KR20230110972A - Anticancer composition comprising MARCH6 inhibitor - Google Patents
Anticancer composition comprising MARCH6 inhibitor Download PDFInfo
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- KR20230110972A KR20230110972A KR1020220006597A KR20220006597A KR20230110972A KR 20230110972 A KR20230110972 A KR 20230110972A KR 1020220006597 A KR1020220006597 A KR 1020220006597A KR 20220006597 A KR20220006597 A KR 20220006597A KR 20230110972 A KR20230110972 A KR 20230110972A
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- South Korea
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- cancer
- marh6
- activity
- ferrotosis
- march6
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Abstract
본 발명은 MARCH6 활성 또는 발현 저해제를 포함하는 항암용 조성물에 관한 것이다. 본 발명의 MARCH6 활성 또는 발현 저해제는 암세포의 페로토시스 민감도를 증가시켜, 페로토시스를 통한 암 세포 사멸 및 암세포 성장 억제를 유도할 수 있다. 따라서 본 발명은 페로토시스 유도 항암제로 널리 활용될 수 있다. The present invention relates to an anti-cancer composition comprising a MARCH6 activity or expression inhibitor. The MARCH6 activity or expression inhibitor of the present invention can increase the sensitivity of cancer cells to ferrotosis, thereby inducing cancer cell death and suppression of cancer cell growth through ferrotosis. Therefore, the present invention can be widely used as a ferrotosis-inducing anticancer agent.
Description
본 발명은 MARCH6 활성 또는 발현 저해제를 포함하는 항암용 조성물에 관한 것이다. The present invention relates to an anti-cancer composition comprising a MARCH6 activity or expression inhibitor.
세포사멸 방법 중 하나인 페로토시스는 과도한 철 이온에 의한 지질의 과산화반응을 통해 유도된다. 페로토시스는 기본적으로 철 이온, 활성 산소 종, 지질의 과산화, 과산화 지질을 중화하는 시스템 등에 의해 조절되며, 세포 주변의 환경이나 세포 대사의 변화는 페로토시스를 일으키는 신호가 될 수 있다. 최근 연구들을 통해 페로토시스와 여러 질병 사이의 연관성이 밝혀지게 되었는데, 그 질병의 범주가 암 질환, 뇌 질환, 장기 손상, 면역 및 염증 관련 질환 등을 포함할 만큼 페로토시스의 생리학적 및 병리학적 중요성이 떠오르고 있다. Ferrotosis, one of the cell death methods, is induced through lipid peroxidation by excessive iron ions. Ferrotosis is basically regulated by iron ions, reactive oxygen species, lipid peroxidation, and a system that neutralizes lipid peroxidation, and changes in the cell environment or cell metabolism can be signals that cause ferrotosis. Recent studies have revealed the connection between ferrotosis and various diseases, and the physiological and pathological importance of ferrotosis is emerging to the extent that the disease categories include cancer diseases, brain diseases, organ damage, immune and inflammatory diseases, etc.
MARH6(MARCHF6/MARH6/TEB4/RNF176)는 소포체 막에 위치한 E3 유비퀴틴 리가아제 중 하나로, 소포체 막이나 세포질에 있는 단백질 분해에 관여한다. MARH6에 의해 분해되는 표적 단백질로는 MARH6 자체, 콜레스테롤 합성에 관여하는 SM(Squalene monooxygenase), 혈압 조절 단백질인 RGS2(Regulator of G-protein signaling 2), 지질 방울 형성에 관여하는 Plin2(Perilipin 2) 등이 있다. 최근 연구들에서 MARH6와 지질 및 콜레스테롤 대사와의 연관성이 밝혀졌지만, MARH6의 생리학적 역할은 명확하게 규정되지 않은 상태이다.MARH6 (MARCHF6/MARH6/TEB4/RNF176) is one of the E3 ubiquitin ligases located in the endoplasmic reticulum membrane and is involved in protein degradation in the endoplasmic reticulum membrane or cytoplasm. Target proteins degraded by MARH6 include MARH6 itself, squalene monooxygenase (SM) involved in cholesterol synthesis, regulator of G-protein signaling 2 (RGS2), and Plin2 (Perilipin 2) involved in lipid droplet formation. Although recent studies have revealed an association between MARH6 and lipid and cholesterol metabolism, the physiological role of MARH6 has not been clearly defined.
본 발명자들은 최근 다양한 생리, 병리학적 증상과 연관되어 있는 것으로 밝혀진 페로토시스를 조절하기 위한 타깃 및 방법에 관하여 연구하던 중, 그 동안 그 역할이 명확하게 규명되지 않았던 MARCH6 가 페로토시스 조절 메커니즘에 관여하고, 이를 저해하면 암 세포에서 페로토시스에 대한 민감도를 증가시켜 암세포 성장 억제 효과를 나타낼 수 있음을 확인하고 본 발명을 완성하였다. While studying targets and methods for regulating ferrotosis, which have recently been found to be associated with various physiological and pathological symptoms, the present inventors have confirmed that MARCH6, whose role has not been clearly identified, is involved in the ferrotosis regulatory mechanism, and inhibiting it can increase the sensitivity to ferrotosis in cancer cells and exhibit a cancer cell growth inhibitory effect, thereby completing the present invention.
따라서 본 발명의 목적은 MARCH6 활성 또는 발현 저해제를 포함하는 암 예방 또는 치료용 약학적 조성물을 제공하는 것이다. Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer containing an inhibitor of MARCH6 activity or expression.
따라서 본 발명은 MARCH6 활성 또는 발현 저해제를 포함하는 암 예방 또는 치료용 약학적 조성물을 제공한다. Accordingly, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an inhibitor of MARCH6 activity or expression.
또한 본 발명은 MARCH6 활성 또는 발현 저해제를 포함하는 항암 보조제 조성물을 제공한다In addition, the present invention provides an anti-cancer adjuvant composition comprising a MARCH6 activity or expression inhibitor
또한 본 발명은 MARCH6 활성 또는 발현 저해제를 포함하는 암 예방 또는 개선용 건강기능 식품 조성물을 제공한다. In addition, the present invention provides a health functional food composition for preventing or improving cancer containing an inhibitor of MARCH6 activity or expression.
또한 본 발명은 1) 시료에 후보물질을 처리하는 단계; 2) 후보물질 처리 후 MARCH6 의 활성 또는 발현의 변화를 측정하는 단계; 및 3) 상기 2) 단계의 MARCH6 활성 또는 발현 수준이 감소되면, 상기 후보물질을 항암제로 판정하는 단계; 를 포함하는 페로토시스 유도 항암제의 스크리닝 방법을 제공한다. In addition, the present invention comprises the steps of 1) treating a candidate substance to a sample; 2) measuring the change in activity or expression of MARCH6 after treatment with the candidate substance; and 3) determining the candidate substance as an anticancer drug when the activity or expression level of MARCH6 in step 2) is reduced; It provides a screening method for ferrotosis-inducing anticancer agents comprising a.
본 발명의 MARCH6 활성 또는 발현 저해제는 암세포의 페로토시스 민감도를 증가시켜, 페로토시스를 통한 암 세포 사멸 및 암세포 성장 억제를 유도할 수 있다. 따라서 본 발명은 페로토시스 유도 항암제로 널리 활용될 수 있다. The MARCH6 activity or expression inhibitor of the present invention can increase the sensitivity of cancer cells to ferrotosis, thereby inducing cancer cell death and suppression of cancer cell growth through ferrotosis. Therefore, the present invention can be widely used as a ferrotosis-inducing anticancer agent.
도 1의 A 는 48시간 동안 MARH6 siRNA 및 대조군으로 처리한 HeLa 세포에서의 지질 ROS 수준을 3번의 독립 실험을 통해 확인한 결과를 나타낸 도이다. 좌측은 증가된 형광을 나타내는 세포의 비율을 나타낸 도이다. 우측은 C11-BODIPY 를 이용하여 측정된 지질 ROS 정량값의 mean ± SD을 나타낸 도이다 (Two-tailed t-test. p**** < 0.0001).
도 1 의 B 및 C 는 야생형 및 MARH6 KO HeLa 세포(B) 또는 HEK293T 세포(C)에서 erastin (5 μM) 24시간 처리/미처리 후 지질 ROS 수준을 3번의 독립 실험을 통해 확인한 결과를 나타낸 도이다. 좌측은 증가된 형광을 나타내는 세포의 비율을 나타낸 도이다. 우측은 C11-BODIPY 를 이용하여 측정된 지질 ROS 정량값의 mean ± SD을 나타낸 도이다 (one-way ANOVA with Tukey's HSD post-hoc test. **p<0.01 and ****p<0.0001).
도 1D 는 야생형 RSL3 (0.1 μM) 12시간 처리/미처리 후 MARH6 KO HEK293T 세포에서 지질 ROS 수준을 3번의 독립 실험을 통해 확인한 결과를 나타낸 도이다. 좌측은 증가된 형광을 나타내는 세포의 비율을 나타낸 도이다. 우측은 C11-BODIPY 를 이용하여 측정된 지질 ROS 정량값의 mean ± SD을 나타낸 도이다 (one-way ANOVA with Tukey's HSD post-hoc test. **p<0.01 and ****p<0.0001).
