신경세포의 Sgk1 저해제 처리에 따른 TH 발현율을 확인한 결과이다.14 is a result of confirming the TH expression rate according to Sgk1 inhibitor treatment of dopamine neurons.
도 15a는 신경세포에 Sgk1 저해제를 처리한 후 Parkin, Paris 및 PGC1a의 발현 정도를 확인한 결과이다.15A is a result of confirming the expression levels of Parkin, Paris, and PGC1a after treatment with an Sgk1 inhibitor on neurons.
도 15b는 SH-SY5Y 세포에 Sgk1 저해제를 처리한 후 Parkin의 발현 정도를 확인한 결과이다.15b is a result of confirming the expression level of Parkin after treatment with the Sgk1 inhibitor in SH-SY5Y cells.
도 15c는 Sgk1 저해제가 미토콘드리아의 ROS 생성을 감소시키는 결과를 나타낸 것이다.Figure 15c shows the results of reducing the Sgk1 inhibitor ROS production of mitochondria.
도 15d는 Sgk1 저해제가 α-synuclein의 응집을 억제시키는 결과를 나타낸 것이다.Figure 15d shows the results of the Sgk1 inhibitor inhibiting the aggregation of α-synuclein.
도 16은 MPTP-유도 파킨슨병 마우스 모델의 제작 과정을 나타낸 것이다.Figure 16 shows the manufacturing process of the MPTP-induced Parkinson's disease mouse model.
도 17은 도 16의 마우스 모델에 대해 균형 실험(beam test)과 막대 실험(pole test)을 수행한 결과이다.17 is a result of performing a beam test and a pole test for the mouse model of FIG. 16.
도 18은 도 16의 마우스 모델을 MPTP 처리 후 1개월 후에 희생시켜 얻은 SN 부위 조직의 RT-PCR을 수행한 결과이다.18 is a result of performing RT-PCR of the SN region tissue obtained by sacrificing the mouse model of FIG. 16 1 month after MPTP treatment.
도 19는 도 16의 마우스 모델을 MPTP 처리 후 1개월 후에 희생시켜 얻은 SN 주변부 조직의 RT-PCR을 수행한 결과이다.19 is a result of performing RT-PCR of tissues around the SN obtained by sacrificing the mouse model of FIG. 16 1 month after MPTP treatment.
도 20은 도 16의 마우스 모델의 SN 부위의 도파민 신경세포를 TH 염색을 통해 확인한 결과이다.20 is a result of confirming the dopamine neurons in the SN region of the mouse model of FIG. 16 through TH staining.
도 21은 도 16의 마우스 모델의 SN 부위의 도파민 신경세포의 개수를 측정한 결과이다.21 is a result of measuring the number of dopamine neurons in the SN region of the mouse model of FIG. 16.
도 22는 in vitro 상에서 Sgk1 저해제의 처리에 따른 α-synuclein 응집 방지 및 응집 제거 효과가 있는지 확인하기 위해 α-synuclein이 과발현된 중뇌 유래의 신경전구세포와 성상교세포의 공동 배양을 도식화한 것이다.22 is a schematic diagram of co-culture of neural progenitor cells and astrocytes derived from the midbrain overexpressing α-synuclein in order to confirm whether there is an effect of preventing α-synuclein aggregation and removing aggregation according to the treatment of the Sgk1 inhibitor in vitro.
도 23은 α-synuclein이 과발현된 중뇌 유래의 신경전구세포를 3주간 분화시키면서 Sgk1 저해제를 처리한 후에 면역염색 및 웨스턴 블롯을 수행하여 α-synuclein의 응집 정도를 확인한 것이다.FIG. 23 shows the degree of aggregation of α-synuclein by performing immunostaining and western blot after treatment with an Sgk1 inhibitor while differentiating the neural progenitor cells derived from the midbrain overexpressing α-synuclein for 3 weeks.
도 24는 α-synuclein이 과발현된 중뇌 유래의 신경전구세포와 성상교세포를 공동 배양하여 Sgk1 저해제를 처리한 후에 면역염색 및 웨스턴 블롯을 수행하여 α-synuclein의 응집 정도를 확인한 것이다.24 shows the degree of aggregation of α-synuclein by performing immunostaining and western blot after co-culturing α-synuclein-derived neural progenitor cells and astrocytes derived from the midbrain and astrocytes co-cultured with an Sgk1 inhibitor.
도 25는 마우스 ICR 종의 SN 부위에 pre-formed Fibril(PFF)와 AAV2 α-synuclein 바이러스를 주입한 후, Sgk1 저해제를 매일 복강 주사한 후의 마우스 뇌 조직을 형광 염색하는 과정을 도식화한 것이다.FIG. 25 is a schematic diagram of a process of fluorescently staining mouse brain tissue after injection of pre-formed Fibril (PFF) and AAV2 α-synuclein virus into the SN site of a mouse ICR species, and then daily intraperitoneal injection of an Sgk1 inhibitor.
도 26은 도 25의 Sgk1 저해제를 복강 주사한 마우스 뇌의 형광 염색 결과를 나타낸 것으로, phospho-α-synuclein의 발현 정도를 관찰한 결과이다.FIG. 26 is a result of fluorescence staining of a mouse brain intraperitoneally injected with the Sgk1 inhibitor of FIG. 25, and is a result of observing the level of expression of phospho-α-synuclein.
도 27은 도 25의 Sgk1 저해제를 복강 주사한 마우스 뇌의 형광 염색 결과를 나타낸 것으로, 미세아교세포(microglia)의 M1 타입의 마커인 CD16/32의 발현 정도를 관찰한 결과이다.FIG. 27 shows the result of fluorescence staining of the mouse brain injected with the Sgk1 inhibitor of FIG. 25 intraperitoneally, and is a result of observing the expression level of CD16/32, an M1 type marker of microglia.
도 28은 도 25의 Sgk1 저해제를 복강 주사한 마우스의 동물행동 실험(beam test 및 pole test) 결과를 나타낸 것이다.FIG. 28 shows the results of animal behavior experiments (beam test and pole test) of mice intraperitoneally injected with the Sgk1 inhibitor of FIG. 25.
이하, 본 출원을 실시예를 통해 상세히 설명한다. 하기 실시예는 본 출원을 예시하는 것일 뿐 본 출원의 범위가 하기 실시예에 한정되는 것은 아니다. Hereinafter, the present application will be described in detail through examples. The following examples are merely illustrative of the present application, and the scope of the present application is not limited to the following examples.
[실시예] [Example]
1. 세포 배양1. Cell culture
1-1. 신경전구세포 배양1-1. Neural precursor cell culture
도파민성 신경전구세포(Neural progenitor cells)는 마우스(imprinting control region, ICR)의 경우 임신 10.5일의 중뇌 부위에서 수득하였으며, 래트(Sprague-Dawley rat, SD)의 경우, 임신 12일째 주의 중뇌 부위에서 수득하였다. 중뇌성 신경전구세포는 혈청이 없는 N2 배지를 이용하여 배양하였으며, 분열을 촉진시키기 위해 배양배지에 bFGF(basic fibroblast growth factor; 20 ng/ml; R&D Systems, Minneapolis, MN, USA)와 epithelial growth factor (EGF; 20 ng/ml; R&D Systems, 마우스 세포 배양시)를 첨가하여 배양하였다. 이후, 세포 밀집도(confluence)가 70%에 다다랐을 때 계대배양하였다.Dopaminergic neuronal progenitor cells were obtained from the midbrain region at 10.5 days gestation in the case of mice (imprinting control region, ICR), and in the midbrain region at the 12th day gestation in rats (Sprague-Dawley rat, SD). Obtained. Mesocerebral neural progenitor cells were cultured using serum-free N2 medium, and bFGF (basic fibroblast growth factor; 20 ng/ml; R&D Systems, Minneapolis, MN, USA) and epithelial growth factor were cultured in the culture medium to promote division. (EGF; 20 ng/ml; R&D Systems, mouse cell culture) was added and cultured. Thereafter, when the cell density (confluence) reached 70%, it was subcultured.
신경전구세포의 세포 증식 이후, 세포들은 실험을 위해 수득하거나, 분열촉진인자를 제외한 배양 배지를 이용하여 세포를 분화시켰다. 또한, 도파민성 신경세포로 분화하지 않는 피질 부위의 신경전구세포는 임신 12일의 마우스와 임신 14일의 래트로부터 수득하여 실험을 진행하였다. After cell proliferation of the neural progenitor cells, the cells were obtained for an experiment, or the cells were differentiated using a culture medium excluding a mitogenic factor. In addition, neuroprogenitor cells in the cortical region that do not differentiate into dopaminergic neurons were obtained from mice at the 12th day of pregnancy and rats at the 14th day of pregnancy, and the experiment was conducted.
1-2. 신경교세포와의 공배양1-2. Co-culture with glial cells
성상세포(astrocyte)는 생후 1~5일의 마우스 또는 래트의 중뇌 또는 피질 부위로부터 수득하였다. 간략히 설명하면, 중뇌 조직을 파이펫을 이용하여 잘게 쪼갠 후, 10%의 소태아혈청(Fetal bovine serum; FBS; Gibco)을 함유한 Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Camarillo, CA, USA)배지를 이용하여 75-cm 2 T-플라스크에 배양시켰다. 세포 밀집도가 75-cm 2 T-플라스크의 80 내지 90% 정도 찼을 때 0.1%의 트립신(trypsin)을 이용하여 poly-D-lysine(PDL; MilliporeSigma)로 코팅된 플레이트로 계대배양하였다.Astrocytes were obtained from midbrain or cortical regions of mice or rats 1-5 days old. Briefly, after dividing the midbrain tissue with a pipette, Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Camarillo, CA, USA) containing 10% Fetal bovine serum (FBS; Gibco) It was cultured in a 75-cm 2 T-flask using a medium. When the cell density was about 80 to 90% of the 75-cm 2 T-flask, it was subcultured to a plate coated with poly-D-lysine (PDL; MilliporeSigma) using 0.1% trypsin.
