KR20230115493A - Composition for preventing or treating stathmin1 related diseases comprising the selective PI3K p110δ inhibitor - Google Patents
Composition for preventing or treating stathmin1 related diseases comprising the selective PI3K p110δ inhibitor Download PDFInfo
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- KR20230115493A KR20230115493A KR1020220012061A KR20220012061A KR20230115493A KR 20230115493 A KR20230115493 A KR 20230115493A KR 1020220012061 A KR1020220012061 A KR 1020220012061A KR 20220012061 A KR20220012061 A KR 20220012061A KR 20230115493 A KR20230115493 A KR 20230115493A
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Abstract
본 발명은 특이적 PI3K p110δ 억제제를 유효성분으로 함유하는 스타스민1 관련 질환 예방 또는 치료용 조성물에 관한 것으로, 보다 상세하게는 상기 특이적 PI3K p110δ 억제제가 STMN1 단백질의 인산화를 억제시킴으로써, 스타스민1 관련 질환 예방 또는 치료용 조성물로 유용하게 활용될 수 있다.The present invention relates to a composition for preventing or treating Stasmin 1-related diseases, containing a specific PI3K p110δ inhibitor as an active ingredient, and more particularly, by inhibiting the phosphorylation of STMN1 protein by the specific PI3K p110δ inhibitor, Stasmin 1 It can be usefully used as a composition for preventing or treating related diseases.
Description
본 발명은 특이적 PI3K p110δ 억제제, 특히 IC87114를 유효성분으로 함유하는 스타스민1 관련 질환 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing or treating stamin 1-related diseases, containing a specific PI3K p110δ inhibitor, particularly IC87114, as an active ingredient.
대식세포는 병원체, 세포 자멸사 또는 괴사 세포 조각과 같은 유해한 자극에 반응한다. 대식세포의 활성화는 Toll like receptor (TLR)과 사이토카인 수용체의 하위 신호 전달에 의해 매개된다. 대식세포가 활성화되면 전염증성 사이토카인 및 케모카인이 생성되고 이동 및 손상의 제거로 이어진다.Macrophages respond to noxious stimuli such as pathogens, apoptotic or necrotic cell fragments. Activation of macrophages is mediated by downstream signaling of Toll like receptors (TLRs) and cytokine receptors. When macrophages are activated, pro-inflammatory cytokines and chemokines are produced and lead to migration and clearance of damage.
대식세포의 활성화는 M1과 M2 단계로 나누며 나누는 기준은 자극에 따라 정의된다. M1의 자극은 IFN-γ와 리포폴리사카라이드(lipopolysaccharide; LPS)에 의해 구분되며 M2는 인터류킨-4, 면역복합체(immune complex), 글루코코르티코이드, 전환성장인자-β(transforming growth Factor-β; TGF-β)의 조합에 따라 정의된다. 이러한 분류는 다양한 병원체의 제거에서 사멸 세포의 식균작용과 조직 재건에 이르기까지 다양한 기능을 설명한다. 대식세포의 활성화는 수용체 자극 혹은 세포 내 조절 단백질에 의해 유도되는 여러 신호 전달 캐스케이드에 의해 엄격하게 조절된다. 그 중 포스파티딜이노시티드 3-키나제(phosphatidylinositide 3-kinase; PI3K) / 단백질 카이네이즈 B(PKB, AKT) 경로와 그 하류 신호는 대식세포에서 대표적 활성화 신호이다.Activation of macrophages is divided into M1 and M2 stages, and the criteria for dividing are defined according to stimuli. M1 stimulation is distinguished by IFN-γ and lipopolysaccharide (LPS), and M2 is interleukin-4, immune complex, glucocorticoid, transforming growth factor-β (TGF). -β) is defined according to the combination. These classifications account for a variety of functions, from elimination of various pathogens to phagocytosis of apoptotic cells and tissue reconstruction. Activation of macrophages is tightly regulated by several signal transduction cascades induced by receptor stimulation or intracellular regulatory proteins. Among them, the phosphatidylinositide 3-kinase (PI3K) / protein kinase B (PKB, AKT) pathway and its downstream signals are representative activation signals in macrophages.
PI3K는 막에 있는 포스파티딜이노시톨(PI)의 3'-OH 그룹을 인산화할 수 있는 지질 효소의 큰 복합체이며 대사, 염증, 세포 생존, 운동성 및 암 진행과 같은 여러 세포 과정에서 PI3K 신호 전달은 핵심 역할을 하였다. PI3K는 면역 신호 전달에서 중요하며 조절되지 않는 PI3K 신호 전달은 대부분 암의 형태와 연관 있다. PI3K/AKT 신호 전달은 PI3K와 그 하류 분자 AKT의 두 부분으로 구성된 중요한 신호 경로이다. PI3K is a large complex of lipid enzymes capable of phosphorylating the 3'-OH group of membrane phosphatidylinositol (PI), and PI3K signaling plays a key role in several cellular processes such as metabolism, inflammation, cell survival, motility and cancer progression. did PI3K is important in immune signaling, and dysregulated PI3K signaling is associated with most forms of cancer. PI3K/AKT signaling is an important signaling pathway composed of two parts: PI3K and its downstream molecule AKT.
PI3K는 세 가지의 class(class I, class II 및 class III)로 나뉜다. Class IA는 수용체 단백질 티로신 카이네이즈(receptor protein tyrosine kinase; RTK)에 의해 활성화되며, 촉매 서브유닛인 p110α, p110β 및 p110δ와 조절 서브유닛인 p85α, p85β, p55γ, p55α, p50α으로 구성된 이종이량체 효소이다. PI3K is divided into three classes (class I, class II and class III). Class IA is a heterodimer enzyme that is activated by receptor protein tyrosine kinase (RTK) and is composed of catalytic subunits p110α, p110β, and p110δ and regulatory subunits p85α, p85β, p55γ, p55α, and p50α. .