도 1 의 E 및 F 는 야생형 및 MARH6 KO A549 세포 (E) 또는 HCT116 세포(F)에서 erastin (5 μM) 24시간 처리/미처리 후 지질 ROS 수준을 3번의 독립실험을 통해 확인한 결과를 나타낸 도이다. 좌측은 증가된 형광을 나타내는 세포의 비율을 나타낸 도이다. 우측은 C11-BODIPY 를 이용하여 측정된 지질 ROS 정량값의 mean ± SD을 나타낸 도이다 (Two-tailed t-test. p** < 0.05, p*** < 0.001, p**** < 0.0001).
도 2는 MARH6 -/- 마우스에 페로토시스 유도제인 에라스틴을 처리한 후 유기체 수준의 지질 ROS 축적을 확인한 결과를 나타낸 도이다. 각 결과는 3번 독립 실험의 mean ± SD로 표시하였다(One-way ANOVA with Tukey's HSD post-hoc test: p** < 0.01; p*** < 0.001; p**** < 0.0001).
도 3은 야생형, MARH6 KO HeLa, HEK293T, A549, 또는 HCT116 세포에 페로토시스 유도제인 에라스틴 또는 RSL3를 농도별로 처리하고 이에 따른 상대적인 생존능을 확인한 결과를 나타낸 도이다.
도 4a는 공백터, MARH63f, 및 MARH63f C9A 를 발현시킨 MARH6 KO HeLa 세포에서 RSL3 및 에라스틴에 대한 민감도를 MARH6 KO HeLa 의 상대적 생존능을 통해 확인한 결과를 나타낸 도이다 (two-tailed t-test. p**** < 0.0001).
도 4b는 RSL3 를 mock, DFO, Fer-1, Z-VAD 또는 Nec-1과 함께 MARH6 KO HeLa 또는 MARH6 KO HEK293T 에 처리하고, 상대적인 세포 생존능을 확인한 결과를 나타낸 도이다 (one-way ANOVA with Tukey's HSD post-hoc test. p * < 0.05, p**** < 0.0001).
도 5는 페로토시스 유도제인 에라스틴, RSL3 처리에 따른 MARH6 기질 SM 안정화 효과를 확인한 결과를 나타낸 도이다 (도 5A는 에라스틴 처리군, 도 5B는 RSL3 처리군).
도 6은 MARH6 KO HeLa 세포에서 야생형 대조군 대비 하향 조절된 유전자들 및 그의 기능을 DEG(differentially expressed genes) 를 통해 확인한 결과를 나타낸 도이다.
도 7은 야생형과 MARH6 KO HeLa 세포에서 NADP(H) 또는 NADPH 의 상대적인 양 을 확인한 결과를 나타낸 도이다 (mean ± SD (three biological replicates), Two-tailed t-test. p*** < 0.001).
도 8은 MARH6의 구조 및 인간을 포함하는 다양한 생물종에서 MARH6 FRR 의 시퀀스 얼라인먼트를 나타낸 도이다.
도 9는 MARH6 KO HeLa 세포에 전장 또는 일부 서열이 결실된 MARH6 를 발현시킨 후, 페로토시스 유도제 처리에 따른 세포 생존율을 확인한 결과를 나타낸 도이다(mean ± SD (three independent experiments), One-way ANOVA with Tukey's HSD post-hoc test. p* < 0.05, p**** < 0.0001).
도 10은 MARH63f 1-900와 전장 MARH63f 1-910 의 분해 속도를 CHX 추적 분석을 통해 확인하고 이를 정량화한 결과를 나타낸 도이다.
도 11은 링 도메인과 FRR 도메인의 상호작용 부위를 확인하기 위한 GST-pulldown 어세이 결과를 나타낸 도이다.
도 12는 링 도메인과 FRR 도메인의 상호작용을 확인하기 위한 in vitro 유니퀴틴화 어세이 수행 결과(a) 및 연결 체인의 종류를 확인한 결과(b)를 나타낸 도이다.
도 13은 NADPH 와 FRR 결합을 확인하기 위한 이용한 2',5'-adenosine diphosphate (ADP)-agarose 친화도 크로마토그래피 결과를 나타낸 도이다.
도 14는 NADPH를 인식하는 FRR 영역을 매핑하기 위한 2',5'-ADP-agarose 친화도 크로마토그래피 결과 (A), GST 또는 GST-FRR 의 GST 풀다운 어세이 결과(B) 및 NADPH 또는 NADH 존재에 따른 유비퀴틴화 분석 결과 (C) 를 나타낸 도이다.
도 15는 야생형 및 MARH6 KO HeLa 또는 A549 세포에서 MARH6 유전자 유무에 따른 페로토시스 조절 단백질 ACSL4(A), ALOX5(B), TfR1(C), p53 (D) 단백질 분해 패턴을 CHX 추적 어세이를 통해 확인한 결과를 나타낸 도이다.
도 16은 MARH6 결실에 따른 pro-ferroptosis 단백질 및 항-페로토시스 단백질의 발현을 확인한 결과를 나타낸 도이다.
도 17은 에라스틴 처리/미처리 모체로부터 분리된 MARH6-/- 배아 및 야생형의 간 조직; 및 야생형 및 MARH6 KO A549 세포 유래 이종이식 종양에서 pro-ferroptosis 단백질과 항-페로토시스 단백질 발현 변화를 확인한 결과를 나타낸 도이다.
도 18은 MARH6의 종양 성장 억제 효과를 확인하기 위한 이종이식 실험 모식도 (A) 및 종양 성장 억제효과를 확인한 결과 (B) 를 나타낸 도이다.
도 19는 비타민 E 식이에 따른 MARH6-/- 새끼 마우스의 생존율 확인을 위한 교배 모식도(A) 및 생존율을 확인한 결과(B)를 나타낸 도이다.
도 20은 본 발명에 따른 MARH6의 페로토시스 조절기전을 나타낸 모식도이다. Figure 1A is a diagram showing the results of confirming lipid ROS levels in HeLa cells treated with MARH6 siRNA and control for 48 hours through three independent experiments. The left side is a diagram showing the percentage of cells exhibiting increased fluorescence. The right side is a diagram showing the mean ± SD of lipid ROS quantitative values measured using C11-BODIPY (Two-tailed t -test. p**** < 0.0001).
1B and C are diagrams showing the results of lipid ROS levels in wild-type and MARH6 KO HeLa cells (B) or HEK293T cells (C) treated/untreated with erastin (5 μM) for 24 hours through three independent experiments. The left side is a diagram showing the percentage of cells exhibiting increased fluorescence. The right side is a diagram showing the mean ± SD of lipid ROS quantitative values measured using C11-BODIPY (one-way ANOVA with Tukey's HSD post-hoc test. **p<0.01 and ****p<0.0001).
FIG. 1D is a diagram showing the results of three independent experiments on lipid ROS levels in MARH6 KO HEK293T cells after 12 hours of treatment/untreatment with wild-type RSL3 (0.1 μM). The left side is a diagram showing the percentage of cells exhibiting increased fluorescence. The right side is a diagram showing the mean ± SD of lipid ROS quantitative values measured using C11-BODIPY (one-way ANOVA with Tukey's HSD post-hoc test. **p<0.01 and ****p<0.0001).
1E and F are diagrams showing the results of three independent experiments on lipid ROS levels after treatment/untreatment with erastin (5 μM) for 24 hours in wild-type and MARH6 KO A549 cells (E) or HCT116 cells (F). The left side is a diagram showing the percentage of cells exhibiting increased fluorescence. The right side is a diagram showing the mean ± SD of lipid ROS quantitative values measured using C11-BODIPY (Two-tailed t -test. p** < 0.05, p*** < 0.001, p**** < 0.0001).
Figure 2 is a diagram showing the result of confirming the accumulation of lipid ROS at the organism level after treating MARH6 -/- mice with the ferrotosis inducer elastin. Each result was expressed as mean ± SD of 3 independent experiments (One-way ANOVA with Tukey's HSD post-hoc test: p** <0.01; p*** <0.001; p**** < 0.0001).
3 is a diagram showing the result of confirming the relative viability of wild-type, MARH6 KO HeLa, HEK293T, A549, or HCT116 cells treated with the ferrotosis inducer Erastin or RSL3 at different concentrations.
Figure 4a is a diagram showing the results of confirming the sensitivity to RSL3 and erastin in MARH6 KO HeLa cells expressing blank, MARH63f, and MARH6 3f C9A through the relative viability of MARH6 KO HeLa (two-tailed t -test. p**** < 0.0001).
Figure 4b is a diagram showing the results of treating RSL3 with mock, DFO, Fer-1, Z-VAD or Nec-1 in MARH6 KO HeLa or MARH6 KO HEK293T and confirming the relative cell viability (one-way ANOVA with Tukey's HSD post-hoc test. p * < 0.05, p **** < 0.0001).
FIG. 5 is a view showing the results confirming the effect of stabilizing MARH6 substrate SM according to the treatment of elastin and RSL3, which are ferrotosis inducers (FIG. 5A shows an erastin-treated group, and FIG. 5B shows an RSL3-treated group).