1-3. 신경세포의 초대 배양1-3. Primary culture of nerve cells
도파민 신경세포의 초대 배양은 마우스의 중뇌 조직으로부터 수득하여 수행하였다. 먼저, 임신 14일의 래트 중뇌 조직을 파파인(papain; Worthington, Ls003126)을 이용하여 잘게 쪼갠 후, NB 배지(neurobasal media; Life Technologies, Carlsbad, CA), B27 및 L-글루타민(L-glutamine; Life Technologies)을 혼합한 배양 배지를 이용하여 Poly-L-Ornithine(PLO; Sigma, P3655)와 FN(Fibronectin; Sigma, F1141)이 코팅된 플레이트에 배양시켰다. 이후, 분화 3~5일째에 β-D-arabinofuranoside(Ara-C; Sigma) 3μM 를 배지에 첨가하여 증식하는 성상세포들을 제거하였다. Primary culture of dopamine neurons was performed by obtaining from mouse midbrain tissue. First, the rat midbrain tissue on the 14th day of pregnancy was finely split using papain (Worthington, Ls003126), and then NB medium (neurobasal media; Life Technologies, Carlsbad, CA), B27 and L-glutamine (L-glutamine; Life) Technologies) was cultured on a plate coated with Poly-L-Ornithine (PLO; Sigma, P3655) and FN (Fibronectin; Sigma, F1141). Thereafter, 3 μM of β-D-arabinofuranoside (Ara-C; Sigma) was added to the medium on days 3 to 5 of differentiation to remove proliferating astrocytes.
1-4. α-synuclein이 과발현된 중뇌 유래 신경전구세포의 배양 및 성상교세포와의 공동 배양1-4. Cultivation of neural progenitor cells derived from midbrain overexpressing α-synuclein and co-culture with astrocytes
CMV promoter가 달린 Lenti α-synuclein virus를 중뇌 유래의 신경전구세포에 과발현시킨 후, 배양 또는 성상교세포와 공동 배양하여 3주간 분화시키면서 Sgk1 inhibitor(GSK-650394)를 배지에 섞어서 처리하였다. 이 후 α-synuclein 응집체 형성이 방지되는지를 확인하기 위해 western blotting과 phosphor-alpha synuclein 형광 염 실험을 진행했다.After overexpressing the Lenti α-synuclein virus with the CMV promoter on neural progenitor cells derived from midbrain, cultured or co-cultured with astrocytes and differentiated for 3 weeks, followed by treatment with Sgk1 inhibitor (GSK-650394) mixed in the medium. After that, western blotting and phosphor-alpha synuclein fluorescent salt experiments were conducted to determine whether the formation of α-synuclein aggregates was prevented.
2. 바이러스 생성2. Virus generation
CMV 프로모터의 조절 하에, Nurr1, Foxa2 및 Sgk1을 발현하는 렌티바이러스 벡터들은 각각의 cDNA를 pCDH(System Biosciences, Mountain View, CA)의 다중클로닝 부위에 삽입함으로써 생성하였다. pGIPZ-shSgk1 렌티바이러스 벡터는 Open Biosystems (Rockford, IL)에서 구입하였다. 이때 음성 대조군으로는 비어있는 기본 벡터(pCDH 또는 pGIPZ)를 사용하였다. 렌티바이러스의 역가는 QuickTiterTM HIV Lentivirus Quantitation Kit(Cell Biolabs, San Diego, CA, USA)를 이용하여 측정하였고, 10 6개의 형질도입 유닛(transducing unit, TU)/ml을 포함한 Nurr1 및 Foxa2 각각의 바이러스 20 ㎕는 2 ml의 배지와 혼합한 후, 형질도입 반응을 위해 1-1.5 X 10 6 세포/6cm-dish에 첨가하였다. Under the control of the CMV promoter, lentiviral vectors expressing Nurr1, Foxa2 and Sgk1 were generated by inserting each cDNA into the multicloning site of pCDH (System Biosciences, Mountain View, CA). The pGIPZ-shSgk1 lentiviral vector was purchased from Open Biosystems (Rockford, IL). At this time, an empty basic vector (pCDH or pGIPZ) was used as a negative control. Lentivirus titers were measured using the QuickTiterTM HIV Lentivirus Quantitation Kit (Cell Biolabs, San Diego, CA, USA), and each virus of Nurr1 and Foxa2 including 10 6 transducing units (TU)/ml 20 Μl was mixed with 2 ml of medium, and added to 1-1.5 X 10 6 cells/6cm-dish for transduction reaction.
3. 면역 염색3. Immunostaining
배양된 세포는 PBS(phosphate-buffered saline) 내 4%의 파라포름알데하이드(PFA)를 이용하여 고정하고, PBS에 1%의 소혈청알부민(bovine serum albumin)과 0.3%(또는 0.6%)의 Triton X-100이 첨가된 용액으로 블록킹(blocking) 시켰다. 이후 블록킹된 용액에 1차 항체를 첨가하여 4℃에서 오버나이트시켰다. 이때 1차 항체로는 TH(1:1000, 토끼, Pel-Freez, Rogers, AR), GFAP(1:200, 마우스, MP Biodmedicals, Santa Ana, CA), MAPS(microtubule-associated protein 2; 1:1000, 마우스, Sigma)를 사용하였고, 2차 항체로는 Cy3(1:200, Jackson Immunoresearch Laboratories, West Grove, PA, USA) 또는 Alexa Fluor 488(1:200, Life Technologies)를 사용하였다. 염색된 세포는 DAPI 계수 용액(mounting solution)이 있는 VECTASHIELD를 사용하여 계수하고 형광 현미경(Leica, Heidelberg, Germany)을 이용하여 형광 촬영하였다. Cultured cells were fixed using 4% paraformaldehyde (PFA) in PBS (phosphate-buffered saline), and 1% bovine serum albumin and 0.3% (or 0.6%) Triton in PBS. It was blocked with a solution to which X-100 was added. Thereafter, a primary antibody was added to the blocked solution and overnighted at 4°C. At this time, the primary antibodies include TH (1:1000, rabbit, Pel-Freez, Rogers, AR), GFAP (1:200, mouse, MP Biodmedicals, Santa Ana, CA), microtubule-associated protein 2 (MAPS); 1: 1000, mouse, Sigma) was used, and Cy3 (1:200, Jackson Immunoresearch Laboratories, West Grove, PA, USA) or Alexa Fluor 488 (1:200, Life Technologies) was used as a secondary antibody. The stained cells were counted using VECTASHIELD with a DAPI mounting solution, and fluorescence was photographed using a fluorescence microscope (Leica, Heidelberg, Germany).
4. mRNA 발현 분석4. mRNA expression analysis
RNA 분리 프로토콜에 따라 Trizol Reagent (Invitrogen, Carlsbad, CA, USA)를 사용하여 세포로부터 전체 RNA를 수득하였다. 이후 슈퍼스크립트 키트 (superscript kit; Invitrogen)를 사용하여 cDNA를 합성하였다. 실시간 PCR은 iQ TM SYBR green supermix (Bio-Rad, Hercules, CA, USA)를 사용하는 CFX95 TM Real-Time System에서 진행하였으며, 유전자 발현 수준은 GAPDH로 보정하였다. 프라이머에 대한 정보는 다음 표 1에 기재하였다. Total RNA was obtained from cells using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the RNA isolation protocol. Thereafter, cDNA was synthesized using a superscript kit (Invitrogen). Real-time PCR was performed in a CFX95 TM Real-Time System using iQ TM SYBR green supermix (Bio-Rad, Hercules, CA, USA), and the gene expression level was corrected with GAPDH. Information on the primers are shown in Table 1 below.
SEQ ID NO.SEQ ID NO. 유전자(Gene)Gene 핵산서열Nucleic acid sequence
1One Sgk1-ForwardSgk1-Forward GTA CCC TGC TCT CGC CTGGTA CCC TGC TCT CGC CTG
22 Sgk1-ReverseSgk1-Reverse ATC TGT ATC CCG ATC CGC CTATC TGT ATC CCG ATC CGC CT
33 IL-1b-ForwardIL-1b-Forward CTG TGA CTC GTG GGA TGA TGCTG TGA CTC GTG GGA TGA TG
44 IL-1b-ReverseIL-1b-Reverse GGG ATT TTG TCG TTG CTT GTGGG ATT TTG TCG TTG CTT GT
55 TNF-a-ForwardTNF-a-Forward AGA TGT GGA ACT GGC AGA GGAGA TGT GGA ACT GGC AGA GG
66 TNF-a-ReverseTNF-a-Reverse CCC ATT TGG GAA CTT CTC CTCCC ATT TGG GAA CTT CTC CT
77 iNOS-ForwardiNOS-Forward CAC CTT GGA GTT CAC CCA GTCAC CTT GGA GTT CAC CCA GT
88 iNOS-ReverseiNOS-Reverse ACC ACT CGT ACT TGG GAT GCACC ACT CGT ACT TGG GAT GC
5. 활성산소종(reactive oxygen species, ROS) 활성 측정5. Measurement of reactive oxygen species (ROS) activity
5-1. 활성산소종(reactive oxygen species, ROS) 활성 측정(DCF-DA)5-1. Active oxygen species (ROS) activity measurement (DCF-DA)
세포 내 ROS 활성을 측정하기 위해, 세포를 5-(및-6)-클로로메틸-20, 70-디클로로디하이드로플루오레세인 디아세테이트(5-(and-6)-chloromethyl-20, 70-dichlorodihydrofluorescein diacetate) [CM-H2DCF-DA(여기서는 DCF라 함) (Life Technologies) 10 μM로 10분간 배양하였다. 이후 세포들을 D-PBS(mM 단위로: 2.68 KCl, 1.47 KH 2PO 4, 136.89 NaCl 및 8.1 Na 2HPO 4)를 이용하여 배지를 여러 번 교체해주고, DAPI가 포함된 VECTASHIELD(Vector Laboratories, CA, USA)로 세포를 염색 고정하고 형광 현미경으로 촬영하였다. To measure intracellular ROS activity, cells were treated with 5-(and-6)-chloromethyl-20, 70-dichlorodihydrofluorescein diacetate (5-(and-6)-chloromethyl-20, 70-dichlorodihydrofluorescein). diacetate) [CM-H2DCF-DA (referred to herein as DCF) (Life Technologies) 10 μM for 10 minutes. Thereafter, the cells were replaced with D-PBS (in mM: 2.68 KCl, 1.47 KH 2 PO 4 , 136.89 NaCl and 8.1 Na 2 HPO 4 ) several times to replace the medium several times, and VECTASHIELD (Vector Laboratories, CA, containing DAPI) USA) stained and fixed and photographed under a fluorescence microscope.