PI3K 신호 전달에 있어서 전달 경로는 잘 알려져 있지만, 선택적 PI3K 아이소폼 선택적 억제제를 사용하지 않은 것은 신호 전달에 관여하는 촉매 서브유닛의 특성에 대한 연구를 방해해 왔다. Class IA PI3K의 구성요소인 PI3K α, PI3K β 그리고 PI3K δ 중에서 PI3K α와 PI3K β는 발현이 광범위 하지만, class IA PI3K δ와 class IB PI3K의 PI3K γ는 면역 기능과 면역 관련된 작용에 국한된다. PI3K 아이소폼에 대한 연구는 PI3K의 기능과 면역 관련 체계를 밝히는데 중요한 역할을 할 것이다. 그러므로 PI3K 아이소폼에 대한 연구는 매우 의미가 있고, 각각의 억제제를 이용하여 개별적인 억제를 통해 각 PI3K 아이소폼에 관련하여 조절되는 단백질들을 발굴하고 기능을 조사할 수 있다.Although the transduction pathways in PI3K signal transduction are well known, the absence of selective PI3K isoform-selective inhibitors has hampered the characterization of the catalytic subunits involved in signal transduction. Among the components of Class I PI3K, PI3K α, PI3K β, and PI3K δ, PI3K α and PI3K β have a wide range of expression, but class IA PI3K δ and class IB PI3K PI3K γ are limited to immune function and immune-related actions. Research on PI3K isoforms will play an important role in elucidating PI3K functions and immune-related systems. Therefore, research on PI3K isoforms is very meaningful, and it is possible to discover proteins regulated in relation to each PI3K isoform through individual inhibition using each inhibitor and investigate their functions.
따라서, class I PI3K 아이소폼들에 대한 각각의 억제제를 사용하여 각 아이소폼에 의하여 조절되는 단백질을 발굴하고 발굴한 단백질이 세포 내에서 어떠한 기능을 하는지를 조사할 필요가 있다. Therefore, it is necessary to discover proteins regulated by each isoform using inhibitors for each of the class I PI3K isoforms and investigate what functions the discovered proteins perform in cells.
본 발명의 목적은 특이적 PI3K p110δ 억제제를 유효성분으로 함유하는 스타스민1(stathmin1; STMN1) 관련 질환 예방 또는 치료용 약학 조성물을 제공하는 데에 있다.An object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease associated with stathmin1 (STMN1) containing a specific PI3K p110δ inhibitor as an active ingredient.
본 발명의 또 다른 목적은 특이적 PI3K p110δ 억제제를 유효성분으로 함유하는 스타스민1 관련 질환 개선 또는 예방용 건강식품 조성물을 제공하는 데에 있다.Another object of the present invention is to provide a health food composition for improving or preventing stamin 1-related diseases containing a specific PI3K p110δ inhibitor as an active ingredient.
본 발명의 또 다른 목적은 특이적 PI3K p110δ 억제제를 유효성분으로 함유하는 스타스민1 단백질 인산화 억제용 시약조성물을 제공하는 데에 있다.Another object of the present invention is to provide a reagent composition for inhibiting Stasmin 1 protein phosphorylation containing a specific PI3K p110δ inhibitor as an active ingredient.
본 발명의 또 다른 목적은 특이적 PI3K p110δ 억제제를 세포에 처리하는 단계를 포함하는 스타스민1 단백질 인산화 억제 방법을 제공하는 데에 있다.Another object of the present invention is to provide a method for inhibiting stamin 1 protein phosphorylation, comprising treating cells with a specific PI3K p110δ inhibitor.
상기 목적을 달성하기 위하여, 본 발명은 특이적 PI3K p110δ 억제제를 유효성분으로 함유하는 스타스민1 관련 질환 예방 또는 치료용 약학 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating stamin 1-related diseases containing a specific PI3K p110δ inhibitor as an active ingredient.
또한, 본 발명은 특이적 PI3K p110δ 억제제를 유효성분으로 함유하는 스타스민1 관련 질환 개선 또는 예방용 건강식품 조성물을 제공한다.In addition, the present invention provides a health food composition for improving or preventing stamin 1-related diseases, containing a specific PI3K p110δ inhibitor as an active ingredient.
또한, 본 발명의 또 다른 목적은 특이적 PI3K p110δ 억제제를 유효성분으로 함유하는 스타스민1 단백질 인산화 억제용 시약조성물을 제공한다.Another object of the present invention is to provide a reagent composition for inhibiting stamin 1 protein phosphorylation containing a specific PI3K p110δ inhibitor as an active ingredient.
또한, 본 발명의 또 다른 목적은 특이적 PI3K p110δ 억제제를 세포에 처리하는 단계를 포함하는 스타스민1 단백질 인산화 억제 방법을 제공한다.Another object of the present invention is to provide a method for inhibiting stamin 1 protein phosphorylation, comprising treating cells with a specific PI3K p110δ inhibitor.
본 발명은 특이적 PI3K p110δ 억제제를 유효성분으로 함유하는 조성물에 관한 것으로, 보다 상세하게는, 상기 특이적 PI3K p110δ 억제제가 STMN1 단백질의 인산화를 억제시킴으로써, 스타스민1 관련 질환 예방 또는 치료용 조성물로 유용하게 활용될 수 있다.The present invention relates to a composition containing a specific PI3K p110δ inhibitor as an active ingredient, and more particularly, to a composition for preventing or treating stasmin 1-related diseases by inhibiting phosphorylation of STMN1 protein by the specific PI3K p110δ inhibitor. can be put to good use.
도 1은 LPS에 자극된 RAW 264.7 대식세포에서 PI3K 아이소폼 선택적 억제제에 의한 AKT의 인산화 분석 결과이다.