6 is a diagram showing the results of confirming down-regulated genes and their functions in MARH6 KO HeLa cells compared to wild-type control through DEG (differentially expressed genes).
7 is a diagram showing the results of confirming the relative amount of NADP(H) or NADPH in wild-type and MARH6 KO HeLa cells (mean ± SD (three biological replicates), two-tailed t -test. p*** < 0.001).
8 is a diagram showing the structure of MARH6 and the sequence alignment of MARH6 FRR in various species, including humans.
Figure 9 is a diagram showing the results of confirming the cell viability according to the ferrotosis inducer treatment after expressing the full-length or partial sequence-deleted MARH6 in MARH6 KO HeLa cells (mean ± SD (three independent experiments), One-way ANOVA with Tukey's HSD post-hoc test. p* < 0.05, p**** < 0.0001).
10 is a diagram showing the results of confirming and quantifying the degradation rates of MARH6 3f 1-900 and full-length MARH6 3f 1-910 through CHX tracking analysis.
11 is a diagram showing the results of a GST-pulldown assay for confirming an interaction site between a ring domain and an FRR domain.
12 is a diagram showing the result of performing an in vitro uniquitination assay (a) and the result of confirming the type of linking chain (b) for confirming the interaction between the ring domain and the FRR domain.
13 is a diagram showing the results of 2',5'-adenosine diphosphate (ADP)-agarose affinity chromatography used to confirm the binding between NADPH and FRR.
14 is a diagram showing 2',5'-ADP-agarose affinity chromatography results (A), GST pull-down assay results of GST or GST-FRR (B), and ubiquitination analysis results according to the presence of NADPH or NADH (C).
15 is a diagram showing the results of confirming proteolysis patterns of ferrotosis regulatory proteins ACSL4 (A), ALOX5 (B), TfR1 (C), and p53 (D) according to the presence or absence of the MARH6 gene in wild-type and MARH6 KO HeLa or A549 cells through a CHX tracking assay.
16 is a view showing the results of confirming the expression of pro-ferroptosis protein and anti-ferrotosis protein according to MARH6 deletion.
Figure 17: Liver tissues of MARH6 -/- embryos and wild type isolated from Erastin treated/untreated mothers; and wild-type and MARH6 KO A549 cell-derived xenograft tumors showing pro-ferroptosis protein and anti-ferroptosis protein expression changes.
18 is a schematic view of xenotransplantation experiment for confirming the tumor growth inhibitory effect of MARH6 (A) and a diagram showing the result of confirming the tumor growth inhibitory effect (B).
19 is a diagram showing a mating schematic diagram (A) and a result (B) of confirming the survival rate for confirming the survival rate of MARH6 -/- baby mice according to the vitamin E diet.
20 is a schematic diagram showing the ferrotosis regulation mechanism of MARH6 according to the present invention.
본 발명은 MARCH6 활성 또는 발현 저해제를 포함하는 암 예방 또는 치료용 약학적 조성물에 관한 것이다. The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising an inhibitor of MARCH6 activity or expression.
이하, 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명은 MARCH6 활성 또는 발현 저해제를 포함하는 암 예방 또는 치료용 약학적 조성물 및 이를 이용한 암 예방 또는 치료방법에 관한 것이다. The present invention relates to a pharmaceutical composition for preventing or treating cancer containing a MARCH6 activity or expression inhibitor and a method for preventing or treating cancer using the same.
본 발명에 따르면, MARCH6 활성 또는 발현을 저해하는 경우, 암세포에서 페로토시스가 유도되거나 페로토시스에 대한 민감도를 증진시켜 페로토시스에 의한 사멸을 유도할 수 있다. According to the present invention, when the activity or expression of MARCH6 is inhibited, ferrotosis is induced in cancer cells or the sensitivity to ferrotosis is enhanced to induce death by ferrotosis.
발명의 MARH6는 E3 유비퀴틴-단백질 리가아제로 MARH6 유전자에 의하여 암호화되는 효소이다. MARH6는 MARCHF6, MARCH6, TEB4, 또는 RNF176 이라는 이름으로도 알려져 있으며, ER 막에 존재하는 단백질이다. MARH6 는 14개의 막-투과 영역, 세포질로 향한 RING 부위를 함유하는 N-말단과 C-말단 영역을 포함한다(도 8 참조). MARH6 는 다양한 동물에서 발견되고 이들의 C-말단은 특히 척추 동물에서 보존되어 있는 것으로 알려져 있다. 인간 MARH6 (GenBank: AAI36462.1) 는 총 910 개 아미노산으로 이루어져 있다. 용어 '페로토시스 (Ferrotosis)' 는 철 매개 지질과산화(iron-mediated lipid peroxidation)에 의한 세포사멸 방식으로, 철이 활성 산소(reactive oxygen species; ROS)와 만나 펜톤 반응(Fenton reaction)이 유도되고, 이를 통해 지질과산화물이 세포 내에 축적되어 세포가 사멸하는 방식이다. 즉, 페로토시스란 철-의존성 세포 사멸로 철 의존적인 활성 산소종의 발생, 지질 과산화의 발생 및 축적에 의해 발생하는 세포 사멸을 지칭할 수 있다. 최근 이러한 페로토시스를 이용한 암세포 사멸 방법에 대한 연구가 보고된 바 있다. MARH6 of the present invention is an E3 ubiquitin-protein ligase and is an enzyme encoded by the MARH6 gene. MARH6, also known as MARCHF6, MARCH6, TEB4, or RNF176, is a protein present in the ER membrane. MARH6 contains 14 transmembrane domains, N-terminal and C-terminal domains containing RING sites directed to the cytoplasm (see FIG. 8). MARH6 is found in various animals and its C-terminus is known to be particularly conserved in vertebrates. Human MARH6 (GenBank: AAI36462.1) consists of a total of 910 amino acids. The term 'Ferrotosis' is a method of cell death by iron-mediated lipid peroxidation, in which iron meets reactive oxygen species (ROS) to induce a Fenton reaction, through which lipid peroxides are accumulated in cells and cells die. That is, ferrotosis is iron-dependent cell death, and may refer to cell death caused by generation and accumulation of iron-dependent reactive oxygen species and lipid peroxidation. Recently, a study on a method of killing cancer cells using such ferrotosis has been reported.
발명에 있어서, 페로토시스를 유도할 수 있는 MARH6 활성 또는 발현 저해제는 MARH6 유전자의 mRNA에 상보적으로 결합하는 안티센스 뉴클레오티드, 짧은 헤어핀 RNA(small hairpin RNA, shRNA), 작은 간섭 RNA(small interfering RNA, siRNA), 리보자임(ribozyme), MARH6 단백질에 특이적으로 결합하는 화합물, 펩티드, 펩티드 모방체, 기질 유사체, 앱타머 및 항체로 이루어진 군에서 선택된 1종 이상일 수 있다. In the present invention, the MARH6 activity or expression inhibitor capable of inducing ferrotosis may be at least one selected from the group consisting of an antisense nucleotide, a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a ribozyme, a compound that specifically binds to the MARH6 protein, a peptide, a peptide mimetic, a substrate analog, an aptamer, and an antibody.
상기 활성 또는 발현 저해제는 MARH6 가 페로토시스 단계에서 수행하는 역할을 억제함으로써, 페로토시스를 더욱 촉진시키는 물질을 말한다. 예컨대 MARH6 활성 또는 발현 저해제는 개체의 페로토시스에 대한 민감도를 증가시켜 페로토시스에 의한 세포 사멸을 촉진시킬 수 있다. 따라서 본 발명의 MARCH6 활성 또는 발현 저해제는 페로토시스 유도용인 것을 특징으로 한다. The activity or expression inhibitor refers to a substance that further promotes ferrotosis by inhibiting the role that MARH6 plays in the ferrotosis step. For example, MARH6 activity or expression inhibitors can increase the sensitivity of an individual to ferrotosis and promote cell death by ferrotosis. Therefore, the MARCH6 activity or expression inhibitor of the present invention is characterized in that it is for inducing ferrotosis.
또한 본 발명의 상기 활성 또는 발현 저해제는 MARH6와 NADPH 의 결합 또는 MARH6 링 도메인과 FRR (Ferroptosis regulatory region) 도메인의 결합을 억제하는 것일 수 있다. MARH6 의 C-말단 꼬리 부위의 페로토시스 조절 영역(Ferroptosis regulatory region, FRR) 영역이 최초로 확인되었으며, NADPH 는 상기 FRR 영역에 결합하여 빠른 유비퀴틴화를 촉진한다. 따라서 페로토시스를 유도하기 위한 목적으로 MARH6의 FRR 영역과 NADPH 의 결합을 억제하는 물질을 MARH6 의 활성 또는 발현 저해제로 사용할 수 있다. 상기 MARH6 링 도메인은 인간 MARH6 (GenBank: AAI36462.1)를 기준으로 MARH6 N 말단의 1 내지 69 아미노산 영역일 수 있으며, 상기 MARH6의 FRR 도메인은 MARH6 의 상기 링 도메인과 결합하는 870-910 영역일 수 있다. FRR 은 RING 도메인에 결합하여 MARH6 의 효소적 RING 을 활성화시키므로, 이들의 결합을 억제하는 물질은 MARH6 활성 또는 발현 저해제로 사용할 수 있다. In addition, the activity or expression inhibitor of the present invention may inhibit the binding of MARH6 and NADPH or the binding of MARH6 ring domain and ferroptosis regulatory region (FRR) domain. The ferroptosis regulatory region (FRR) region of the C-terminal tail of MARH6 was identified for the first time, and NADPH binds to the FRR region to promote rapid ubiquitination. Therefore, for the purpose of inducing ferrotosis, a substance that inhibits the binding between the FRR region of MARH6 and NADPH may be used as an inhibitor of MARH6 activity or expression. The MARH6 ring domain may be 1 to 69 amino acids from the N-terminus of MARH6 based on human MARH6 (GenBank: AAI36462.1), and the FRR domain of MARH6 may be a region 870-910 that binds to the ring domain of MARH6. Since FRR activates enzymatic RING of MARH6 by binding to the RING domain, substances that inhibit their binding can be used as MARH6 activity or expression inhibitors.