5-2. 미토콘드리아 활성산소종 활성 측정(Mito-Sox)5-2. Measurement of mitochondrial reactive oxygen species activity (Mito-Sox)
미토콘드리아의 활성산소종(ROS)은 형광 탐침이 있는 Mito-Sox(Life Technologies)를 이용하여 확인하였다. 세포는 기본 배지 용액으로 2 μM의 Mito-Sox를 만들어 5% (v/v) CO 2 세포 배양기에서 37℃, 30분간 배양하였다. 그 다음 PBS를 이용하여 세포를 세척하고, 4%의 파라포름알데하이드(PFA)로 세포를 고정하였다. Mito-Sox 수준은 flow cytometry(arbitrary units)와 DAPI가 포함된 용액인 VECTASHIELD (Vector Laboratories, CA, USA)를 이용하여 형광 촬영하였다. Reactive oxygen species (ROS) of mitochondria was identified using Mito-Sox (Life Technologies) with a fluorescent probe. Cells were prepared with 2 μM of Mito-Sox in a basic medium solution and incubated for 30 minutes at 37° C. in a 5% (v/v) CO 2 cell incubator. Then, the cells were washed with PBS, and the cells were fixed with 4% paraformaldehyde (PFA). Mito-Sox levels were fluoresced using flow cytometry (arbitrary units) and VECTASHIELD (Vector Laboratories, CA, USA), a solution containing DAPI.
6. 세포 계수 및 통계 분석6. Cell counting and statistical analysis
면역 염색되고, DAPI 염색된 세포들의 수는 접안 격자를 이용하여 200X 또는 400X 배율에서 각 배양의 커버슬립에 9-20개의 무작위로 선정된 부위 내에서 집계되었다. 모든 데이터는 평균(mean) ± SEM과 적절하게 통계 검정하여 표시하였다. 통계 비교는 스튜던트 T-검정(unpaired)이나 한 방향 ANOVA(one-way ANOVA)를 사용하였다. The number of immunostained and DAPI-stained cells was counted within 9-20 randomly selected sites on the coverslip of each culture at 200X or 400X magnification using an eyepiece grid. All data were expressed by means ± SEM and appropriate statistical tests. For statistical comparison, Student's T-test (unpaired) or one-way ANOVA (one-way ANOVA) was used.
7. 글루타메이트 흡수 활성7. Glutamate absorption activity
준비된 세포를 tissue buffer(5 mM Tris, 320 mM sucrose, pH 7.4)를 이용하여 2번 세척한 후, 10 μM의 글루타메이트(glutamate)에 Na +가 포함된 Krebs buffer (120 mM NaCl, 25 mM NaHCO 3, 5 mM KCl, 2 mM CaCl 2, 1 mM KH 2PO 4, 1 mM MgSO 4 및 10% glucose) 또는 Na +-free Kreb buffer (120 mM choline-Cl 및 25 mM Tris-HCl)을 이용하여 37℃에서 10분간 노출시켰다. 글루타메이트의 흡수는 세포를 얼음 위에 위치시킨 후, wash buffer (5 mM Tris/160 mM NaCl, pH 7.4)를 이용하여 2번 세척하여 수행하였다. 이후 100 ㎕의 assay buffer를 이용하여 세포를 수집하고 균질화한 후, 글루타메이트 분석 키트(Abcam, Cambridge, MA, USA, ab83389)를 통해 글루타메이트 흡수량을 확인하였다. Na + 의존적 흡수는 남아있는 현재 Na + 양에서 자유로워진 Na + 양을 제외하여 계산하였다.After washing the prepared cells twice with tissue buffer (5 mM Tris, 320 mM sucrose, pH 7.4), Krebs buffer (120 mM NaCl, 25 mM NaHCO 3 containing Na + in 10 μM glutamate) , 5 mM KCl, 2 mM CaCl 2 , 1 mM KH 2 PO 4 , 1 mM MgSO 4 and 10% glucose) or Na + -free Kreb buffer (120 mM choline-Cl and 25 mM Tris-HCl) 37 It was exposed for 10 minutes at °C. The absorption of glutamate was performed by placing the cells on ice and then washing them twice with a wash buffer (5 mM Tris/160 mM NaCl, pH 7.4). Thereafter, the cells were collected and homogenized using 100 µl of an assay buffer, and the amount of glutamate uptake was confirmed through a glutamate assay kit (Abcam, Cambridge, MA, USA, ab83389). Na + dependent absorption was calculated by excluding the amount of Na + freed from the remaining current Na + amount.
8. SA-ß(senescence-associated beta-galactosidase) 염색8. SA-ß (senescence-associated beta-galactosidase) staining
SA-ß 염색은 [Dimri et al., 1995 (Abcam)]과 동일하게 진행하였다. 24 웰 배양 접시를 이용하여 한 웰당 4.0 × 10 4 (4.0 × 10 4 cells/well)이 되도록 배양시키고, SA-ß 염색약을 이용하여 12 내지 18시간 동안 배양시켰다. 이후 파란색으로 염색된 세포들을 계수하여 전체 세포에 대한 백분율로 표현하였다.SA-ß staining was performed in the same manner as [Dimri et al., 1995 (Abcam)]. It was cultured to be 4.0 × 10 4 (4.0 × 10 4 cells/well) per well using a 24-well culture dish, and incubated for 12 to 18 hours using SA-ß dye. Thereafter, cells stained in blue were counted and expressed as a percentage of the total cells.
참고문헌: Dimri et al., Cellular Senescence Is Induced by the Environmental Neurotoxin Paraquat and Contributes to Neuropathology Linked to Parkinson's Disease (Cell Rep. 2018 January 23; 22(4): 930-940. doi:10.1016/j.celrep.2017.12.092.)Reference: Dimri et al., Cellular Senescence Is Induced by the Environmental Neurotoxin Paraquat and Contributes to Neuropathology Linked to Parkinson's Disease (Cell Rep. 2018 January 23; 22(4): 930-940.doi:10.1016/j.celrep.2017.12.092.)
9. CCK 세포의 생존력 분석9. Viability analysis of CCK cells
신경줄기세포를 96 웰 배양 접시에 1.0 × 10 4 cells/well로 배양한 후, 각각 다른 농도의 Sgk1 저해제를 처리하여 2일 내지 4일 후에 10 ㎕의 CCK-8 (D-Plus TM CCK cell viability assay kit, DonginBio, Korea) 용액을 각 웰에 첨가하여 37℃ 세포 배양기에서 30분 이상 배양하였다. 이후 세포 생존력을 450 nm 파장대의 분광 광도계(Bio-Rad Laboratories, Inc., Hercules, CA, USA)를 이용하여 측정하였다. 실험은 3번 이상 반복하였다.After culturing neural stem cells in a 96-well culture dish at 1.0 × 10 4 cells/well, each treatment with different concentrations of Sgk1 inhibitors, 10 μl of CCK-8 (D-Plus TM CCK cell viability assay) 2 to 4 days later. kit, DonginBio, Korea) solution was added to each well and incubated for at least 30 minutes in a cell incubator at 37°C. Thereafter, cell viability was measured using a spectrophotometer in the 450 nm wavelength band (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The experiment was repeated at least 3 times.
10. 염증조절 복합체(inflammasome)/caspase-1 활성 분석10. Inflammasome/caspase-1 activity assay
NLRP3 염증조절 복합체를 활성화하기 위해, 혼합된 신경교세포(성상세포+미세아교세포)에 0.25 ㎍/ml의 LPS를 3시간 처리한 후, 2 내지 2.5 mM의 ATP를 30 내지 45분동안 처리하였다. 염증조절 복합체의 활성은 배양액 내 활성화된 caspase-1 p10과 IL-1ß의 양을 면역 블롯으로 확인하였고, 세포외 IL-1ß 및 IL-6은 ELISA 분석을 통해 정량화하였다(E. Lee et al., 2018)In order to activate the NLRP3 inflammation-regulating complex, the mixed glial cells (astrocytic cells + microglia) were treated with 0.25 μg/ml of LPS for 3 hours, followed by treatment with 2 to 2.5 mM ATP for 30 to 45 minutes. The activity of the inflammation-regulating complex was confirmed by immunoblot of activated caspase-1 p10 and IL-1ß in the culture medium, and extracellular IL-1ß and IL-6 were quantified through ELISA analysis (E. Lee et al. , 2018)
참고문헌: Lee, E. et al., MPTP-driven NLRP3 inflammasome activation in microglia plays a central role in dopaminergic neurodegeneration, Cell Death Differ, doi:10.1038/s41418-018-0124-5 (2018)Reference: Lee, E. et al., MPTP-driven NLRP3 inflammasome activation in microglia plays a central role in dopaminergic neurodegeneration, Cell Death Differ, doi:10.1038/s41418-018-0124-5 (2018)
11. RNA-sequencing 분석11. RNA-sequencing analysis
RNA 서열분석은 E-biogen (Seoul, Korea)에서 실시하였다. 서열분석은 FastQC를 이용하여 질적 점수에 따라 잘라낸 후, Bowtie를 이용하여 미스매치 정도를 확인하였다.RNA sequencing was performed by E-biogen (Seoul, Korea). After sequencing was cut according to the qualitative score using FastQC, the degree of mismatch was confirmed using Bowtie.
모든 RNA 서열분석 데이터는 STAR을 사용하여 마우스 참조 게놈(GRCm38/mm10)을 이용하여 제작하였다. 마우스 게놈(NCBI RefSeq 13 annotations Release 105: Feb. 2015) 46,432 개 유전자들과, 1,076,310 개 유전체, 그리고 1,276,131 개 단백질의 발현양을 측정하기 위해, Htseq-count를 이용하여 유전자의 읽혀진 엑손을 확인하고, FRKM(Fragments Per Kilobase of exon per Million fragments 15 mapped)으로 표현하였다. 또한, 각 그룹당 데이터를 표준화하여 오차 값을 줄였다. 모든 데이터는 GEO 데이터베이스에 등록되어 있다(GEO: 17 GSE106216).All RNA sequencing data were prepared using the mouse reference genome (GRCm38/mm10) using STAR. Mouse genome (NCBI RefSeq 13 annotations Release 105: Feb. 2015) To measure the expression level of 46,432 genes, 1,076,310 genomes, and 1,276,131 proteins, the read exons of the gene were identified using Htseq-count, It was expressed as FRKM (Fragments Per Kilobase of exon per Million fragments 15 mapped). In addition, the error value was reduced by standardizing the data for each group. All data are registered in the GEO database (GEO: 17 GSE106216).