도 2의 왼쪽 그림은 LPS에 자극된 RAW 264.7 대식세포에서 인산화 조절된 단백질의 heatmap clustering 분석 결과이고, 오른쪽 그림은 LPS에 자극된 RAW 264.7 대식세포에서 PI3K 아이소폼 선택적 억제제에 의해 인산화 감소된 단백질의 heatmap clustering 분석 결과이다.
도 3은 LPS에 자극된 RAW 264.7 대식세포에서 PI3K 아이소폼 선택적 억제제에 의한 STMN1의 인산화 분석결과이다.
도 4는 PI3K δ 억제제에 의해 인산화 감소된 STMN1 단백질과 다른 단백질 간 상호작용을 나타낸 STRING 분석결과이다.Figure 1 shows the results of phosphorylation analysis of AKT by PI3K isoform-selective inhibitors in LPS-stimulated RAW 264.7 macrophages.
The left figure of FIG. 2 shows the results of heatmap clustering analysis of phosphorylation-regulated proteins in LPS-stimulated RAW 264.7 macrophages, and the right figure shows the results of protein phosphorylation reduced by a PI3K isoform-selective inhibitor in LPS-stimulated RAW 264.7 macrophages. This is the result of heatmap clustering analysis.
Figure 3 shows the results of phosphorylation analysis of STMN1 by PI3K isoform-selective inhibitors in LPS-stimulated RAW 264.7 macrophages.
4 is a STRING analysis result showing the interaction between the STMN1 protein phosphorylated by the PI3K δ inhibitor and other proteins.
본 발명자는 Class I PI3K 관련 신호 단백질을 발굴하기 위해 PI3K 아이소폼 선택적 억제제를 처리하여 각각의 아이소폼들이 특징적으로 조절하는 단백질을 확인한 결과, 스타스민1(stathmin1; STMN1) 단백질의 인산화는 PI3K p110δ 억제제의 처리에 의해서만 크게 감소하는 것을 확인함으로써 본 발명을 완성한 것이다.In order to discover Class I PI3K-related signaling proteins, the present inventors treated PI3K isoform-selective inhibitors to identify proteins that each isoform characteristically regulates. The present invention was completed by confirming that it greatly decreased only by the treatment of.
본 발명에서 사용된 용어 "예방"은 특이적 PI3K p110δ 억제제에 의해 스타스민1 관련 질환을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" refers to any activity that suppresses or delays the onset of stasmin1-related diseases by specific PI3K p110δ inhibitors.
본 발명에서 사용된 용어 "치료"는 특이적 PI3K p110δ 억제제에 의해 스타스민1 관련 질환의 증세가 호전되거나 이롭게 변경하는 모든 행위를 의미한다.As used herein, the term "treatment" refers to any activity that improves or beneficially alters the symptoms of stasmin 1-related diseases by a specific PI3K p110δ inhibitor.
STMN1은 C-말단 나선 도메인이 고도로 보존된 인산화 단백질의 일종으로, 주로 세포질에서 발현되며 튜블린 이량체와 결합하여 복합체를 형성하여 미세소관의 중합을 차단하거나 미세소관 구조를 직접 파괴하여 미세소관을 조절하며, 세포 이동, 이동, 세포 주기 조절 및 기타 과정에서 중요한 역할을 한다. 미세소관 안정성을 조절하는 것 외에도 STMN1은 세포의 다양한 신호 전달 경로와 상호 작용하여 종양 세포 증식을 촉진하고 종양 세포 사멸을 억제할 수 있다. 즉, 종양 유전자로 알려진 STMN1의 증가는 여러 암에서 세포 분화, 증식 및 이동을 유도하는 작용을 미치는 것으로 잘 알려져 있다(STMN1 upregulation mediates hepatocellular carcinoma and hepatic stellate cell crosstalk to aggravate cancer by triggering the MET pathway. Cancer Sci 2020; 111(2): 406-417). STMN1 is a type of phosphorylated protein with a highly conserved C-terminal helix domain. It is mainly expressed in the cytoplasm and binds to tubulin dimer to form a complex to block microtubule polymerization or directly destroy microtubule structure to release microtubules. It plays an important role in cell migration, migration, cell cycle regulation and other processes. In addition to regulating microtubule stability, STMN1 can promote tumor cell proliferation and inhibit tumor cell death by interacting with various signaling pathways in cells. That is, the increase of STMN1, known as an oncogene, is well known to induce cell differentiation, proliferation, and migration in various cancers (STMN1 upregulation mediates hepatocellular carcinoma and hepatic stellate cell crosstalk to aggravate cancer by triggering the MET pathway. Cancer Sci 2020;111(2): 406-417).
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 특이적 PI3K p110δ 억제제를 유효성분으로 함유하는 스타스민1 관련 질환 예방 또는 치료용 약학 조성물을 제공한다. The present invention provides a pharmaceutical composition for preventing or treating stamin 1-related diseases, containing a specific PI3K p110δ inhibitor as an active ingredient.
상세하게는, 상기 특이적 PI3K p110δ 억제제는 IC87114, 이델라리시브( idelalisib), TGR-1202, AMG-319 및 INCB040093으로 이루어지는 군으로부터 선택될 수 있지만, 이에 한정되는 것은 아니다.Specifically, the specific PI3K p110δ inhibitor may be selected from the group consisting of IC87114, idelalisib, TGR-1202, AMG-319 and INCB040093, but is not limited thereto.
상기 특이적 PI3K p110δ 억제제는 스타스민1 단백질의 인산화를 억제함으로써 다양한 암질환을 치료하거나 개선할 수 있다.The specific PI3K p110δ inhibitor can treat or ameliorate various cancer diseases by inhibiting phosphorylation of stasmin 1 protein.