본 발명의 MARH6 활성 또는 발현 저해제는 ACSL4, ALOX5, TFR1및 p53로 이루어진 군에서 선택된 1종 이상의 프로-페로토시스(pro-ferroptosis) 단백질의 분해를 억제; 또는 SLC7A11 (cystine/glutamate antiporter xCT), GPX4, 및 NRF2 로 이루어진 군에서 선택 1종 이상의 항-페로토시스 인자들의 발현을 하향 조절하는 것일 수 있다. The MARH6 activity or expression inhibitor of the present invention inhibits degradation of one or more pro-ferroptosis proteins selected from the group consisting of ACSL4, ALOX5, TFR1 and p53; Alternatively, it may down-regulate the expression of at least one anti-ferrotosis factor selected from the group consisting of SLC7A11 (cystine/glutamate antiporter xCT), GPX4, and NRF2.
본 발명에서는 MARH6 가 부재한 상황에서 ACSL4, ALOX5 및 TfR1과 squalene monooxygenase SM과 같은 주요 pro-ferroptosis 효과기들이 분해 속도가 감소되어 안정화되어 있음을 확인하였고, 항-페로토시스 단백질인 SLC7A11 (cystine/glutamate antiporter xCT), GPX4, 및 NRF2의 수준은 하향 조절되어 있는 것을 확인하였다. 따라서 MARH6 활성 또는 발현저해제는 pro-ferroptosis의 분해를 억제하거나, 항-페로토시스 인자들의 발현을 하향 조절하는 것일 수 있다. In the present invention, in the absence of MARH6, it was confirmed that major pro-ferroptosis effectors such as ACSL4, ALOX5, and TfR1 and squalene monooxygenase SM were stabilized by a reduced degradation rate, and anti-ferrotosis proteins SLC7A11 (cystine/glutamate antiporter xCT), GPX4, and NRF2 were found to be downregulated. Therefore, inhibitors of MARH6 activity or expression may inhibit the degradation of pro-ferroptosis or down-regulate the expression of anti-ferroptosis factors.
본 발명에 있어, 암은 페로토시스 유도에 의하여 사멸을 유도할 수 있는 암종을 모두 제한없이 포함할 수 있으나, 예컨대 유방암, 대장암, 직장암, 폐암, 결장암, 갑상선암, 구강암, 인두암, 후두암, 자궁경부암, 뇌암, 난소암, 방광암, 신장암, 간암, 췌장암, 전립선암, 피부암, 혀암, 자궁암, 위암, 골암, 및 혈액암으로 이루어진 군으로부터 선택된 어느 하나 이상인 것을 특징으로 할 수 있다. In the present invention, cancer may include all carcinomas capable of inducing death by ferrotosis induction without limitation, but may be, for example, at least one selected from the group consisting of breast cancer, colon cancer, rectal cancer, lung cancer, colon cancer, thyroid cancer, oral cancer, pharynx cancer, laryngeal cancer, cervical cancer, brain cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, uterine cancer, stomach cancer, bone cancer, and blood cancer. .
본 발명에 있어서, “예방(prevention)”이란 본 발명에 따른 조성물의 투여에 의해 암 등의 질환을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.In the present invention, “prevention” refers to any activity that suppresses or delays the onset of diseases such as cancer by administration of the composition according to the present invention.
본 발명에 있어서, “치료(treatment)“란 본 발명에 따른 조성물의 투여에 의해 암 등의 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다. In the present invention, "treatment" refers to all activities that improve or beneficially change symptoms such as cancer by administration of the composition according to the present invention.
본 발명에 있어서, “개체(individual)”란 본 발명의 조성물이 투여될 수 있는 대상을 말하며, 그 대상에는 제한이 없다.In the present invention, "individual" refers to a subject to which the composition of the present invention can be administered, and the subject is not limited.
본 발명에 있어, “약학적 조성물(pharmaceutical composition)”이란 캡슐, 정제, 과립, 주사제, 연고제, 분말 또는 음료 형태임을 특징으로 할 수 있으며, 상기 약학적 조성물은 인간을 대상으로 하는 것을 특징으로 할 수 있다. 상기 약학적 조성물은 이들로 한정되는 것은 아니지만, 각각 통상의 방법에 따라 산제, 과립제, 캡슐, 정제, 수성 현탁액 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 본 발명의 약학적 조성물은 약제적으로 허용가능한 담체를 포함할 수 있다. 약제학적으로 허용되는 담체는 경구투여시에는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등을 사용할 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등을 혼합하여 사용할 수 있으며, 국소투여용의 경우에는 기제, 부형제, 윤활제, 보존제 등을 사용할 수 있다. 본 발명의 약제학적 조성물의 제형은 상술한 바와 같은 약제학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구투여시에는 정제, 트로키, 캡슐, 엘릭서(elixir), 서스펜션, 시럽, 웨이퍼 등의 형태로 제조할 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수회 투약 형태로 제조할 수 있다. 기타, 용액, 현탁액, 정제, 캡슐, 서방형 제제 등으로 제형할 수 있다.In the present invention, "pharmaceutical composition" may be in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be for humans. The pharmaceutical compositions are not limited thereto, but may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories and sterile injection solutions, respectively, according to conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. For oral administration, pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc. for oral administration. The dosage form of the pharmaceutical composition of the present invention may be variously prepared by mixing with a pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in the form of unit dose ampoules or multiple doses. In addition, it may be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, and the like.
한편, 제제화에 적합한 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말디톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유 등이 사용될 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다.On the other hand, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, and propylhydroxybenzoate. Acetic acid, talc, magnesium stearate or mineral oil may be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like may be further included.
본 발명에 따른 약학적 조성물의 투여 경로는 이들로 한정되는 것은 아니지만 구강, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장이 포함된다. 경구 또는 비경구 투하가 바람직하다. 본원에 사용된 용어 "비경구"는 피하, 피내, 정맥내, 근육내, 관절내, 활액낭내, 흉골내, 경막내, 병소내 및 두개골내 주사 또는 주입기술을 포함한다. 본 발명의 약학적 조성물은 또한 직장 투여를 위한 좌제의 형태로 투여될 수 있다.Routes of administration of the pharmaceutical composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal. Oral or parenteral administration is preferred. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrabursal, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.
본 발명의 약학적 조성물은 사용된 특정 화합물의 활성, 연령, 체중, 일반적인 건강, 성별, 정식, 투여시간, 투여경로, 배출율, 약물 배합 및 예방 또는 치료될 특정 질환의 중증을 포함한 여러 요인에 따라 다양하게 변할 수 있고, 상기 약학적 조성물의 투여량은 환자의 상태, 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만 당업자에 의해 적절하게 선택될 수 있고, 1일 0.0001 내지 50 mg/kg 또는 0.001 내지 50 mg/kg으로 투여할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. 본 발명에 따른 의약 조성물은 환제, 당의정, 캡슐, 액제, 겔, 시럽, 슬러리, 현탁제로 제형화될 수 있다.The pharmaceutical composition of the present invention may vary depending on various factors including activity, age, weight, general health, sex, dosage, administration time, route of administration, excretion rate, drug formulation and severity of a specific disease to be prevented or treated, and the dosage of the pharmaceutical composition varies depending on the patient's condition, body weight, degree of disease, drug type, administration route and period, but can be appropriately selected by those skilled in the art, and can be appropriately selected from 0.0001 to 50 mg/kg or 0.0 per day. 01 to 50 mg/kg may be administered. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated into a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.
또한 본 발명은 MARCH6 활성 또는 발현 저해제를 포함하는 항암 보조제 조성물을 제공한다. In addition, the present invention provides an anticancer adjuvant composition comprising a MARCH6 activity or expression inhibitor.