12. 동물 실험 112. Animal Experiment 1
마우스 ICR 종의 SN 부위에 PFF와 α-synuclein 바이러스 벡터를 주입하였다. 이후 마우스의 안정을 위해 일주일이 지난 다음에 Sgk1 저해제를 3mg/ml로 매일 복강 주사하였다. 주사 1주 후부터 동물 행동실험을 수행하였으며, 총 3개월이 경과되었을 때 마우스의 뇌 조직 고정과 형광 염색을 진행하였다.PFF and α-synuclein virus vectors were injected into the SN site of mouse ICR species. After a week for stability of the mouse, the Sgk1 inhibitor was injected daily intraperitoneally at 3 mg/ml. Animal behavioral experiments were performed from 1 week after injection, and when a total of 3 months had elapsed, brain tissue was fixed and fluorescent staining was performed.
13. 동물 실험 213. Animal Experiment 2
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)에 의해 유도된 아만성적(subchronic) 파킨슨병 마우스 모델을 제작하기 위해, 마우스에 5일간 30mg/kg 만큼 30㎕로 복강 주사하였다. 이후 일주일간 행동실험을 연습시킨 후 균형 막대 실험, 막대 실험 및 운동성 실험을 매주 2번씩 3번 반복실험하여 실험 결과를 기록하였다. Sgk1 저해제(GSK-650394)는 MPTP를 주사하는 시점부터 매일 3mg/kg으로 복강 주사하였다. 전 과정을 도 16에 나타내었다.To create a subchronic Parkinson's disease mouse model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the mice were intraperitoneally in 30 µl for 5 days at 30 mg/kg. Injected. After practicing the behavioral experiment for a week, the balance rod experiment, the rod experiment, and the motility experiment were repeated three times a week, twice a week, and the experimental results were recorded. The Sgk1 inhibitor (GSK-650394) was injected intraperitoneally at 3 mg/kg every day from the time of injection of MPTP. The entire process is shown in FIG. 16.
14. 거동 검사(행동실험)14. Behavior test (behavioral test)
14-1. 균형 막대 실험(Balance beam test)14-1. Balance beam test
약 30cm 정도의 얇은 원통형 나무막대를 수직으로부터 15cm 정도 떨어진 위치에 수평이 되도록 설치한 후, 동물들을 출발지로부터 반대편까지 가도록 하여 시간을 측정하였다. 쥐가 떨어지거나 막대를 벗어나 움직이지 않는다면 다시 실시하였다. 측정 전 2번의 테스트를 거친 후, 3번의 반복실험을 통해서 평균값을 계산하였다.After installing a thin cylindrical wooden rod of about 30 cm horizontally at a location 15 cm away from the vertical, the animals were taken from the starting point to the other side to measure the time. If the rat fell or did not move away from the rod, it was repeated. After two tests before measurement, the average value was calculated through three repeated experiments.
14-2. 막대 실험(pole test)14-2. Pole test
동물들을 머리가 위로 가도록 하여, 수직으로 세운 길이 50cm(지름 1cm)의 나무 막대의 꼭대기에 놓았다. 막대의 바닥은 우리 속에 두었다. 막대 위에 동물들을 놓으면, 동물들은 아래로 몸을 향해 막대를 따라 아래로 내려가 그들의 우리로 향하게 하였다. 모든 동물들은 2일동안 훈련시키고, 그 훈련은 세션당 5회 수행하였다. 검사 당일, 실험은 5회 반복하였고, 아래로 몸을 향할 때까지 소요된 시간을 측정하였다.The animals were placed on top of a 50 cm long (1 cm diameter) wooden bar erected upright with their heads up. The bottom of the rod was placed in the cage. When the animals were placed on the rod, the animals moved down the rod toward the body and directed to their cages. All animals were trained for 2 days, and the training was performed 5 times per session. On the day of the test, the experiment was repeated 5 times, and the time taken to face the body downward was measured.
14-3. 운동성 실험(locomotor test)14-3. Locomotor test
동물들을 길이 42cm, 너비 42cm 및 높이 42cm로 이루어진 흑색 플라스틱 박스에 넣고 5분간 그 환경에 익숙해지도록 방치한 후, 10분동안 운동 활성을 측정하였다. 운동 활성은 박스 안에서 움직인 총 거리(cm)와 움직인 총 시간(분)을 CCD 카메라에 기반하여 움직임을 추적하는 장치 및 소프트웨어(EthoVision 3.1, Noldus information Technology, Netherlands)를 이용해 측정하였다. The animals were placed in a black plastic box consisting of 42 cm long, 42 cm wide and 42 cm high, and left to get used to the environment for 5 minutes, and then the motor activity was measured for 10 minutes. The motor activity was measured using a device and software (EthoVision 3.1, Noldus information Technology, Netherlands) that tracks the motion based on a CCD camera by measuring the total distance (cm) and the total time (minutes) moved in the box.
[결과 및 고찰][Results and Discussion]
본 발명자들은 이전 연구(KR 공개특허 제10-2017-01018131호)를 통해 중뇌 발현 인자인 Nurr1(N)과 Foxa2(F)가 신경교세포에서 염증성 전사 인자의 발현을 억제함으로써 항염증 기능을 갖고 있음을 밝혔다. 또한, 마우스 피질에서 얻은 신경교세포의 1차 배양 세포를 마이크로어레이 분석한 결과, Nurr1과 Foxa2의 과발현에서 가장 특이적으로 발현이 감소된 인자는 serum-and-glucocorticoid-inducible kinase-1 (Sgk1) (N과 F의 복합 효과로 9.3배 감소됨)인 것을 알 수 있었다(도 1a). 마찬가지로 N+F가 과발현된 신경교세포(마우스 중뇌 부위로부터 얻은)의 RNA 서열분석 데이터에서도 Sgk1이 감소된 것을 확인할 수 있었다(도 1b). 이에, qPCR 데이터에서도 N+F에 의해서 Sgk1이 감소되는지를 알아보기 위해, Nurr1과 Foxa2를 각각 단독으로 처리했을 때와 동시에 처리했을 때의 발현양을 비교한 결과, Nurr1과 Foxa2를 동시에 처리한 군에서 Sgk1이 가장 많이 감소된 것을 확인할 수 있었다(도 1c). Through a previous study (KR Patent Publication No. 10-2017-01018131), the present inventors found that the midbrain expression factors Nurr1(N) and Foxa2(F) have anti-inflammatory functions by inhibiting the expression of inflammatory transcription factors in glial cells. Revealed. In addition, as a result of microarray analysis of primary cultured cells of glial cells obtained from the mouse cortex, the most specifically reduced factor in overexpression of Nurr1 and Foxa2 was serum-and-glucocorticoid-inducible kinase-1 (Sgk1) ( It was found that the combination of N and F was reduced by 9.3 times) (Fig. 1a). Similarly, it was confirmed that Sgk1 was reduced in RNA sequencing data of N+F-overexpressed glial cells (obtained from the mouse midbrain region) (Fig. 1b). Therefore, in order to find out whether Sgk1 is reduced by N+F in qPCR data, the expression levels when Nurr1 and Foxa2 were treated alone and at the same time were compared. As a result, the group treated with Nurr1 and Foxa2 at the same time It was confirmed that Sgk1 was reduced the most in (Fig. 1c).
Sgk1은 염증성 사이토카인 생산의 주요 경로인 NFκB의 세포 내 신호를 활성화시킨다고 알려져 있다. NFκB 신호 전달은 억제성 IκB-NFκB 복합체로부터 NFκB를 제거함으로써 활성화된다. Sgk1은 잠재적으로 억제성 복합체의 해리에 필요한 IκB kinase beta (IKKβ) 키나아제를 인산화할 수 있는 것으로 알려져 있다. 실제로 Western blot 분석 결과, 배양된 신경교세포(마우스 중뇌, GFAP+성상교세포> 80%, Iba+미세아교세포 < 5%, H 2O 2(150 μM, 8 시간 처리)에서 Nurr1과 Foxa2의 과발현시, 잠재적 하위 분자인 Sgk1의 감소와 동시에 IKKβ와 IκB의 인산화가 감소되었다(도 2a). 결과적으로, Nurr1 및/또는 Foxa2가 발현된 성상교세포에서의 NFκB 단백질 양은 세포질 IκB가 비활성화된 복합체와 존재할 때 증가되며, 반대로 핵 내에서의 활성화된 NFκB의 양이 감소된 것을 확인할 수 있다(도 2a). Sgk1 is known to activate intracellular signaling of NFκB, a major pathway for inflammatory cytokine production. NFκB signaling is activated by removing NFκB from the inhibitory IκB-NFκB complex. It is known that Sgk1 can potentially phosphorylate IκB kinase beta (IKKβ) kinase, which is required for dissociation of inhibitory complexes. In fact, as a result of Western blot analysis, when overexpression of Nurr1 and Foxa2 in cultured glial cells (mouse midbrain, GFAP+astroglia> 80%, Iba+ microglia <5%, H 2 O 2 (150 μM, 8 hours treatment), potential The phosphorylation of IKKβ and IκB was decreased simultaneously with the decrease of the submolecule Sgk1 (Fig. 2A). As a result, the amount of NFκB protein in astrocytes expressing Nurr1 and/or Foxa2 increased when the cytoplasmic IκB was present in the inactive complex On the contrary, it can be seen that the amount of activated NFκB in the nucleus is reduced (Fig. 2a).
비활성화된 NFκB 신호 전달은 인산화된(활성화된) p65 (주된 NFκB의 형태(p-p65))의 발현량 감소를 확인함으로써 명확히 알 수 있었다. 또한, TLR 리간드인 LPS을 신경교세포에 처리함으로써 NFκB 활성을 유도한 후, 신경교세포 내의 NFκB의 활성을 확인했는데, Sgk1 저해제(GSK-650394)가 처리된 신경교세포에서 NFκB 활성이 감소되어 있었다(도 2b).Inactivated NFκB signaling was clearly seen by confirming the decrease in the expression level of phosphorylated (activated) p65 (the main form of NFκB (p-p65)). In addition, after inducing NFκB activity by treating glial cells with a TLR ligand LPS, the activity of NFκB in glial cells was confirmed, and NFκB activity was decreased in glial cells treated with the Sgk1 inhibitor (GSK-650394) (Fig. 2b).