상기 암질환은 고형암 또는 혈액암일 수 있고, 보다 상세하게는 뇌암, 식도암, 위암, 간암, 췌장암, 신장암, 소장암, 대장암, 직장암, 폐암, 유방암, 자궁암, 난소암, 백혈병 및 전립선암으로 이루어지는 군으로부터 선택될 수 있지만, 이에 한정되는 것은 아니다.The cancer disease may be solid cancer or blood cancer, and more specifically, brain cancer, esophageal cancer, stomach cancer, liver cancer, pancreatic cancer, kidney cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, breast cancer, uterine cancer, ovarian cancer, leukemia and prostate cancer. It may be selected from the group consisting of, but is not limited thereto.
상기 약학 조성물은 상기 유효성분 이외에 약제학적으로 허용되는 담체를 포함할 수 있는데, 이러한 약제학적으로 허용되는 담체는 약품 제제 시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘, 미네랄 오일 등을 포함할 수 있으나, 이에 한정되는 것은 아니다. 또한, 상기 약학 조성물은 첨가제 및 보조제로서 충진제, 중량제, 결합제, 윤활제, 습윤제, 붕해제, 감미제, 향미제, 유화제, 현탁제, 방향제, 보존제 등을 추가로 포함할 수 있다.The pharmaceutical composition may include a pharmaceutically acceptable carrier in addition to the active ingredient. Such a pharmaceutically acceptable carrier is commonly used in pharmaceutical formulations, and includes lactose, dextrose, sucrose, sorbitol, mannitol, Starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, Magnesium stearate, mineral oil, and the like may be included, but are not limited thereto. In addition, the pharmaceutical composition may further include fillers, weighting agents, binders, lubricants, wetting agents, disintegrants, sweeteners, flavoring agents, emulsifiers, suspending agents, fragrances, preservatives, and the like as additives and adjuvants.
상기 약학 조성물은 정제, 환제, 산제, 과립제, 캡슐제, 현탁제, 내용액제, 유제, 시럽제, 멸균된 수용액, 비수성용제, 유제, 동결건조제제, 좌제 및 멸균 주사용액으로 제형화할 수 있다. 본 발명의 약학 조성물은 정맥내 투여, 동맥내 투여, 복강내 투여, 근육내 투여, 흉골내 투여, 피하 투여, 피내 투여, 비내 투여, 폐내 투여, 직장 내 투여, 국소 투여, 경구 투여 및 흡입을 통해 통상적인 방식으로 투여할 수 있다The pharmaceutical composition may be formulated into tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, emulsions, lyophilized formulations, suppositories and sterile injection solutions. The pharmaceutical composition of the present invention can be administered by intravenous administration, intraarterial administration, intraperitoneal administration, intramuscular administration, intrasternal administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, intrarectal administration, topical administration, oral administration and inhalation. It can be administered in the usual way through
상기 약학 조성물은 증상 정도에 따라 투여 방법이 결정되는데, 통상적으로는 국소 투여 방식이 바람직하다. 또한, 상기 약학 조성물의 유효성분의 유효량은 질환의 예방 또는 치료 요구되는 양을 의미한다. 따라서, 질환의 종류, 질환의 중증도, 조성물에 함유된 유효 성분 및 다른 성분의 종류 및 함량, 제형의 종류 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료 기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다. The method of administration of the pharmaceutical composition is determined according to the degree of symptoms, and a topical administration method is usually preferred. In addition, the effective amount of the active ingredient of the pharmaceutical composition means an amount required for preventing or treating a disease. Therefore, the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, weight, general health condition, sex and diet, administration time, administration route and composition It can be controlled according to various factors including secretion rate, duration of treatment, and drugs used concurrently.
본 발명은 특이적 PI3K p110δ 억제제를 유효성분으로 함유하는 스타스민1 관련 질환 개선 또는 예방용 건강식품 조성물을 제공한다.The present invention provides a health food composition for improving or preventing stamin 1-related diseases, containing a specific PI3K p110δ inhibitor as an active ingredient.
상세하게는, 상기 특이적 PI3K p110δ 억제제는 IC87114, 이델라리시브( idelalisib), TGR-1202, AMG-319 및 INCB040093으로 이루어지는 군으로부터 선택될 수 있지만, 이에 한정되는 것은 아니다.Specifically, the specific PI3K p110δ inhibitor may be selected from the group consisting of IC87114, idelalisib, TGR-1202, AMG-319 and INCB040093, but is not limited thereto.
상기 건강식품 조성물은 분말, 과립, 정제, 캡슐, 시럽 또는 음료의 형태로 제공될 수 있으며, 상기 건강식품 조성물은 상기 유효성분 이외에 다른 식품 또는 식품 첨가물과 함께 사용되고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 그의 사용 목적 예를 들어 예방, 건강 또는 치료적 처치에 따라 적합하게 결정될 수 있다.The health food composition may be provided in the form of a powder, granule, tablet, capsule, syrup or beverage, and the health food composition may be used with other foods or food additives in addition to the active ingredient, and may be appropriately used according to a conventional method. can The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use thereof, for example, prevention, health or therapeutic treatment.
상기 건강식품 조성물에 함유된 상기 유효성분의 유효용량은 상기 약학 조성물의 유효용량에 준해서 사용할 수 있으나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있음은 확실하다.The effective dose of the active ingredient contained in the health food composition may be used according to the effective dose of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it should be less than the above range. It is certain that the active ingredient can be used in an amount greater than the above range because there is no problem in terms of safety.
상기 건강식품의 종류에는 특별한 제한이 없고, 예로는 육류, 소세지, 빵, 초콜릿, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등을 들 수 있다.There is no particular limitation on the type of health food, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, Drinks, alcoholic beverages, vitamin complexes, and the like are exemplified.