항암 보조제 조성물이란, 다른 항암제와 함께 병용하여 투여되는 것을 목적으로 하는 조성물을 의미하며, 당 분야에 공지되어 있는 다양한 종류의 항암제와 MARCH6 활성 또는 발현 저해제를 함께 투여하여 항암제가 가진 효과를 극대화할 수 있는 것을 의미한다. 특히 본 발명의 MARCH6 활성 또는 발현 저해제를 페로토시스 유도 기전을 통한 암세포 사멸 효과를 목적으로 하는 항암제와 함께 투여하면, 페로토시스 민감도를 증진시킬 수 있고, 페로토시스 저항성 암에 본 발명의 MARCH6 활성 또는 발현 저해제를 투여하는 경우, 페로토시스에 대한 민감성이 개선될 수 있으므로, 보다 효과적인 항암 효과를 달성할 수 있다. The anticancer adjuvant composition refers to a composition intended to be administered in combination with other anticancer agents, and the effect of the anticancer agent is maximized by administering together various types of anticancer agents known in the art and a MARCH6 activity or expression inhibitor. In particular, when the MARCH6 activity or expression inhibitor of the present invention is administered together with an anticancer agent for the purpose of killing cancer cells through a ferrotosis induction mechanism, ferrotosis sensitivity can be enhanced, and when the MARCH6 activity or expression inhibitor of the present invention is administered to a ferrotosis-resistant cancer, since sensitivity to ferrotosis can be improved, a more effective anticancer effect can be achieved.
또한 본 발명은 MARCH6 활성 또는 발현 저해제를 포함하는 암 예방 또는 개선용 건강기능 식품 조성물에 관한 것이다. In addition, the present invention relates to a health functional food composition for preventing or improving cancer, including an inhibitor of MARCH6 activity or expression.
본 명세서에 있어서, '건강기능식품'이란 식품에 물리적, 생화학적, 생물공학적 수법 등을 이용하여 해당 식품의 기능을 특정 목적에 작용, 발현하도록 부가가치를 부여한 식품군이나 식품 조성이 갖는 생체방어리듬조절, 질병 방지와 회복 등에 관한 체내조절기능을 생체에 대하여 충분히 발현하도록 설계하여 가공한 식품으로, 장기적으로 복용하였을 때 인체에 무해하여야 한다.In the present specification, 'health functional food' refers to food designed and processed to sufficiently express the body's regulatory functions related to biological defense rhythm control, disease prevention and recovery, etc. to the living body of a food group or food composition that has added value to act or express the function of the food for a specific purpose using physical, biochemical, or bioengineering methods, etc., and must be harmless to the human body when taken for a long time.
상기 건강기능식품에는 식품학적으로 허용 가능한 식품 보조 첨가제를 포함할 수 있으며, 건강기능식품의 제조에 통상적으로 사용되는 적절한 담체, 부형제 및 희석제를 더욱 포함할 수 있다. 상기 건강기능식품 조성물을 식품 첨가물로 사용할 경우, 상기 조성물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조 시에 본 발명의 조성물은 원료에 대하여 15 중량% 이하, 바람직하게는 10 중량% 이하의 양으로 첨가된다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.The health functional food may include food additives acceptable in food science, and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of health functional foods. When the health functional food composition is used as a food additive, the composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when producing food or beverage, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, it may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.
상기 건강기능식품의 종류에 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 건강기능식품의 예로는 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 해당 식품의 기능을 특정 목적에 작용, 발현하도록 부가가치를 부여한 식품군이나 식품 조성이 갖는 생체방어리듬조절, 질병 방지와 회복 등에 관한 체내조절기능을 생체에 대하여 충분히 발현하도록 설계된 식품을 모두 포함한다. There is no particular limitation on the type of health functional food. Examples of health functional foods to which the substance can be added include dairy products including ice creams, various soups, beverages, teas, drinks, alcoholic beverages, and vitamin complexes, and include all foods designed to sufficiently express the body's regulatory functions related to biological defense rhythm control, disease prevention and recovery, etc. of food groups or food compositions with added value so as to act or express the function of the corresponding food for a specific purpose in the usual sense.
상기 외에 본 명세서 내 개시된 건강기능식품 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 포함할 수 있다. 그 밖에 본 발명의 식품 조성물은 천연 과일주스, 과일주스 음료 및 야채 음료의 제조를 위한 과육을 포함할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the health functional food composition disclosed herein may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. In addition, the food/composition of the present invention may include fruit flesh for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.
또한 본 발명은 1) 시료에 후보물질을 처리하는 단계; 2) 후보물질 처리 후 MARCH6 의 활성 또는 발현의 변화를 측정하는 단계; 및 3) 상기 2) 단계의 MARCH6 활성 또는 발현 수준이 감소되면, 상기 후보물질을 항암제로 판정하는 단계; 를 포함하는 페로토시스 유도 항암제의 스크리닝 방법을 제공한다. In addition, the present invention comprises the steps of 1) treating a candidate substance to a sample; 2) measuring the change in activity or expression of MARCH6 after treatment with the candidate substance; and 3) determining the candidate substance as an anticancer drug when the activity or expression level of MARCH6 in step 2) is reduced; It provides a screening method for ferrotosis-inducing anticancer agents comprising a.
또한 본 발명은 1) 시료에 후보물질을 처리하는 단계; 2) 후보물질 처리 후 MARCH6 의 활성 또는 발현의 변화를 측정하는 단계; 및 3) 상기 2) 단계의 MARCH6 활성 또는 발현 수준이 감소되면, 상기 후보물질을 항암제 보조제로 판정하는 단계; 를 포함하는 페로토시스 유도 항암제 보조제의 스크리닝 방법을 제공한다.In addition, the present invention comprises the steps of 1) treating a candidate substance to a sample; 2) measuring the change in activity or expression of MARCH6 after treatment with the candidate substance; and 3) determining the candidate substance as an anticancer drug adjuvant when the activity or expression level of MARCH6 in step 2) is reduced; It provides a screening method for a ferrotosis-inducing anti-cancer agent adjuvant comprising a.
상기 후보물질은 MARCH6 의 발현 또는 활성을 억제할 것으로 예상되는 미지의 물질로, 화합물, 단백질, 천연물, 추출물, 유전자 등을 제한없이 포함할 수 있다. The candidate substance is an unknown substance expected to inhibit the expression or activity of MARCH6, and may include compounds, proteins, natural products, extracts, genes, and the like without limitation.
상기 MARCH6 의 활성 또는 발현 변화의 측정은 당분야에 공지된 방법을 모두 제한없이 사용하여 측정할 수 있고 예컨대 중합효소반응(PCR), 역전사 중합효소반응(RT-PCR), 경쟁적 역전사 중합효소반응(Competitive RT-PCR), 실시간 역전사 중합효소반응(Realtime RT-PCR), RNase 보호분석법(RPA; RNase protection assay), 노던 블랏팅(northern blotting), 또는 DNA 마이크로어레이 분석법 등, 웨스턴 블랏팅(western blotting), ELISA(enzyme linked immunosorbent assay), 방사선면역분석법(Radioimmunoassay), 방사면역 확산법(Radioimmunodiffusion), 오우크테로니(Ouchterlony) 면역 확산법, 로케트(Rocket) 면역전기영동, 조직면역염색, 면역 침전분석법(immunoprecipitation assay), 보체 고정 분석법(complete fixation assay), 유세포분석법(Fluorescence Activated Cell Sorter, FACS) 또는 단백질 칩(protein chip) 분석법 등을 사용할 수 있으나, 이에 제한되지 않는다. The measurement of the activity or expression change of MARCH6 can be measured using any method known in the art without limitation, such as polymerase reaction (PCR), reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Realtime RT-PCR), RNase protection assay (RPA; RNase protection assay), northern blotting (northern blotting), or DNA microarray analysis Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion method, Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation complete (fixation assay), flow cytometry (F Luorescence Activated Cell Sorter (FACS) or protein chip analysis may be used, but is not limited thereto.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited by the following examples.
실시예 1. MARH6 넉아웃에 따른 지질 ROS 증가확인Example 1. Confirmation of lipid ROS increase according to MARH6 knockout
페로토시스와 MARH6의 관련성을 확인하기 위하여, HEK293T, HeLA, A549 또는 HCT116 세포를 이용한 실험을 수행하였다. 먼저 siRNA를 이용한 MARH6 넉다운 세포와 대조군에서 지질 과산화 센서 C11-BODIPY581/591 를 이용한 유세포 분석을 통해 지질 ROS 수준을 비교하였다. 또한 페로토시스 유도제 에라스틴(erastin, 시스틴/글루타메이트 항포터 억제제) 또는 RSL3(글루타티온 퍼옥시다제 4(GPX4) 억제제) 를 야생형 또는 MARH6 넉아웃 세포에 처리하고 지질 ROS 수준의 변화를 측정하였다. 그 결과를 도 1에 나타내었다. In order to confirm the relationship between ferrotosis and MARH6, experiments using HEK293T, HeLA, A549 or HCT116 cells were performed. First, lipid ROS levels were compared in MARH6 knockdown cells using siRNA and control cells through flow cytometry using the lipid peroxidation sensor C11-BODIPY 581/591 . In addition, wild-type or MARH6 knockout cells were treated with the ferrotosis inducer erastin (a cystine/glutamate antiporter inhibitor) or RSL3 (a glutathione peroxidase 4 (GPX4) inhibitor), and changes in lipid ROS levels were measured. The results are shown in Figure 1.