또한 Sgk1의 발현 억제를 위해 다양한 넉다운(knock-down) 기법들, shRNA, siRNA, 그리고 특이적인 저해제(GSK-650394, EMD-638683)를 신경교세포에 처리하여 반응을 보았는데, 주요 염증성 사이토카인인 IL-1ß와 TNF-α가 일관되게 감소됨을 확인하였고(도 3), 반면에 Sgk1이 과발현된 세포에서는 반대 효과를 보였다. 이러한 결과들은 Nurr1+Foxa2에 매개된 항염증성 기능이 Sgk1 신호 경로를 막음으로써 재현 가능한 것으로 볼 수 있으며, 이러한 Sgk1 저해가 파킨슨병과 같은 신경 염증에 의한 신경 퇴행성 질환 치료에도 적용될 수 있음을 보여준다. 따라서 다음 실험으로는 이러한 Sgk1 저해가 파킨슨병의 치료제로서 활용 가능한지를 알아보기 위해 실험을 진행하였다. In addition, to suppress the expression of Sgk1, various knock-down techniques, shRNA, siRNA, and specific inhibitors (GSK-650394, EMD-638683) were treated to glial cells to react. IL, a major inflammatory cytokine It was confirmed that -1ß and TNF-α were consistently decreased (FIG. 3), on the other hand, in cells overexpressing Sgk1, the opposite effect was shown. These results can be seen that the anti-inflammatory function mediated by Nurr1+Foxa2 is reproducible by blocking the Sgk1 signaling pathway, and that Sgk1 inhibition can be applied to the treatment of neurodegenerative diseases caused by neuroinflammation such as Parkinson's disease. Therefore, as the next experiment, an experiment was conducted to find out whether such Sgk1 inhibition can be used as a therapeutic agent for Parkinson's disease.
먼저, Sgk1 조절에 의한 세포 간 상호 작용 효과를 알아보기 위해, Sgk1 발현이 감소된 성상교세포와 Sgk1이 과발현된 성상교세포(모두 마우스 중뇌 부위에서 수득함)를 중뇌성 도파민 신경세포와 공배양을 하였다(도 4a). 그 결과, 활성산소종(ROS)을 생산하는 H 2O 2의 처리 하에서, shSgk1이 처리된 신경교세포와의 공동 배양이 sh-control이 처리된 신경교세포에 비해 세포사멸(TH+ 세포들)과 신경돌기의 퇴화로부터 보호되었음을 알 수 있었으며(도 4b), 반대로 Sgk1이 과발현된 신경교세포에서는 중뇌성 도파민 신경세포의 신경 보호 효과가 감소되었다(도 4c). 상기 결과에서, Sgk1 감소는 신경세포의 신경보호와 신경염증의 감소 역할을 함을 보여준다.First, in order to investigate the effect of Sgk1 regulation on intercellular interactions, astrocytes with reduced Sgk1 expression and astrocytes overexpressing Sgk1 (both obtained from the mouse midbrain region) were co-cultured with mesocerebral dopamine neurons. (Fig. 4a). As a result, under the treatment of H 2 O 2 producing reactive oxygen species (ROS), co-culture with shSgk1-treated glial cells compared to sh-control-treated glial cells apoptosis (TH+ cells) and neurons. It can be seen that it was protected from the degeneration of the protrusion (FIG. 4B ), and on the contrary, the neuroprotective effect of the mid-cerebral dopamine neurons was reduced in the glial cells overexpressing Sgk1 (FIG. 4C ). From the above results, it is shown that Sgk1 reduction plays a role in neuroprotection of neurons and reduction of neuroinflammation.
또한, 확인된 Sgk1 억제 효과에 대한 각 세포 유형(미세아교세포 또는 성상교세포)의 기여도를 확인하기 위해서, 신경교세포인 미세아교세포(microglia)와 성상교세포(astrocyte)를 분리 배양하여 Sgk1 발현양을 확인하였다. 그 결과, Sgk1은 신경세포보다 신경교세포에서 발현양이 높았으며, 특히 미세아교세포에서 Sgk1 발현양이 높은 것을 확인할 수 있었다(도 5a). 또한 스트레스 반응 유전자인 Sgk1은 ROS를 유발하는 독소인 H 2O 2의 처리 하에 발현양이 증가하였다(도 5b). 같은 방법으로, H 2O 2(250 μM, 4 시간)가 처리된 중뇌 부위의 성상교세포(VM glia*)와 피질 부위의 성상교세포(CTX glia*) 모두 Sgk1의 형광 발현 강도가 증가된 것을 확인하였다(도 5c). 또한, 상기 도 5a에서 도파민 신경세포보다 신경교세포에서의 Sgk1 mRNA 레벨이 높았으며, 특히 미세아교세포에서 높았다. 보통 미세아교세포에서 신경염증이 시작되고, 이후 성상교세포로 전이되어 신경독성이 퍼진다고 알려져 있지만, 반대로 성상교세포에서 미세아교세포로 신경독성이 전이된다고도 알려져 있다.In addition, in order to confirm the contribution of each cell type (microglia or astrocyte) to the confirmed Sgk1 inhibitory effect, the amount of Sgk1 expression was determined by separating and culturing microglia and astrocytes, which are glial cells. Confirmed. As a result, it was confirmed that the amount of Sgk1 expression was higher in glial cells than in neurons, and in particular, the amount of Sgk1 expression was higher in microglia (FIG. 5A ). In addition, the amount of expression of Sgk1, which is a stress response gene, was increased under the treatment of H 2 O 2 , a toxin causing ROS (FIG. 5B). In the same way, both H 2 O 2 (250 μM, 4 hours)-treated astrocytes in the midbrain region (VM glia*) and astrocytes in the cortical region (CTX glia*) both showed increased fluorescence intensity of Sgk1. (Fig. 5c). In addition, in FIG. 5A, the Sgk1 mRNA level in glial cells was higher than in dopamine neurons, especially in microglia. It is known that neuroinflammation begins in microglia and then is transferred to astrocytes and neurotoxicity is spread, but it is also known that neurotoxicity is transferred from astrocytes to microglia.
이후, 성상교세포에서 Sgk1 저해 효과에 대해 더욱 자세히 알아보기 위해, 중뇌 유래 신경교세포를 배양하여 Sgk1 저해제(GSK-650394)의 유무에 따른 RNA 서열분석을 하였다(도 6). 앞서 기술한 결과들과 일관되게, Sgk1 저해제가 처리된 군(FPKM>1, log2>1)에서 하향 조절된 유전자 그룹은 '염증반응'과 'NFκB 경로'였다. 실제로 하향 조절된 유전자 그룹 10개 중 5개 이상이 염증/면역과 관련된 그룹이었다(도 6에 표시함). '염증 반응'의 유전자 카테고리에 포함된 3,000 개의 유전자 중 516 개가 Sgk1 억제제가 처리된 신경교세포에서 처리되지 않은 대조군에 비해 하향 조절되었다. 여기서 염증/면역과 관련된 유전자 그룹은 heat-map을 통해서 나타내었다(도 7).Thereafter, in order to learn more about the Sgk1 inhibitory effect in astrocytes, the midbrain-derived glial cells were cultured to perform RNA sequence analysis according to the presence or absence of the Sgk1 inhibitor (GSK-650394) (FIG. 6). Consistent with the above-described results, the downregulated gene groups in the Sgk1 inhibitor-treated group (FPKM>1, log2>1) were'inflammatory response' and'NFκB pathway'. In fact, 5 or more out of 10 downregulated gene groups were related to inflammation/immunity (shown in FIG. 6). Of the 3,000 genes included in the'inflammatory response' gene category, 516 were downregulated in glial cells treated with the Sgk1 inhibitor compared to the untreated control group. Here, the group of genes related to inflammation/immunity is shown through heat-map (FIG. 7).
또한, IL-1ß와 IL-18의 활성은 NF-kB 경로와 매개된 전-염증성 사이토카인 발현과 염증조절 복합체(inflammasome)의 활성과도 관련이 있는데, 이것은 염증 반응의 전형적인 다른 활성 분자이다. 외인성 또는 내인성 자극에 따라, NOD-like receptor family, pyrin domain-containing 3(NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) 및 procaspase-1을 함유하는 세포 사멸 관련 센서(sensor) 분자들은 복잡한 다중 단백질 복합체인 '염증조절 복합체(inflammasome)'를 생성하고 이후 즉각적으로 활성화되어 전-염증성 관련 사이토카인 개시의 중요한 역할을 하는 caspase-1과 IL-1ß을 방출하게 된다. 이러한 NRLP3 염증조절 복합체의 활성은 알츠하이머 병, 파킨슨병과 같은 신경 퇴행성 질환의 병적 메커니즘과 관련이 있으며, NLRP3 염증조절 복합체를 제거하게 되면 신경 퇴행성 질환 치료에 도움이 될 수 있다고 알려져 있다. 특히 신경교세포 RNA 서열분석 데이터에서 Sgk1 저해제를 처리했을 때 염증조절 복합체의 구성 성분들인 NRLP3, ASC 및 procaspase-1은 모두 현저히 감소되었다. Sgk1 저해가 NRLP3 염증조절 복합체의 활성에 미치는 영향을 자세히 알아보기 위해, 중뇌 유래 신경교세포에 Sgk1 저해제(GSK-650394)를 4일간 처리하여 배양하였고, 이후 LPS (0.25 ug/ml, 3시간)와 바로 ATP(2.5mM, 30 분)를 처리해주어 신경교세포에서 염증조절 복합체의 활성을 더욱 자극 시켰다. 대조군에서 순차적인 LPS-ATP 처리에 의해 caspase-1과 IL-1β가 활성화되는 것을 확인할 수 있었으며, 이는 활성화된 caspase-1 (p10), 및 성숙된 IL-1β가 세포 배양액에 남아 있는지를 확인함으로써 결정하였다(도 8a). 반대로, 활성화된 caspase-1 (p10), 및 성숙된 IL-1β를 Sgk1 저해제가 처리된 신경교세포의 배양액에서는 확인할 수 없었는데, 이것은 Sgk1 저해가 NRLP3 염증조절 복합체의 활성을 막는다는 것을 보여준다. 또한, IL-1β와 신경염증 및 신경 노화와 관련된 IL-6 모두 Sgk1 저해제가 처리된 중뇌 유래 신경교세포 세포 배양액에서 현저하게 감소되어 있었다(도 8b). In addition, the activities of IL-1ß and IL-18 are also related to pro-inflammatory cytokine expression mediated by the NF-kB pathway and the activity of the inflammasome, which is another active molecule typical of the inflammatory response. According to exogenous or endogenous stimulation, apoptosis-related sensor containing NOD-like receptor family, pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and procaspase-1 ) Molecules create a complex multi-protein complex, an'inflammasome', which is then immediately activated to release caspase-1 and IL-1ß, which play an important role in initiating pro-inflammatory related cytokines. The activity of the NRLP3 inflammation-regulating complex is related to the pathological mechanism of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and it is known that removal of the NLRP3 inflammation-regulating complex can help treat neurodegenerative diseases. In particular, in the glial RNA sequencing data, when the Sgk1 inhibitor was treated, NRLP3, ASC, and procaspase-1, which are components of the inflammation control complex, were significantly reduced. To learn more about the effect of Sgk1 inhibition on the activity of the NRLP3 inflammation-regulating complex, midbrain-derived glial cells were cultured with Sgk1 inhibitor (GSK-650394) for 4 days, followed by LPS (0.25 ug/ml, 3 hours) and Immediately, ATP (2.5mM, 30 minutes) was treated to further stimulate the activity of the inflammation control complex in glial cells. In the control group, it was confirmed that caspase-1 and IL-1β were activated by sequential LPS-ATP treatment, which was confirmed by confirming whether activated caspase-1 (p10) and mature IL-1β remained in the cell culture. Was determined (Fig. 8a). In contrast, activated caspase-1 (p10) and mature IL-1β could not be identified in the culture medium of glial cells treated with the Sgk1 inhibitor, which shows that Sgk1 inhibition prevents the activity of the NRLP3 inflammatory regulatory complex. In addition, both IL-1β and IL-6 related to neuroinflammation and neuroaging were significantly reduced in the midbrain-derived glial cell culture medium treated with the Sgk1 inhibitor (FIG. 8B).