본 발명은 특이적 PI3K p110δ 억제제를 유효성분으로 함유하는 스타스민1 단백질 인산화 억제용 시약조성물을 제공한다.The present invention provides a reagent composition for inhibiting phosphorylation of Stasmin 1 protein containing a specific PI3K p110δ inhibitor as an active ingredient.
상세하게는, 상기 특이적 PI3K p110δ 억제제는 IC87114, 이델라리시브( idelalisib), TGR-1202, AMG-319 및 INCB040093으로 이루어지는 군으로부터 선택될 수 있지만, 이에 한정되는 것은 아니다.Specifically, the specific PI3K p110δ inhibitor may be selected from the group consisting of IC87114, idelalisib, TGR-1202, AMG-319 and INCB040093, but is not limited thereto.
본 발명은 특이적 PI3K p110δ 억제제를 세포에 처리하는 단계를 포함하는 스타스민1 단백질 인산화 억제 방법을 제공한다.The present invention provides a method for inhibiting stamin 1 protein phosphorylation comprising treating cells with a specific PI3K p110δ inhibitor.
상세하게는, 상기 세포는 인간 세포를 제외한 포유류 세포일 수 있다.Specifically, the cells may be mammalian cells other than human cells.
이하에서는 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
[실시예][Example]
1. 재료 및 실험방법1. Materials and test methods
1) 세포 배양1) Cell culture
대표적인 마우스 대식세포인 RAW 264.7 세포를 사용하였다. RAW 264.7 세포는 1% penicillin과 10% fetal bovine serum (FBS, Hyclone, USA)가 포함되어 있는 DMEM (Hyclone, USA) 배지를 사용하여 37℃와 5% CO2가 유지되는 배양기에서 배양하였다. RAW 264.7 cells, which are representative mouse macrophages, were used. RAW 264.7 cells were cultured in DMEM (Hyclone, USA) medium containing 1% penicillin and 10% fetal bovine serum (FBS, Hyclone, USA) in an incubator maintained at 37°C and 5% CO 2 .
2) 사용 물질2) Substances used
PI3K 선택적 억제제 중 isoform 선택적 억제제인 A66 (α 억제제), TGX221 (β 억제제), AS252424 (γ 억제제), IC87114 (δ 억제제)와 범용 억제제인 GDC0941 (범용 억제제)는 Selleckchem (USA)으로부터 구입하였다. 모든 억제제들은 디메틸설폭사이드 (DMSO, Thermo Fisher Scientific, USA)에 녹여 사용하였다.Among the PI3K-selective inhibitors, isoform-selective inhibitors A66 (α inhibitor), TGX221 (β inhibitor), AS252424 (γ inhibitor), IC87114 (δ inhibitor) and GDC0941 (universal inhibitor) were purchased from Selleckchem (USA). All inhibitors were dissolved in dimethyl sulfoxide (DMSO, Thermo Fisher Scientific, USA).
3) 세포 용해물 준비3) Cell lysate preparation
RAW 264.7 세포를 1x106 cells/ml 농도로 사용하였다. 세포에 0.1% DMSO 또는 500 nM classⅠ PI3K 선택적 억제제 (A66, TGX-221, AS252424, IC87114, GDC0941)를 처리하고, 30분 후 200 ng/ml LPS를 처리한 후 37℃, 5% CO2가 유지되는 배양기에서 30분 동안 배양하였다. 이후 액체 배지를 제거하고 차가운 1X PBS (Hyclone, USA)를 이용하여 두 번 세척 하였다. RIPA buffer (Thermo Fisher scientific, USA)에 100X protease/phosphatase inhibitor cocktail (Thermo Fisher scientific, USA)을 1:100의 비율로 희석하여 제작한 용해 시약을 차갑게 유지하며 100 ㎕/1x106 cells 분주하였다. 이후 차가운 상태를 유지하면서 스크래퍼로 긁고 1.5 ml 용기에 옮겨 담아 세포 용해물을 준비하였다. 준비된 시료의 단백질 농도 측정은 BCA 정량법을 사용하였다. 표준 단백질로 bovine serum albumin (BSA, Thermo Fisher scientific, USA)을 이용하였고, BCA protein assay kit (Thermo Fisher scientific, USA)와 multi detection microplate reader spectromax M2e (Molecular Devices, USA)를 이용하였다. RAW 264.7 cells were used at a concentration of 1x10 6 cells/ml. Cells were treated with 0.1% DMSO or 500 nM class I PI3K selective inhibitor (A66, TGX-221, AS252424, IC87114, GDC0941), treated with 200 ng/ml LPS after 30 minutes, and maintained at 37°C and 5% CO 2 Incubated for 30 minutes in an incubator. Then, the liquid medium was removed and washed twice using cold 1X PBS (Hyclone, USA). A lysis reagent prepared by diluting 100X protease/phosphatase inhibitor cocktail (Thermo Fisher scientific, USA) in RIPA buffer (Thermo Fisher scientific, USA) at a ratio of 1:100 was dispensed at 100 μl/1x10 6 cells while keeping it cold. Thereafter, the cell lysate was prepared by scraping with a scraper while maintaining a cold state and transferring to a 1.5 ml container. The protein concentration of the prepared sample was measured using the BCA quantification method. As a standard protein, bovine serum albumin (BSA, Thermo Fisher scientific, USA) was used, BCA protein assay kit (Thermo Fisher scientific, USA) and multi detection microplate reader spectromax M2e (Molecular Devices, USA) were used.