도 1에 나타낸 바와 같이, MARH6 넉아웃은 지질 ROS 수준을 증가시켰으며, 이는 야생형과 CRISPR/Cas9 시스템 기반 MARH6 KO HeLa, HEK293T, A549 또는 HCT116 암 세포주의 여러 쌍에서 일관된 결과를 나타내었다. 특히, 페로토시스 유도제 에라스틴(erastin, 시스틴/글루타메이트 항포터 억제제) 또는 RSL3(글루타티온 퍼옥시다제 4(GPX4) 억제제) 로 처리된 MARH6 KO 세포에서 지질 ROS 생산이 증가한 것으로 나타났다. As shown in Figure 1, MARH6 knockout increased lipid ROS levels, consistent with several pairs of wild-type and CRISPR/Cas9 system-based MARH6 KO HeLa, HEK293T, A549 or HCT116 cancer cell lines. In particular, lipid ROS production was shown to be increased in MARH6 KO cells treated with the ferrotosis inducer erastin (a cystine/glutamate antiporter inhibitor) or RSL3 (a glutathione peroxidase 4 (GPX4) inhibitor).
추가적으로 CRISPR/Cas9 시스템을 이용하여 제조된 MARH6 -/- 마우스를 이용하여, MARH6가 유기체 수준에서 지질 ROS 축적에 영향을 미치는지 확인하기 위한 실험을 수행하였다. MARH6 -/- 마우스는 성장 지연 및/또는 출생 후 높은 치사율을 보였으므로, 옥수수 오일(대조군) 또는 에라스틴 (체중 당 15mg/kg) 을 매일 복강 내로 7일 동안 임신한 쥐에 주사한 후 한배 새끼의 배아 간에서 지질 ROS 생성을 측정하였고, 그 결과를 도 2에 나타내었다. 지질 ROS의 생성은 지질 과산화의 부산물인 MDA (malondialdehyde)로 평가하였다. Additionally, using MARH6 -/- mice prepared using the CRISPR/Cas9 system, an experiment was performed to confirm whether MARH6 affects lipid ROS accumulation at the organism level. Since MARH6 -/- mice showed growth retardation and/or high postnatal mortality, corn oil (control) or elastin (15 mg/kg per body weight) was daily intraperitoneally injected into pregnant mice for 7 days, then lipid ROS production was measured in the embryonic liver of littermates, and the results are shown in Figure 2. The production of lipid ROS was evaluated by malondialdehyde (MDA), a by-product of lipid peroxidation.
도 2에 나타낸 바와 같이, 옥수수 오일 처리된 E18.5 Marh6 -/- 배아 간에서는 Marh6 +/+ 또는 Marh6+/- 배아의 간과 비교하여, 상향 조절된 지질 ROS 수준이 확인되었다. 인간 세포주에서와 마찬가지로 에라스틴 투여는 Marh6 -/- 배아 간의 지질 ROS 수준을 야생형이나 이형접합군 대비 현저하게 증가시켰다. As shown in Figure 2, upregulated lipid ROS levels were confirmed in the livers of corn oil-treated E18.5 Marh6 -/- embryos compared to livers of Marh6 +/+ or Marh 6 +/- embryos. As in the human cell line, administration of elastin to Marh6 -/- The level of lipid ROS between embryos was significantly increased compared to wild-type or heterozygous group.
실시예 2. MARH6 의 페로토시스 억제능 확인Example 2. Confirmation of ferrotosis inhibitory ability of MARH6
실시예 1을 통해 MARH6 결핍 인간세포주 및 마우스에서 지질 ROS 수준의 증가가 확인되었으므로, 야생형 및 MARH6 KO 세포에서 페로토시스 유도제에 대한 용량-의존적 반응성을 확인하였다. 야생형, MARH6 KO HeLa, HEK293T, A549, 또는 HCT116 세포의 상대적인 생존능을 에라스틴, RSL3 를 24시간 농도별로 처리한 후 확인하였으며, 그 결과를 도 3에 나타내었다. Since an increase in lipid ROS levels was confirmed in MARH6 deficient human cell lines and mice through Example 1, dose-dependent responsiveness to ferrotosis inducers was confirmed in wild-type and MARH6 KO cells. The relative viability of wild-type, MARH6 KO HeLa, HEK293T, A549, or HCT116 cells was confirmed after treatment with erastin and RSL3 for 24 hours at each concentration, and the results are shown in FIG. 3 .
도 3에 나타낸 바와 같이, 다양한 MARH6 KO 세포가 페로토시스 유도제인 에라스틴 또는 RSL3 처리에 대한 민감도가 증가되어, 같은 농도에서 더 많은 세포 사멸이 유도되는 것을 확인하였다. As shown in FIG. 3 , it was confirmed that the sensitivity of various MARH6 KO cells to treatment with elastin or RSL3, which is a ferrotosis inducer, was increased, and more apoptosis was induced at the same concentration.
한편 MARH6 KO 세포의 에라스틴 또는 RSL3에 대한 민감도가 촉매적으로 불활성인 MARH63f C9A 돌연변이체에 의해서 개선되지는 여부를 확인하기 위하여 24시간 동안 에라스틴(15 μM), RSL3(0.15 μM) 처리 하에서 공벡터, MARH63f, 및 MARH63f C9A를 발현시킨 MARH6 KO 세포의 생존능을 확인하였다. 그 결과를 도 4a 에 나타내었다. Meanwhile, in order to confirm whether the sensitivity of MARH6 KO cells to erastin or RSL3 is improved by the catalytically inactive MARH6 3f C9A mutant, the viability of MARH6 KO cells expressing the empty vector, MARH63f, and MARH6 3f C9A was examined under treatment with erastin (15 μM) and RSL3 (0.15 μM) for 24 hours. The results are shown in Figure 4a.
도 4a 에 나타낸 바와 같이, 에라스틴 또는 RSL3에 대한 민감도가 촉매적으로 불활성인 MARH63f C9A 돌연변이체에서는 개선되지 않았다. C-말단 삼중 flag-태그된 야생형 MARH63f의 이소성 발현과 달리 MARH63f C9A 돌연변이체에서 개선되지 않는다는 이러한 결과는 MARH6의 E3 Ub 리가아제 활성이 페로토시스 억제에 필요하다는 것을 보여주는 결과이다.As shown in Figure 4a, sensitivity to either erastin or RSL3 was not improved in the catalytically inactive MARH6 3f C9A mutant. Unlike the ectopic expression of the C-terminal triple flag-tagged wild-type MARH6 3f , this result does not improve in the MARH6 3f C9A mutant, indicating that the E3 Ub ligase activity of MARH6 is required for ferrotosis inhibition.
RSL3 0.15 μM과 함께 mock, DFO (100 μM), Fer-1 (5 μM), Z-VAD (20 μM), 또는 Nec-1 (40 μM) 를 MARH6 KO HeLa 세포에 24시간 동안 처리하고 세포 생존율을 확인하였다. 또한 RSL3 0.1 μM를 MARH6 KO HEK293T 세포에 처리하고 동일한 실험을 수행하여 세포 생존율을 확인하였다. 세포 생존율을 확인한 결과를 도 4b에 나타내었다. MARH6 KO HeLa cells were treated with mock, DFO (100 μM), Fer-1 (5 μM), Z-VAD (20 μM), or Nec-1 (40 μM) together with 0.15 μM of RSL3 for 24 hours, and cell viability was confirmed. In addition, MARH6 KO HEK293T cells were treated with 0.1 μM of RSL3, and cell viability was confirmed by performing the same experiment. The result of confirming the cell viability is shown in FIG. 4B.
도 4b에 나타낸 바와 같이, 페로토시스 유도제 RSL3에 대한 MARH6 KO 세포의 생존율은 DFO (deferoxamine) 또는 Fer-1 (ferrostatin-1) 에 의하여 상당히 회복되었으나, 카스파아제 억제제인 Z-VAD-FMK 에 의해서는 거의 회복되지 않았고, 네크롭토시스 억제제인 네로스타틴-1(Nec-1)에 의해서도 거의 회복되지 않거나 일부만 낮은 정도로 회복되었다. 이러한 결과는 MARH6가 아폽토시스나 네크롭토시스와 같은 다른 세포 사멸 형태가 아니라, 특히 페로토시스 조절과 관련이 있음을 나타내는 결과이다. As shown in Figure 4b, the viability of MARH6 KO cells against the ferrotosis inducer RSL3 was significantly recovered by deferoxamine (DFO) or ferrostatin-1 (Fer-1), but hardly recovered by the caspase inhibitor Z-VAD-FMK, and was hardly or only partially recovered by the necroptosis inhibitor nerostatin-1 (Nec-1). These results indicate that MARH6 is not related to other forms of cell death such as apoptosis or necroptosis, but is particularly related to ferrotosis regulation.
실시예 3. 페로토시스 유도제에 의한 MARH6 기질의 안정화Example 3. Stabilization of MARH6 substrate by ferrotosis inducer
페로토시스 유도제가 MARH6 기질의 대사 턴오버에 영향을 미치는지 여부를 확인하기 위하여 단백질 분해의 시클로헥시미드(CHX)-추적 분석을 수행하였다. mock, 에라스틴 (5 μM), 또는 RLS3 (0.15 μM) 를 처리하고 HeLa 세포에서 SM 의 CHX 추적분석을 24시간 동안 수행하였고, 그 결과를 도 5에 나타내었다. A cycloheximide (CHX)-tracking assay of proteolysis was performed to determine whether ferrotosis inducers affect the metabolic turnover of MARH6 substrates. After treatment with mock, erastin (5 μM), or RLS3 (0.15 μM), CHX tracking analysis of SM in HeLa cells was performed for 24 hours, and the results are shown in FIG. 5 .