바이러스 감염, 핵 및 미토콘드리아 손상에 의해 도입된 세포질 DNA는 cyclic GMP-AMP 합성 효소 (cGAS)-stimulator of interferon genes (STING) 경로의 자극제를 통해 선천성 면역/염증 반응을 유발한다. 여기서 cGAS는 cytosolic DNA 센서로서 작용한다. 이중 가닥 DNA (dsDNA)에 결합하면 cGAS는 독특한 2차 대사산물인 2′,3′-cyclic GMP-AMP를 생성한 후 중요한 어댑터 ER 막 단백질인 STING에 결합한다. 이후, STING은 TANK binding kinase 1 (TBK1)을 끌어 모아, interferon regulatory factor 3 (IRF-3)을 인산화시키고 나서 이합체화 되어 핵 속으로 들어가 interferon-beta (IFN-β)를 포함한 interferon 자극 유전자의 전사를 개시한다. 최근, cGAS-STING 경로가 Parkin과 PINK1 돌연변이에 의해 파킨슨병 유발에 기여한다고 강조된 바 있다. Cytoplasmic DNA introduced by viral infection, nuclear and mitochondrial damage triggers innate immune/inflammatory responses through stimulators of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. Here, cGAS acts as a cytosolic DNA sensor. When bound to double-stranded DNA (dsDNA), cGAS generates a unique secondary metabolite, 2′,3′-cyclic GMP-AMP, and then binds to STING, an important adapter ER membrane protein. Subsequently, STING collects TANK binding kinase 1 (TBK1), phosphorylates interferon regulatory factor 3 (IRF-3), then dimers and enters the nucleus, transcribing interferon-stimulating genes including interferon-beta (IFN-β). Start. Recently, it has been emphasized that the cGAS-STING pathway contributes to the induction of Parkinson's disease by Parkin and PINK1 mutations.
흥미롭게도, RNA 서열분석 데이터에서 cGAS-STING 경로와 관련된 TLR9, DAI, IFI16, cGAS(DNA sensor)과 TMEM173, NLRC3, IRF3(activator) 모두 Sgk1 저해제가 처리된 신경교세포에서 그렇지 않은 신경교세포보다 눈에 띄게 감소하는 것을 확인할 수 있었다. 또한, 신경교세포에서 Sgk1의 저해가 cGAS-STING 경로 활성을 저해함을 IRF-3, phospho-IRF-3(activated IRF-3) 감소를 통해서 한번 더 확인하였다(도 8c).Interestingly, in the RNA sequencing data, TLR9, DAI, IFI16, cGAS (DNA sensor) related to the cGAS-STING pathway, and TMEM173, NLRC3, and IRF3 (activator) were all noticeable in glial cells treated with Sgk1 inhibitors than glial cells that were not. It could be confirmed that it decreased significantly. In addition, it was confirmed once again that inhibition of Sgk1 in glial cells inhibited the cGAS-STING pathway activity by reducing IRF-3 and phospho-IRF-3 (activated IRF-3) (FIG. 8C).
최근 연구들에서 신경교세포에서 세포내(특히 성상교세포) 노화가 파킨슨병의 신경병리학에 매우 중요한 기여를 한다고 보고되었다. 또한 이러한 노화된 세포를 제거하거나 막는 것이 신경 퇴행성 질환 치료에 한 방향이 될 것이라고 제시하였다. 이러한 세포의 노화 현상은 전-산화 유전자에 의한 ROS에 의해서 발생하게 되는데, Sgk1 저해제가 처리된 세포에서는 세포 노화 현상이 감소되는 것으로 나타났다. 마찬가지로, 세포내 ROS를 측정할 수 있는 DCF-DA 염색을 통해서도 Sgk1 저해제가 처리된 세포에서 ROS 양이 감소된 것을 확인하였다(도 9a). 또한, 세포의 ROS 생산에 있어서 미토콘드리아가 중요한 역할을 하는데, 미토콘드리아에 의한 ROS 형성은 미토콘드리아의 기능 저하로 인해서 증가하게 된다. In recent studies, it has been reported that intracellular (especially astrocyte) aging in glial cells plays a very important role in the neuropathology of Parkinson's disease. In addition, it was suggested that removing or blocking these aged cells would be one direction in the treatment of neurodegenerative diseases. The senescence of these cells is caused by ROS by the pre-oxidation gene, and it was found that the cell senescence was reduced in the cells treated with the Sgk1 inhibitor. Likewise, it was confirmed that the amount of ROS was decreased in the cells treated with the Sgk1 inhibitor through DCF-DA staining, which can measure intracellular ROS (FIG. 9A). In addition, mitochondria play an important role in the production of ROS in cells, and the formation of ROS by mitochondria increases due to a decrease in mitochondrial function.
RNA 서열분석 데이터에서 PGC1a의 발현이 Sgk1 저해제가 처리된 신경교세포에서 증가되는 것을 확인할 수 있는데, PGC1a는 미토콘드리아의 생합성 증가와 항산화 능력을 통해 ROS 형성을 줄여주기 때문에 흥미로웠다. 또한 염증조절 복합체와 STING-cGAS 경로는 미토콘드리아 기능 저하와 서로 관련이 있다. 따라서 신경교세포에 Sgk1 저해제를 처리하면, 염증조절 복합체와 STING-cGAS 경로를 막아줌으로써 신경염증을 줄여줘 결론적으로 미토콘드리아의 ROS를 줄여줄 것이라 생각했다. 실제로, 면역염색법과 FACS을 통해 Mito-Sox+세포가 Sgk1 저해제가 처리된 신경교세포에서 줄어들어 있는 것을 확인했다(도 9a). 게다가, % B-galactosidase+ 세포는 세포화가 진행된다고 보는데 이 또한 Sgk1 저해에 의해서 현저히 감소되었다(도 9b).From the RNA sequencing data, it can be confirmed that the expression of PGC1a is increased in glial cells treated with the Sgk1 inhibitor, which is interesting because PGC1a reduces ROS formation through increased mitochondrial biosynthesis and antioxidant capacity. In addition, the inflammation control complex and the STING-cGAS pathway are correlated with mitochondrial function decline. Therefore, it was thought that treatment of glial cells with Sgk1 inhibitor would reduce neuroinflammation by blocking the inflammation-regulating complex and the STING-cGAS pathway, consequently reducing mitochondrial ROS. In fact, it was confirmed that Mito-Sox+ cells were reduced in glial cells treated with the Sgk1 inhibitor through immunostaining and FACS (FIG. 9A). In addition,% B-galactosidase+ cells are considered to undergo cellularization, which was also significantly reduced by Sgk1 inhibition (Fig. 9b).
반대로 Sgk1 억제에 의해 상향 조절된 유전자를 이용한 그룹별 온톨로지(ontology) 분석에서 상위 2위는 '세포 부착/세포 외 기질 (ECM)'이었다. 신경교세포는 중추신경계에서 세포 부착/세포 외 기질(ECM)에 있어서 매우 중요한 역할을 하며, 성상교세포에 의해 분비되는 여러 분자들을 신경영양성/신경재생 능력에 도움을 주는 것으로 본다.Conversely, in the group ontology analysis using genes upregulated by Sgk1 inhibition,'cell adhesion/extracellular matrix (ECM)' ranked second. Glial cells play a very important role in cell adhesion/extracellular matrix (ECM) in the central nervous system, and several molecules secreted by astrocytes are considered to help neurotrophic/neuronal regeneration.
또한 Sgk1 저해제가 처리된 세포 배양에서의 세포 부착/세포 외 기질 관련 유전자 발현을 heat-map을 통해서 나타내었다(도 10). 또 다른 신경교세포에 의한 신경보호 메커니즘은 글루타메이트에 의한 독성을 제거하는데 있다. 글루타메이트 흡수는 글루타메이트 운송 유전자인 GLAST와 GLT-1(SLC1A3 또는 SLC1A2라 불림)의 발현이 매우 중요한데, Sgk1 저해제가 처리된 신경교세포에서 대조군 세포보다 상기 두 유전자가 증가되어 있었으며, 실제로 글루타메이트 흡수량 또한 높게 나타났다(도 10). 특히, Sgk1 저해제를 2 μM만큼 신경교세포에 처리했을 때 대조군에 비해서 글루타메이트 흡수량이 매우 증가됐으며, 이러한 글루타메이트 제거 효과는 신경보호에 크게 기여한다고 볼 수 있다.In addition, cell adhesion/extracellular matrix-related gene expression in cell culture treated with the Sgk1 inhibitor was shown through heat-map (FIG. 10). Another neuroprotective mechanism by glial cells is to eliminate the toxicity caused by glutamate. Glutamate uptake is very important in the expression of glutamate transport genes GLAST and GLT-1 (called SLC1A3 or SLC1A2).In glial cells treated with Sgk1 inhibitor, the above two genes were increased compared to control cells, and the amount of glutamate uptake was also higher. (Fig. 10). In particular, when the Sgk1 inhibitor was treated to glial cells by 2 μM, the absorption of glutamate was significantly increased compared to the control, and this glutamate removal effect can be considered to contribute significantly to neuroprotection.