4) TMT 표지 및 인산화 단백질 농축4) TMT labeling and enrichment of phosphoproteins
정량적 단백질체의 경우 각 분해된 단백질 샘플은 제조업체가 제공한 프로토콜에 따라 적절한 TMT 시약(Thermo Fisher Scientific, USA)으로 표지되었다. TMT 반응을 풀어주기 위해 5% 하이드록실아민을 각 샘플에 첨가하고 실온에서 15분 동안 배양하였다. 모든 표지된 펩타이드를 동일한 양으로 혼합하고 speed vacuum system을 사용하여 건조되었다. 펩타이드 혼합물을 제조사의 절차에 따라 high-pH reverse-phase peptide fractionation kit (Thermo Fisher Scientific, USA)를 사용하여 분리하였다. 분리된 펩타이드는 염을 제거하기 위해 ZipTip 피펫 팁(Millipore)을 사용하였고, speed vacuum system을 통해 완전히 건조되었다. For quantitative proteomics, each digested protein sample was labeled with the appropriate TMT reagent (Thermo Fisher Scientific, USA) according to the protocol provided by the manufacturer. To release the TMT reaction, 5% hydroxylamine was added to each sample and incubated for 15 minutes at room temperature. All labeled peptides were mixed in equal amounts and dried using a speed vacuum system. The peptide mixture was separated using a high-pH reverse-phase peptide fractionation kit (Thermo Fisher Scientific, USA) according to the manufacturer's procedure. The separated peptides were dried using a ZipTip pipette tip (Millipore) to remove salt and completely dried using a speed vacuum system.
인산화 단백질 농축을 위해 TiO2 enrichment를 하였다. 단백질 샘플을 56℃에서 30분 동안 5 mm 디티오트레이톨(dithiothreitol)에서 환원시키고 실온에서 45분 동안 15 mm 요오도아세트산(iodoacetic acid)에서 알킬화했다. 그런 다음 단백질 샘플을 37℃에서 12시간 동안 50:1 (w/w)의 비율로 트립신(Thermo Fisher Scientific, USA)으로 펩타이드를 생성시켰다. 1% 트리플루오로아세트산(trifluoroacetic acid; TFA)을 첨가하여 트립신 반응을 멈추고, 생성된 펩타이드를 C18 Sep-Pack cartridge (Waters, Milford, MA, USA)를 사용하여 탈염하였다. TiO2 enrichment kit (GL Science, Tokyo, Japan)를 사용하고 SDB 및 GC 팁(GL Science, Tokyo, Japan)를 통한 탈염을 하여 인산 펩타이드의 농축 전에 펩타이드는 high-pH reversed-phase peptide 분획 키트(Thermo Fisher Scientific, USA)를 사용하여 분별되었다. TiO 2 enrichment was performed for phosphorylated protein enrichment. Protein samples were reduced in 5 mM dithiothreitol for 30 minutes at 56°C and alkylated in 15 mM iodoacetic acid for 45 minutes at room temperature. Then, peptides were generated from the protein samples by trypsin (Thermo Fisher Scientific, USA) at a ratio of 50:1 (w/w) for 12 hours at 37°C. The trypsin reaction was stopped by adding 1% trifluoroacetic acid (TFA), and the resulting peptides were desalted using a C18 Sep-Pack cartridge (Waters, Milford, MA, USA). Prior to concentration of phosphopeptides by using TiO 2 enrichment kit (GL Science, Tokyo, Japan) and desalting through SDB and GC tips (GL Science, Tokyo, Japan), peptides were purified using a high-pH reversed-phase peptide fractionation kit (Thermo Fisher Scientific, USA).
5) LC-MS/MS 분석5) LC-MS/MS analysis
준비된 인산 펩타이드는 용액 A (2% acetonitrile in 0.1% 포름산)에 용해되었고 75분에 걸쳐 acetonitrile gradient (5-28%)로 녹여서 분리된 C12 (Proteo C12 4 ㅅm beads, 90-ㅕ pore 크기, Phenomenex, Torrance, CA)를 모세관 컬럼((길이: 10 cm; OD: 75 ㅅm; ID: 150 ㅅm; Molex, Lisle, IL, USA)에 주입했다. LTQ Orbitrap Velos spectrometer (Thermo Fisher Scientific, USA)와 연결된 nano-LC system (Eksigent, Dublin, CA, USA)은 양이온 모드에서 검출되었다. 분석정보는 충돌 유도된 dissociation/fragmentation 모드를 사용하여 m/z 350에서 1800까지 수집되었다. 해상도은 7500이었고 AGC 목표 값은 1.0 x 106이었다. 최대 IT는 전체 스캔에서 100 ms, MS2에서 60 ms였다.The prepared phosphate peptide was dissolved in solution A (2% acetonitrile in 0.1% formic acid) and dissolved in an acetonitrile gradient (5-28%) over 75 minutes to obtain isolated C12 (Proteo C12 4 μm beads, 90-ㅕ pore size, Phenomenex , Torrance, CA) was injected into a capillary column ((length: 10 cm; OD: 75 μm; ID: 150 μm; Molex, Lisle, IL, USA). LTQ Orbitrap Velos spectrometer (Thermo Fisher Scientific, USA) A nano-LC system (Eksigent, Dublin, CA, USA) coupled with was detected in positive ion mode Analytical information was collected using collision induced dissociation/fragmentation mode from m/z 350 to 1800. The resolution was 7500 and the AGC target The value was 1.0 x 10 6. The maximum IT was 100 ms for full scan and 60 ms for MS2.