도 5에 나타낸 바와 같이, HeLa 세포에서 에라스틴 또는 RSL3 처리는 degron-bearing MARH6 기질인 내인성 SM 을 강하게 안정화시켰다. As shown in Fig. 5, treatment with erastin or RSL3 in HeLa cells strongly stabilized endogenous SM, a degron-bearing MARH6 substrate.
실시예 4. MARH6의 NADPH 대사 조절 확인 Example 4. Confirmation of NADPH metabolism regulation of MARH6
MARH6의 페로토시스 조절 메커니즘을 확인하기 위해 야생형 및 MARH6 KO HeLa 세포의 전체 게놈 RNA 시퀀싱 분석을 수행하였다. WikiPathways enrichment 를 이용하여 야생형과 MARH6 KO HeLa 세포에서 DEG(differentially expressed genes) 를 확인하고 이들의 기능을 분석하였으며, MARH6 KO HeLa 세포에서 하향 조절된 유전자들 중 우선 순위에 있는 유전자들을 확인하였고, 그 결과를 도 6 및 도 7에 나타내었다. Whole genome RNA sequencing analysis of wild-type and MARH6 KO HeLa cells was performed to confirm the mechanism of MARH6 regulating fertility. Using WikiPathways enrichment, differentially expressed genes (DEGs) were identified in wild-type and MARH6 KO HeLa cells and their functions were analyzed, and genes in priority among downregulated genes in MARH6 KO HeLa cells were identified. The results are shown in FIGS. 6 and 7 .
도 6에 나타낸 바와 같이, MARH6가 NAD(P), 스테롤, 비타민 대사, 조절된 괴사, 염증 등과 관련이 있음을 확인하였고, 특히 NAD(P) 생산과의 관련성에 대한 중요성을 시사하였다. 도 7에 나타낸 바와 같이, 야생형과 MARH6 KO HeLa 세포에서 NADP(H) 또는NADPH의 상대적 양을 확인한 결과 MARH6 KO HeLa 세포에서 해당 대사 산물들이 유의하게 하향 조절되어 있음을 확인하였다. As shown in FIG. 6 , it was confirmed that MARH6 is related to NAD(P), sterols, vitamin metabolism, regulated necrosis, inflammation, etc., suggesting the importance of its relationship with NAD(P) production in particular. As shown in Figure 7, as a result of confirming the relative amount of NADP(H) or NADPH in wild-type and MARH6 KO HeLa cells, it was confirmed that the corresponding metabolites were significantly down-regulated in MARH6 KO HeLa cells.
실시예 5. MARH6 기질 분해에 대한 페로토시스 조절 영역 (FRR) EXAMPLE 5. MARH6 Ferrotosis Regulatory Region (FRR) for Substrate Degradation
MARH6의 구조 및 인간을 포함하는 다양한 생물종에서 MARH6 FRR 의 시퀀스 얼라인먼트를 도 8에 나타내었다. The structure of MARH6 and the sequence alignment of MARH6 FRR in various species including humans are shown in FIG. 8 .
도 8에 나타낸 바와 같이, MARH6는 N 말단에는 E3 활성을 담당하는 링도메인이 존재하고 C-말단에는 페로토시스 조절에 중요한 FRR 이 위치한다. 인간 MARH6는 고도로 보존된 CTE(잔기 877~892) 및 상대적으로 덜 보존된 C-말단 꼬리를 갖는 페로토시스 조절 영역 (Ferroptosis regulatory region, FRR)을 포함한다. As shown in FIG. 8, MARH6 has a ring domain responsible for E3 activity at the N-terminus and FRR, which is important for ferrotosis regulation, is located at the C-terminus. Human MARH6 contains a highly conserved CTE (residues 877-892) and a Ferroptosis regulatory region (FRR) with a relatively less conserved C-terminal tail.
MARH6 매개 페로토시스 억제에서 FRR의 역할을 조사하기 위해 MARH6 넉아웃 HeLa 세포에 전장 MARH63f 1-910, FRR 결실 (MARH63f 1-869) 및 MARH63f 1-900(마지막 10개 C-말단 잔기 결실)를 벡터를 통해 발현시키고, 페로토시스 유도제인 에라스틴 15 μM 또는 RSL3 0.15 μM를 처리하여 생존율을 확인한 결과를 도 9에 나타내었다. 또한 MARH6 KO HeLa 세포에서 전장 및 MARH63f 1-900 의 CHX 추적 분석을 수행하였고, 이들을 정량화하여 분해속도를 확인하고 그 결과를 도 10에 나타내었다. To investigate the role of FRR in MARH6-mediated fertility inhibition, the full-length MARH6 3f 1-910, FRR deletion (MARH6 3f 1-869), and MARH6 3f 1-900 (deletion of the last 10 C-terminal residues) were expressed through vectors in MARH6 knockout HeLa cells, and 15 μM of ferrotosis inducer elastin or 0.15 μM of RSL3 were used. The results of confirming the survival rate by treatment are shown in FIG. In addition, CHX tracking analysis of full-length and MARH6 3f 1-900 was performed in MARH6 KO HeLa cells, and the degradation rate was confirmed by quantifying them, and the results are shown in FIG. 10 .
도 9에 나타낸 바와 같이, 전장인 MARH63f 1-910의 과발현은 에라스틴 또는 RSL3으로 처리된 MARH6 KO HeLa 세포의 생존율을 실질적으로 증가시킨 반면, MARH63f 1-869의 경우 세포 생존율에 영향을 주지 않는 것을 확인하였다. 한편 예상외로 MARH63f 1-900의 과발현은 전장인 MARH63f 1-910보다 MARH6 KO HeLa 세포에서 페로토시스 유도제에 의해 유발된 세포 사멸을 더 효율적으로 억제하는 것을 확인하였다. As shown in FIG. 9 , overexpression of full-length MARH6 3f 1-910 substantially increased the viability of MARH6 KO HeLa cells treated with erastin or RSL3, whereas it was confirmed that MARH6 3f 1-869 did not affect cell viability. On the other hand, it was unexpectedly confirmed that the overexpression of MARH6 3f 1-900 more efficiently inhibited cell death induced by ferrotosis inducers in MARH6 KO HeLa cells than the full-length MARH6 3f 1-910.
또한, 도 10에 나타낸 바와 같이 MARH63f 1-900은 MARH63f 1-910보다 훨씬 빠르게 분해되었다. 이러한 결과는 C-말단 10개 잔기가 MARH6 활성을 억제함으로써 잠재적으로 페로토시스 촉진을 매개함을 보여주는 결과이다. Also, as shown in FIG. 10, MARH6 3f 1-900 was decomposed much faster than MARH6 3f 1-910. These results show that the C-terminal 10 residues potentially mediate ferrotosis promotion by inhibiting MARH6 activity.
추가적으로 GST-pulldown 어세이를 수행하여 MARH6 활성 조절의 메커니즘을 분석하였다. GST, GST-MARH6870-910, GST-MARH6870-900, GST-MARH6870-891, 또는 GST-MARH6892-910 (각 30 μg)와 RINGha (2 μg) 를 이용하여 GST pulldown 어세이를 수행하였고, MARH6 의 특이적인 상호작용을 확인하였으며, 그 결과를 도 11에 나타내었다. Additionally, a GST-pulldown assay was performed to analyze the mechanism of MARH6 activity regulation. A GST pulldown assay was performed using GST, GST-MARH6 870-910 , GST-MARH6 870-900 , GST-MARH6 870-891 , or GST-MARH6 892-910 (30 μg each) and RINGha (2 μg), and the specific interaction of MARH6 was confirmed. The results are shown in FIG. 11 .
도 11에 나타낸 바와 같이, N 말단 링 함유 단편 (잔기1-91) 이 C 말단 FRR(잔기 870-910)과 특이적으로 상호작용을 하는 것을 확인하였고, C-말단 ha-표지된 RINGha(MARH6 ha 1-71)이 GST-MARH6 870-910, GST-MARH6 870-900 및 GST에 직접 결합하나, GST 및 GST-MARH6 892-910 에는 결합하지 않는 것을 확인하였으며, 이를 통해 RING 결합 영역으로 FRR 내 870-891 잔기를 도출하였다. 즉, 링도메인이 FRR 도메인과 직접적으로 결합하는 것을 확인하였으며, FRR 도메인 (MARH6 870-910) 내에서도 특히 MARH6 970-891 부분이 링 도메인과의 결합에 결정적으로 작용함을 확인하였다. As shown in FIG. 11, it was confirmed that the N-terminal ring-containing fragment (residues 1-91) interacts specifically with the C-terminal FRR (residues 870-910), and the C-terminal ha -labeled RING ha (MARH6 ha 1-71) binds directly to GST-MARH6 870-910, GST-MARH6 870-900 and GST, but GST and GST-MARH6 89 It was confirmed that it did not bind to 2-910, and through this, residues 870-891 in FRR were derived as a RING binding region. That is, it was confirmed that the ring domain directly binds to the FRR domain, and even within the FRR domain (MARH6 870-910), it was confirmed that, in particular, the MARH6 970-891 portion plays a decisive role in binding to the ring domain.