다음으로, 신경세포에서의 Sgk1 저해제(GSK-650394)의 처리에 따른 세포의 성숙과 생존성을 확인하였다(도 11). Sgk1 저해제의 농도에 따른 중뇌 유래 신경세포와 도파민 신경세포의 신경돌기의 길이 및 TH 발현율을 확인한 결과, Sgk1 저해제의 농도가 0.1 μM인 경우에 신경세포의 성숙과 생존성이 가장 우수하게 나타났다(도 11 내지 도 14). 또한, H 2O 2에 의한 산화스트레스가 유발된 세포에 Sgk1 저해제를 처리한 결과, 상기 결과와 마찬가지로 Sgk1 저해제의 농도가 0.1 μM인 경우에 신경세포의 생존율이 가장 높은 것을 확인할 수 있었다. 상기 결과를 통해, Sgk1 저해제가 도파민 신경세포뿐만 아니라 일반 중뇌 유래의 신경세포에도 영향을 미치는 것을 확인함으로써, Sgk1의 발현 저해가 도파민 신경세포가 사멸되는 파킨슨병 외에도 다른 신경 퇴행성 질환에 효과가 있음을 알 수 있다.Next, it was confirmed the maturation and viability of cells according to the treatment of the Sgk1 inhibitor (GSK-650394) in the neurons (Fig. 11). As a result of confirming the length of the neurite and the TH expression rate of midbrain-derived neurons and dopamine neurons according to the concentration of the Sgk1 inhibitor, when the concentration of the Sgk1 inhibitor was 0.1 μM, the maturation and viability of neurons were the best (Fig. 11 to 14). In addition, as a result of treating cells with oxidative stress induced by H 2 O 2 with an Sgk1 inhibitor, as in the above results, when the concentration of the Sgk1 inhibitor was 0.1 μM, it was confirmed that the survival rate of neurons was highest. Through the above results, by confirming that the Sgk1 inhibitor affects not only dopamine neurons but also neurons derived from general midbrain, inhibition of Sgk1 expression is effective in other neurodegenerative diseases besides Parkinson's disease where dopamine neurons are killed. Able to know.
또한, 신경세포에 Sgk1 저해제(GSK-650394)를 처리한 결과, Parkin의 발현이 증가함에 따라 Paris의 발현이 감소하고 PGC1a의 발현이 증가하는 것을 보였다(도 15a). 이는 Sgk1 저해제가 미토콘드리아의 ROS 생성을 감소시키고, 미토콘드리아의 항상성(mito homeostasis) 증진과 세포의 생존력에 영향을 주는 것임을 알 수 있었다. 또한, Sgk1 저해제가 α-synuclein의 응집을 억제시킴으로써 파킨슨병의 치료제로 적용될 수 있는 가능성을 제시하였다. In addition, as a result of treatment of neurons with an Sgk1 inhibitor (GSK-650394), as the expression of Parkin increased, the expression of Paris decreased and the expression of PGC1a increased (FIG. 15A). This suggests that Sgk1 inhibitors reduce mitochondrial ROS production, promote mitochondrial homeostasis, and affect cell viability. In addition, the Sgk1 inhibitor suppresses the aggregation of α-synuclein, thus suggesting the possibility of being applied as a therapeutic agent for Parkinson's disease.
앞선 결과를 토대로, 파킨슨병 동물 모델에서의 Sgk1 저해제의 효과를 확인하였다. 도 16과 같이 MPTP-유도 파킨슨병 마우스 모델을 제작하고, 행동실험을 수행하였다. 행동실험 결과, 도 17 에서 보는 것과 같이 Sgk1 저해제를 처리한 마우스 모델에서 Sgk1 저해제 처리 3주째부터 대조군에 비해 유의하게 행동에 차이를 보이기 시작했으며, 이는 Sgk1 저해제에 의해 MPTP에 의한 도파민 신경세포의 사멸이 방지되어 파킨슨병의 발병이 감소된 것으로 판단하였다. Sgk1 저해제에 따른 파킨슨병의 치료 효과에 대한 더 정확한 결과를 얻기 위해, MPTP 처리 후 1개월 후에 마우스를 희생시켜 SN 부위와 SN 주변부의 조직을 얻어 RT-PCR을 수행하였다. RT-PCR 결과, 도 18 및 도 19에 나타난 것과 같이, SN 부위 및 SN 주변부 조직 모두에서 신경염증 유발이 대조군에 비해 Sgk1 저해제 처리군에서 현저히 감소한 것을 확인할 수 있었다. 이후, SN 부위의 도파민 신경세포의 개수를 측정하고 TH 염색을 진행한 결과(도 20), Sgk1 저해제를 주입한 마우스 모델에서 도파민 신경세포의 개수와 TH의 발현이 대조군에 비해 현저히 높은 것을 확인하였다(도 20 및 도 21). 또한, 대조군(7마리)과 Sgk1 저해제 처리군(9마리)에 대해 운동성 실험(locomotor test)을 진행한 결과, 전체 이동거리는 유의한 차이를 보이지 않았으나, 평균 속도는 Sgk1 저해제 처리군에서 더 빠르게 나타나는 것을 확인할 수 있었다.Based on the previous results, the effect of the Sgk1 inhibitor in the Parkinson's disease animal model was confirmed. As shown in FIG. 16, an MPTP-induced Parkinson's disease mouse model was produced and behavioral experiments were performed. As a result of the behavioral experiment, as shown in FIG. 17, in the mouse model treated with the Sgk1 inhibitor, from the 3rd week of treatment with the Sgk1 inhibitor, compared to the control group, the behavior began to show a significant difference, which is the killing of dopamine neurons by MPTP by the Sgk1 inhibitor. It was determined that this was prevented and the incidence of Parkinson's disease was reduced. In order to obtain a more accurate result of the therapeutic effect of Parkinson's disease according to the Sgk1 inhibitor, the mice were sacrificed 1 month after MPTP treatment to obtain the SN site and the tissues around the SN, and RT-PCR was performed. As a result of RT-PCR, as shown in FIGS. 18 and 19, it was confirmed that the induction of neuroinflammation in both the SN region and the tissues around the SN was significantly reduced in the Sgk1 inhibitor-treated group compared to the control group. Thereafter, as a result of measuring the number of dopamine neurons in the SN region and performing TH staining (FIG. 20), it was confirmed that the number of dopamine neurons and the expression of TH in the mouse model injected with the Sgk1 inhibitor were significantly higher than that of the control group. (FIGS. 20 and 21). In addition, as a result of conducting a locomotor test for the control group (7 animals) and the Sgk1 inhibitor-treated group (9 animals), the total distance did not show a significant difference, but the average speed appeared faster in the Sgk1 inhibitor-treated group. I could confirm that.
나아가, 본 발명은 Sgk1의 발현 저해를 통한 파킨슨병의 치료에 있어서, α-synuclein의 응집 방지와 응집 제거 효과가 있는지 확인하기 위해 α-synuclein이 과발현된 중뇌 유래 신경전구세포를 3주간 분화시키면서 Sgk1 저해제를 같이 처리하는 방식(방식 1)과, α-synuclein이 과발현된 중뇌 유래 신경전구세포를 성상교세포와 공배양하여 Sgk1 저해제를 처리(방식 2)한 이후(도 22), 면역염색법과 western blot을 수행하였다. 그 결과, 먼저 방식 1의 western blot 결과에서는, Sgk1 저해제를 처리한 군에서 insoluble 및 soluble form 모두에서 α-synuclein oligomer가 감소된 것을 확인할 수 있었다(도 23). 그러나, 상기 결과에서 monomer 자체가 줄어든 것으로 보이지는 않았다. 방식 2의 westhern blot 결과에서는, Sgk1 저해제의 처리에 따라 α-synuclein oligomer 뿐만 아니라 monomer까지도 insoluble 및 soluble form 모두에서 감소된 것을 확인할 수 있었다(도 24).Furthermore, in the treatment of Parkinson's disease by inhibiting the expression of Sgk1, the present invention differentiates the midbrain-derived neural progenitor cells overexpressed with α-synuclein for 3 weeks in order to check whether there is an effect of preventing aggregation and removing aggregation of α-synuclein. After treating the inhibitor together (Method 1) and the midbrain-derived neural progenitor cells overexpressing α-synuclein with astrocytes and treating the Sgk1 inhibitor (Method 2) (Fig. 22), immunostaining and western blot Was performed. As a result, first, in the western blot result of method 1, it was confirmed that α-synuclein oligomer was decreased in both insoluble and soluble forms in the group treated with the Sgk1 inhibitor (FIG. 23). However, the monomer itself did not appear to be reduced in the above results. From the westhern blot results of Method 2, it was confirmed that not only the α-synuclein oligomer but also the monomer was reduced in both insoluble and soluble forms according to the treatment of the Sgk1 inhibitor (FIG. 24 ).