6) 포스포프로테옴 분석(Phosphoproteome analysis)6) Phosphoproteome analysis
i) Heatmap clusteringi) Heatmap clustering
Heatmap clustering을 위해 소스 코드로 R 프로그래밍 (https://www.r-project.org) 언어를 사용하여 작성된 오픈 소스 소프트웨어인 R studio (https://www.rstudio.com)를 활용하였다. 분석한 자료를 CSV로의 파일 변환한 후 R studio에 파일을 불러들여 heatmap cluster를 R 패키지를 사용하여 명령어를 입력하였다. (주요 R 패키지 명령어: library, installed packages install packages, read.csv, row.names, gplots, data.matrix, heatmap.2)For heatmap clustering, R studio (https://www.rstudio.com), an open source software written using the R programming (https://www.r-project.org) language, was used as source code. After converting the analyzed data into a CSV file, the file was imported into R studio and commands were entered using the heatmap cluster R package. (Major R package commands: library, installed packages install packages, read.csv, row.names, gplots, data.matrix, heatmap.2)
ii) STRING analysisii) STRING analysis
STRING (https://string-db.org/cgi/input.pl)을 이용하여 후보 단백질 ID를 입력하고 그 중 mus musculus 단백질의 정보를 찾았다. 단백질 간의 상호작용을 나타내는 지도를 통해 단백질 간 상호작용을 분석하였다. Candidate protein IDs were entered using STRING (https://string-db.org/cgi/input.pl), and information on mus musculus proteins was found among them. Interactions between proteins were analyzed through maps showing interactions between proteins.
7) 웨스턴 블롯 분석7) Western blot analysis
세포 용해물에서 얻은 시료를 이용하여 웨스턴 블롯을 수행하였다. 8% 소듐도데실설페이트(sodium dodecyl sulfate; SDS) 폴리아크릴아마이드 겔(polyacrylamide gel)을 제작하여 1X Tris-Glycine SDS Running buffer (Invitrogen, USA)를 넣고 전기영동을 이용하여 단백질을 크기에 따라 분리하였다. 그리고 나이트로셀룰로오즈 멤브레인(nitrocellulose membrane)에 SDS-폴리아크릴아마이드 겔을 1X transfer buffer (BIO RAD, USA)에 20% 메탄올을 넣은 용액을 제작하여 transfer 하였다. 멤브레인을 5% 탈지우유가 들어있는 1X 인산완충액 (PBS, Trans lab, South Korea)에 0.001% Tween 20을 넣은 블로킹 용액을 제작하여 멤브레인을 실온에서 1시간 배양하였다. 첫 번째 항체를 각 항체 희석비율에 맞게 희석하고 4℃ 저온실에서 밤새 배양하였다. 그리고 두 번째 항체 항-mouse 혹은 rabbit IgG (Cell signaling technology)를 이용하여 면역 반응 밴드를 감지하고 ECL system (AI600, Amersham, UK)을 사용하여 밴드를 시각화하였다.Western blotting was performed using samples obtained from cell lysates. An 8% sodium dodecyl sulfate (SDS) polyacrylamide gel was prepared, 1X Tris-Glycine SDS Running buffer (Invitrogen, USA) was added, and proteins were separated according to their size using electrophoresis. . In addition, a solution containing 20% methanol in 1X transfer buffer (BIO RAD, USA) was prepared and transferred to a nitrocellulose membrane with SDS-polyacrylamide gel. The membrane was prepared with a blocking solution containing 0.001% Tween 20 in 1X phosphate buffer containing 5% skim milk (PBS, Trans lab, South Korea), and the membrane was incubated for 1 hour at room temperature. The first antibody was diluted according to each antibody dilution ratio and incubated overnight in a low temperature room at 4°C. Then, an immune response band was detected using a second antibody, anti-mouse or rabbit IgG (Cell signaling technology), and the band was visualized using an ECL system (AI600, Amersham, UK).
2. 실험결과2. Experimental results
1) LPS에 자극된 RAW 264.7 대식세포에서 PI3K isoform 선택적 억제제에 의하여 조절된 AKT의 인산화1) Phosphorylation of AKT regulated by PI3K isoform-selective inhibitors in LPS-stimulated RAW 264.7 macrophages
PI3K 선택적 억제제인 A66 (PI3K α 억제제), TGX221 (PI3K β 억제제), AS252424 (PI3K γ 억제제), IC87114 (PI3K δ 억제제), GDC0941 (PI3K 범용 억제제)는 PI3K isoform에 대한 선택적 또는 범용 억제제이다. LPS 자극을 주지 않은 unstimulated (UN)에서의 웨스턴 블롯 결과는 AKT의 인산화가 거의 되지 않았고, 30분간 LPS 자극만 준 vehicle (VH)에서는 AKT 인산화의 강도가 UN에 비하여 강하게 나타났다. 그리고 PI3K isoform 선택적 억제제를 각각 처리한 결과는 PI3K isoform 선택적 억제제에 의해 AKT의 인산화가 감소하였다(도 1).The PI3K selective inhibitors A66 (PI3K α inhibitor), TGX221 (PI3K β inhibitor), AS252424 (PI3K γ inhibitor), IC87114 (PI3K δ inhibitor), and GDC0941 (PI3K universal inhibitor) are selective or universal inhibitors of the PI3K isoform. Western blot results showed little phosphorylation of AKT in unstimulated (UN) without LPS stimulation, whereas in vehicle (VH) with only LPS stimulation for 30 minutes, the intensity of AKT phosphorylation was stronger than that in UN. And as a result of each treatment with the PI3K isoform-selective inhibitor, the phosphorylation of AKT was reduced by the PI3K isoform-selective inhibitor (FIG. 1).
2) LPS에 자극된 RAW 264.7 대식세포에서 인산화 조절된 단백질의 heatmap clustering 분석2) Heatmap clustering analysis of phosphorylation-regulated proteins in LPS-stimulated RAW 264.7 macrophages
RAW264.7 대식세포에서 LPS 자극이 단백질의 인산화에 미치는 영향을 알아보기 위하여 RAW 264.7 세포(1x106 cells/ml) 배양 배지에 LPS (200 ng/ml)을 30분간 처리한 후 세포 용해물을 추출하여 인산화 단백질 농축 및 LC-MS/MS 분석을 하였다. In order to examine the effect of LPS stimulation on protein phosphorylation in RAW264.7 macrophages, the culture medium of RAW 264.7 cells (1x10 6 cells/ml) was treated with LPS (200 ng/ml) for 30 minutes, and cell lysates were extracted. Then, phosphorylated protein concentration and LC-MS/MS analysis were performed.