마우스 ICR 종을 이용한 in vivo 실험은 도 25와 같은 방법으로 수행하였다. 마우스의 SN 부위에 PFF와 α-synuclein virus를 주입한 후, 수술후 안정을 위해 일주일이 지난 후부터 Sgk1 저해제를 3mg/ml로 매일 복강 주사하였다. 또한, 주사 1주 후부터 동물실험을 측정하였고, 총 3개월이 될 때, 마우스를 희생시켜 SN 부위를 얻어 형광 염색한 결과, 도 26에서는 Sgk1 저해제가 처리된 마우스 뇌에서 phospho-α-synuclein이 거의 발현되지 않는 것을 알 수 있었으며, 마찬가지로 도 27에서는 미세아교세포의 M1(해로운) 타입의 마커인 CD16/32 또한 Sgk1 저해제가 처리된 마우스에서 발현이 비교적 줄어들어 있는 것을 알 수 있었다. 또한, 수술 1개월 후부터 진행된 동물 행동실험은 Sgk1 저해제 처리군(5마리), 대조군(5마리) 모두에서 6주까지는 큰 차이를 보이지 않았으나, 11주 내지 12주부터는 막대 실험과 균형 실험 모두에서 Sgk1 저해제의 처리군이 대조군에 비해 행동이 빠르고 병의 진행이 더딘 것을 확인할 수 있었다(도 28).In vivo experiments using mouse ICR species were performed in the same manner as in FIG. 25. After injecting PFF and α-synuclein virus into the SN site of the mouse, a Sgk1 inhibitor was intraperitoneally injected daily at 3 mg/ml after a week for postoperative stability. In addition, animal experiments were measured from 1 week after injection, and when the total was 3 months, the mice were sacrificed to obtain the SN site and stained with fluorescence. In FIG. 26, phospho-α-synuclein was almost completely in the mouse brain treated with the Sgk1 inhibitor. It was found that it was not expressed, and similarly, in FIG. 27, it was found that the expression of CD16/32, which is a marker of the M1 (harmful) type of microglia, was also relatively reduced in mice treated with the Sgk1 inhibitor. In addition, animal behavior experiments conducted 1 month after surgery did not show a significant difference until 6 weeks in both the Sgk1 inhibitor-treated group (5 animals) and the control group (5 animals), but from 11 weeks to 12 weeks, Sgk1 in both the rod and balance experiments. It was confirmed that the inhibitor-treated group had faster behavior and slower disease progression compared to the control group (FIG. 28).

Claims (12)

  1. Sgk1(serum and glucocorticoid-regulated kinase 1) 유전자에 상보적인 서열을 포함하는 안티센스-올리고뉴클레오타이드, siRNA, shRNA, miRNA 또는 이를 포함하는 벡터; Sgk1 단백질에 특이적인 항체; 또는 하기 화학식 1 또는 화학식 2로 표현되는 Sgk1 효소에 대한 저해 활성을 갖는 화합물; 중 어느 하나인 Sgk1 저해제를 유효성분으로 포함하는 염증성 신경질환의 예방 또는 치료용 약학 조성물:Sgk1 (serum and glucocorticoid-regulated kinase 1) antisense-oligonucleotide comprising a sequence complementary to the gene, siRNA, shRNA, miRNA or a vector containing the same; Antibodies specific for the Sgk1 protein; Or a compound having inhibitory activity against the Sgk1 enzyme represented by the following Formula 1 or Formula 2; Pharmaceutical composition for the prevention or treatment of inflammatory neurological diseases comprising any one of Sgk1 inhibitors as an active ingredient:
    [화학식 1][Formula 1]
    Figure PCTKR2020009141-appb-img-000003
    Figure PCTKR2020009141-appb-img-000003
    [화학식 2][Formula 2]
    Figure PCTKR2020009141-appb-img-000004
    Figure PCTKR2020009141-appb-img-000004
  2. 제1항에 있어서,The method of claim 1,
    Sgk1 저해제는 SEQ ID NO: 9의 핵산서열로 표현되는 shRNA인 염증성 신경질환의 예방 또는 치료용 약학 조성물.The Sgk1 inhibitor is a pharmaceutical composition for the prevention or treatment of inflammatory neurological diseases, which is shRNA represented by the nucleic acid sequence of SEQ ID NO: 9.
  3. 제1항에 있어서,The method of claim 1,
    Sgk1 저해제는 Sgk1에 의한 염증성 사이토카인의 발현을 억제하여 신경염증을 감소시키는 것인 염증성 신경질환의 예방 또는 치료용 약학 조성물.The Sgk1 inhibitor suppresses the expression of inflammatory cytokines by Sgk1 to reduce neuroinflammation. A pharmaceutical composition for preventing or treating inflammatory neuropathy.
  4. 제1항에 있어서,The method of claim 1,
    염증성 신경질환은 파킨슨병, 알쯔하이머병, 다발성 전신 위축, 근 위축성 측삭 경화증(ALS), 뇌경색, 척추손상, 신경병증 통증(neuropathic pain) 및 복합통증증후군(complex region pain syndrome; CRPS)으로 이루어진 군에서 선택된 하나 이상인 염증성 신경질환의 예방 또는 치료용 약학 조성물.Inflammatory neurological diseases were in the group consisting of Parkinson's disease, Alzheimer's disease, multiple systemic atrophy, amyotrophic lateral sclerosis (ALS), cerebral infarction, spinal injury, neuropathic pain, and complex region pain syndrome (CRPS). Pharmaceutical composition for the prevention or treatment of one or more selected inflammatory neurological diseases.
  5. Sgk1(serum and glucocorticoid-regulated kinase 1) 유전자를 후보물질과 인체 외에서 접촉시키고,Sgk1 (serum and glucocorticoid-regulated kinase 1) gene is brought into contact with a candidate substance outside the human body,
    상기 후보물질이 Sgk1 유전자의 발현을 촉진하는지 또는 억제하는지를 판단하는 것을 포함하는 염증성 신경질환의 예방 또는 치료용 약물의 스크리닝 방법.A method for screening a drug for preventing or treating inflammatory neurological diseases, comprising determining whether the candidate substance promotes or inhibits the expression of the Sgk1 gene.
  6. 제5항에 있어서,The method of claim 5,
    후보물질이 Sgk1 유전자의 발현을 억제시키면 염증성 신경질환의 치료제로 판정하는 염증성 신경질환의 예방 또는 치료용 약물의 스크리닝 방법.A method for screening a drug for preventing or treating inflammatory neuropathy, in which a candidate substance inhibits the expression of the Sgk1 gene and is determined as a therapeutic agent for inflammatory neuropathy.
  7. 제5항에 있어서,The method of claim 5,
    염증성 신경질환은 파킨슨병, 알쯔하이머병, 다발성 전신 위축, 근 위축성 측삭 경화증(ALS), 뇌경색, 척추손상, 신경병증 통증(neuropathic pain) 및 복합통증증후군(complex region pain syndrome; CRPS)으로 이루어진 군에서 선택된 하나 이상인 염증성 신경질환의 예방 또는 치료용 약물의 스크리닝 방법.Inflammatory neurological diseases were in the group consisting of Parkinson's disease, Alzheimer's disease, multiple systemic atrophy, amyotrophic lateral sclerosis (ALS), cerebral infarction, spinal injury, neuropathic pain, and complex region pain syndrome (CRPS). Screening method for a drug for preventing or treating one or more selected inflammatory neurological diseases.
  8. Sgk1(serum and glucocorticoid-regulated kinase 1) 단백질을 후보물질과 인체 외에서 접촉시키고,Sgk1 (serum and glucocorticoid-regulated kinase 1) protein is brought into contact with a candidate substance outside the human body,
    상기 후보물질이 Sgk1 단백질의 기능 또는 활성을 증진하는지 또는 억제하는지를 판단하는 것을 포함하는 염증성 신경질환의 예방 또는 치료용 약물의 스크리닝 방법.A method for screening a drug for preventing or treating inflammatory neurological diseases, comprising determining whether the candidate substance enhances or inhibits the function or activity of the Sgk1 protein.
  9. 제8항에 있어서,The method of claim 8,
    후보물질이 Sgk1 단백질의 기능 또는 활성을 억제시키면 염증성 신경질환의 치료제로 판정하는 염증성 신경질환의 예방 또는 치료용 약물의 스크리닝 방법.A method for screening a drug for preventing or treating inflammatory neurological disease, in which a candidate substance inhibits the function or activity of the Sgk1 protein, and is determined as a therapeutic agent for inflammatory neurological disease.
  10. 제8항에 있어서,The method of claim 8,
    염증성 신경질환은 파킨슨병, 알쯔하이머병, 다발성 전신 위축, 근 위축성 측삭 경화증(ALS), 뇌경색, 척추손상, 신경병증 통증(neuropathic pain) 및 복합통증증후군(complex region pain syndrome; CRPS)으로 이루어진 군에서 선택된 하나 이상인 염증성 신경질환의 예방 또는 치료용 약물의 스크리닝 방법.Inflammatory neurological diseases were in the group consisting of Parkinson's disease, Alzheimer's disease, multiple systemic atrophy, amyotrophic lateral sclerosis (ALS), cerebral infarction, spinal injury, neuropathic pain, and complex region pain syndrome (CRPS). Screening method for a drug for the prevention or treatment of one or more selected inflammatory neurological diseases.
  11. 치료상 유효량의 Sgk1 저해제를 이를 필요로 하는 대상체에 투여하는 것을 포함하는 염증성 신경질환의 치료 방법으로서,As a method for treating inflammatory neurological disease comprising administering a therapeutically effective amount of an Sgk1 inhibitor to a subject in need thereof,
    상기 Sgk1 저해제는 Sgk1(serum and glucocorticoid-regulated kinase 1) 유전자에 상보적인 서열을 포함하는 안티센스-올리고뉴클레오타이드, siRNA, shRNA, miRNA 또는 이를 포함하는 벡터; Sgk1 단백질에 특이적인 항체; 또는 하기 화학식 1 또는 화학식 2로 표현되는 Sgk1 효소에 대한 저해 활성을 갖는 화합물; 중 어느 하나인 염증성 신경질환의 치료 방법:The Sgk1 inhibitor may include an antisense-oligonucleotide, siRNA, shRNA, miRNA, or a vector comprising the same, comprising a sequence complementary to the Sgk1 (serum and glucocorticoid-regulated kinase 1) gene; Antibodies specific for the Sgk1 protein; Or a compound having inhibitory activity against the Sgk1 enzyme represented by the following Formula 1 or Formula 2; Any one of the methods of treating inflammatory neurological disease:
    [화학식 1][Formula 1]
    Figure PCTKR2020009141-appb-img-000005
    Figure PCTKR2020009141-appb-img-000005
    [화학식 2][Formula 2]
    Figure PCTKR2020009141-appb-img-000006
    Figure PCTKR2020009141-appb-img-000006
  12. 염증성 신경질환에 대한 치료제의 제조를 위한 Sgk1 저해제의 용도.Use of Sgk1 inhibitors for the manufacture of therapeutic agents for inflammatory neurological diseases.
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