그 결과 heatmap clustering에 나타난 것처럼 대부분의 단백질들이 UN에 비하여 LPS 처리에 의해 높은 인산화를 보였다(도 2 왼쪽 그림).As a result, as shown in the heatmap clustering, most proteins showed higher phosphorylation by LPS treatment than UN (Fig. 2 left figure).
3) LPS에 자극된 RAW 264.7 대식세포에서 PI3K α, β, γ, δ isoform 선택적 억제제에 의해 인산화 감소된 단백질의 heatmap clustering3) Heatmap clustering of proteins with reduced phosphorylation by PI3K α, β, γ, δ isoform selective inhibitors in LPS-stimulated RAW 264.7 macrophages
LPS 자극을 준 RAW 264.7 대식세포에서 PI3K isoform 억제제에 의해 인산화가 조절되는 단백질을 알아보기 위하여 RAW 264.7 세포(1x106 cells/ml) 배양 배지에 PI3K α, β, γ, δ 억제제 (각각 500 nM)을 전처리하고 LPS (200 ng/ml)을 30분간 처리한 후 세포 용해물 추출, 그리고 인산화 단백질 농축 및 LC-MS/MS 분석을 하였다. In order to examine proteins whose phosphorylation is regulated by PI3K isoform inhibitors in LPS-stimulated RAW 264.7 macrophages, PI3K α, β, γ, and δ inhibitors (500 nM each) were added to the culture medium of RAW 264.7 cells (1x10 6 cells/ml). was pre-treated and treated with LPS (200 ng/ml) for 30 minutes, followed by cell lysate extraction, phosphorylated protein concentration, and LC-MS/MS analysis.
그 결과 LC-MS/MS 분석을 통해 얻은 자료에서 PI3K α, β, γ, δ 억제제에 의하여 많은 단백질들의 인산화가 감소하였다. 그리고 그중 특정값(〈0.667)을 기준으로 유효인자 단백질을 선별하였고, PI3K α, β, γ, δ 억제제에 의해 인산화가 감소하는 단백질들을 heatmap clustering 하였다(도 2 오른쪽 그림). As a result, in the data obtained through LC-MS/MS analysis, phosphorylation of many proteins was reduced by PI3K α, β, γ, and δ inhibitors. Among them, effective factor proteins were selected based on a specific value (<0.667), and proteins whose phosphorylation was reduced by PI3K α, β, γ, and δ inhibitors were heatmap clustered ( Fig. 2 right figure).
4) LPS에 자극된 RAW 264.7 대식세포에서 PI3K isoform 선택적 억제제에 의하여 조절된 STMN1의 인산화4) Phosphorylation of STMN1 regulated by PI3K isoform-selective inhibitors in LPS-stimulated RAW 264.7 macrophages
LPS 자극을 준 RAW264.7 대식세포에서 PI3K isoform 선택적 억제제에 의해 인산화가 조절되는 단백질을 알아보기 위하여 RAW 264.7 세포(1x106 cells/ml) 배양 배지에 VH (0.1% DMSO) 또는 PI3K isoform 선택적 억제제(500 nM)을 전처리하고 LPS (200 ng/ml)을 30분간 처리한 후 세포 용해물을 추출, 인산화 단백질 농축, 그리고 LC-MS/MS 분석을 하였다. 수집된 자료에서 특정 PI3K 억제제에서만 감소하는 단백질을 선별하였고, 선별된 단백질들이 어떤 단백질들과 상호작용하는지를 STRING 분석을 통하여 2차 선별하였다. In order to examine proteins whose phosphorylation is regulated by PI3K isoform-selective inhibitors in LPS-stimulated RAW264.7 macrophages, VH (0.1% DMSO) or PI3K isoform-selective inhibitors ( 500 nM) and LPS (200 ng/ml) for 30 minutes, then cell lysates were extracted, phosphorylated proteins were concentrated, and LC-MS/MS analysis was performed. From the collected data, proteins that were reduced only in specific PI3K inhibitors were selected, and the secondary selection was performed through STRING analysis to determine which proteins the selected proteins interact with.
선별을 통하여 선정된 스타스민 1 (STMN1) 단백질의 항체를 이용하여 western blot을 한 결과, PI3K δ 억제제에서 STMN1 단백질의 인산화가 크게 감소하였다(도 3).As a result of western blotting using the stamin 1 (STMN1) protein antibody selected through screening, the phosphorylation of the STMN1 protein was greatly reduced in the PI3K δ inhibitor (FIG. 3).
5) String 분석을 이용한 PI3K δ 억제제에 의하여 인산화 감소된 단백질의 단백질 간 상호작용5) Inter-protein interaction of proteins reduced in phosphorylation by PI3K δ inhibitors using string analysis
LPS 자극을 준 RAW264.7 대식세포에서 PI3K δ 선택적 억제제에 의해 인산화가 조절되는 특정 단백질들이 어떤 단백질들과 매개되어 상호작용하는지 조사하기 위하여 PI3K δ 억제제에서만 감소하는 STMN1 단백질의 STRING 분석하였다(도 4).PI3K δ in LPS-stimulated RAW264.7 macrophages STRING analysis of STMN1 protein, which is reduced only in PI3K δ inhibitors, was performed to investigate which proteins mediated and interacted with specific proteins whose phosphorylation was regulated by selective inhibitors (FIG. 4).
이상으로 본 발명의 특정한 부분을 상세히 기술한 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.Having described specific parts of the present invention in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
본 발명의 범위는 후술하는 특허청구범위에 의하여 나타내어지며, 특허청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.
Claims (9)
A method of inhibiting stamin 1 protein phosphorylation comprising the step of treating cells with a specific PI3K p110δ inhibitor.
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