GST-MARH6870-900 및 GST-MARH6870-891 의 RINGha 에 대한 결합은 GST-MARH6 870-910에 대한 결합과 비교하여 뚜렷하게 강한 결합을 나타내었고, 마지막 C 말단 10개 잔기 스트레치의 강화는 FRR-RING 상호작용을 억제하였다. 이러한 결과는 앞서 확인한 MARH63f 1-900 의 빠른 분해와 일치하는 결과이다. 그러므로 매우 진화적으로 보전된 (특히 척추동물에서) RING-결합/CTE-절편(잔기 870-891) 함유 및 C 말단 10잔기 스트레치 (잔기 901-910)들은, 여기에서 FerR( Fer roptosis R epression)) 도메인 및 FerS ( Fer roptosis S timulation) 도메인을 각각 지칭하여, 이들의 MARH6 활성화를 통한 페로토시스 조절에서의 rheostatic 역할을 반영하였다. 링 도메인과 FRR 도메인의 상호작용을 확인하기 위하여 RINGha-FRR (0.5 μM) 및 Ub, Lys을 포함하지 않는 Ub 돌연변이인인 K0-Ub (25 μM), 단일 Lys을 포함하는 Ub 돌연변이인 K6, K11, K27, K29, K33, K48, K63-Ub를 이용한 유비퀴틴화 어세이 및 항-Ub 면역블랏을 수행하였다. 그 결과를 도 12에 나타내었다. The binding of GST-MARH6 870-900 and GST-MARH6 870-891 to RING ha showed distinctly stronger binding compared to that of GST-MARH6 870-910 , and enhancement of the last C-terminal 10-residue stretch inhibited the FRR-RING interaction. These results are consistent with the rapid degradation of MARH6 3f 1-900 previously identified. Therefore, the highly evolutionarily conserved (particularly in vertebrates) RING-binding/CTE-segment (residues 870-891) containing and C-terminal 10-residue stretches (residues 901-910), here referred to as the Ferroptosis Repression (FerR) domain and the Ferroptosis Stimulation ( FerS ) domain, respectively, reflect their rheostatic role in ferrotosis regulation through MARH6 activation. In order to confirm the interaction between the ring domain and the FRR domain, RINGha-FRR (0.5 μM) and K0-Ub (25 μM), a Ub mutant that does not contain Ub and Lys, and Ub mutants K6, K11, K27, K29, K33, K48, K63-Ub were used for ubiquitination assays and anti-Ub immunoblots. The results are shown in FIG. 12 .
도 12의 a에 나타낸 바와 같이, 정제된 선형의 RINGha-FRR 융합체는 긴 폴리-Ub 체인 형성을 His6-UBE2J2ΔTM 및/또는 His6-UBE2G2 존재 하에서 강하게 촉진한 반면, RINGha는 오직 짧은 Ub 체인만을 생산하였다. 이를 통해 링도메인과 FRR 도메인의 상호작용이 링 도메인의 활성을 높이는데 기여하는 것을 확인하였다. FRR 이 MARH6 의 효소적 RING을 활성화시키고, 이를 통해 E3 리가아제가 Ub-charged E2로부터 free Ub, 단백질에 부착되지 않은 Ub 체인 또는 단백질에 부착된 Ub 체인으로 Ub를 전달하는 것을 촉진시킬 수 있으며 도 12b 에 나타낸 바와 같이, 체인의 종류는 대부분 K48 연결로 이루어져 있음을 확인하였다. As shown in Fig. 12a, the purified linear RING ha -FRR fusions strongly promoted the formation of long poly-Ub chains in the presence of His6-UBE2J2 ΔTM and/or His6-UBE2G2, whereas RING ha produced only short Ub chains. Through this, it was confirmed that the interaction between the ring domain and the FRR domain contributes to increasing the activity of the ring domain. FRR activates the enzymatic RING of MARH6, and through this, E3 ligase can promote the transfer of Ub from Ub-charged E2 to free Ub, Ub chains not attached to proteins, or Ub chains attached to proteins.
실시예 6. NADPH 의 FRR에 대한 직접적인 결합Example 6. Direct binding of NADPH to FRR
FRR이 NADPH 와 직접적으로 상호작용하는지 여부를 확인하기 위하여, 전장 MARH6 3f 1-910 또는 FRR-lacking MARH63f 1-869 를 발현하는 HeLa 세포 추출물을 이용한 2',5'-adenosine diphosphate (ADP)-agarose 친화도 크로마토그래피를 수행하였고, 그 결과를 도 13에 나타내었다. 안정한 NADP(H) analog 2',5'-ADP 는 NADPH-결합 단백질을 확인하는데 일반적으로 사용된다. To confirm whether FRR directly interacts with NADPH, 2',5'-adenosine diphosphate (ADP)-agarose affinity chromatography was performed using HeLa cell extracts expressing full-length MARH6 3f 1-910 or FRR-lacking MARH6 3f 1-869, and the results are shown in FIG. 13 . The stable NADP(H) analog 2',5'-ADP is commonly used to identify NADPH-binding proteins.
도 13에 나타낸 바와 같이, 2',5'-ADP-agarose 친화도 크로마토그래피는 2',5'-ADP가 MARH63f 1-910에는 결합하지만, MARH63f 1-869에는 결합하지 않는 것을 나타내었다. 이러한 결과는 FRR이 MARH6 활성 조절을 위한 NADP(H)의 결합 부위일 가능성을 나타낸다. 또한 정제된 RINGha-FRR또는 GST-FRR 융합체가 직접적으로 2',5'-ADP-agarose에 결합한 반면, RINGha 또는 GST 단독은 이에 결합하지 않았다. 특히 GST-FRR의 2',5'-ADP 에 대한 결합은 1mM NADPH의 경쟁적인 처리에 의하여 사라졌지만, 동일 농도의 NADP+ 처리에서는 부분적으로 유지되었다. 이러한 결과는 FRR 이 NADP+ 보다 NADPH에 대한 결합을 더 선호하는 것을 보여주는 결과이다. As shown in FIG. 13, 2',5'-ADP-agarose affinity chromatography showed that 2',5'-ADP binds to MARH6 3f 1-910, but not to MARH6 3f 1-869. These results indicate the possibility that FRR is a binding site for NADP(H) for regulating MARH6 activity. In addition, the purified RING ha -FRR or GST-FRR fusion directly bound to 2',5'-ADP-agarose, whereas RING ha or GST alone did not bind to it. In particular, the binding of GST-FRR to 2',5'-ADP was abolished by competitive treatment with 1 mM NADPH, but was partially maintained by treatment with the same concentration of NADP+. These results show that FRR prefers binding to NADPH rather than NADP+.
실시예 7. NADPH 의 FRR-RING 상호작용 및 연속적인 MARH6 활성 증가 효과 확인Example 7. FRR-RING interaction of NADPH and confirmation of continuous MARH6 activity increasing effect
NADPH를 인식하는 FRR 영역을 매핑하기 위해 다음과 같은 정제된 GST-FRR 유도체를 사용하여 2',5'-ADP-agarose 친화도 크로마토그래피를 추가로 수행하였다: GST-MARH6 870-910, GST-MARH6 870-900, GST- MARH6 870-891 및 GST-MARH6 892-910. 또한 FRR-RINGha 상호작용에 미치는 NADPH 및 NADH 의 영향을 확인하기 위하여, mock, NADPH, 또는 NADH (각 0.1 mM)과 RINGha (2 μg) 의 존재하에서 GST 또는 GST-FRR (각 40 μg) 의 GST 풀다운 어세이를 수행하였다. To map the FRR region recognizing NADPH, 2',5'-ADP-agarose affinity chromatography was further performed using the following purified GST-FRR derivatives: GST-MARH6 870-910, GST-MARH6 870-900, GST-MARH6 870-891 and GST-MARH6 892-910. In addition, to confirm the effect of NADPH and NADH on the FRR-RING ha interaction, a GST pull-down assay of GST or GST-FRR (40 μg each) was performed in the presence of mock, NADPH, or NADH (each 0.1 mM) and RINGha (2 μg).
도 14A에 나타낸 바와 같이, 2',5'-ADP는 GST-MARH6 870-910 및 GST-MARH6 870-900에 결합하지만 GST-MARH6 870-891 및 GST-MARH6 892-910에는 결합하지 않음을 확인하였다. 이는 FRR 도메인 (MARH6 870-900) 서열이 NADPH와의 결합을 담당하는 것을 나타낸다. 이를 통해 NADPH와 RING은 FRR에 대해 중첩 결합하는 것을 확인하였다. 또한 도 14B 에 나타낸 바와 같이, GST 풀다운 분석을 통해 NADPH가 있는 경우에는 FRR-RINGha 상호작용이 상당히 증가했지만 NADH에서는 그렇지 않은 것을 확인하였다. As shown in FIG. 14A, it was confirmed that 2',5'-ADP binds to GST-MARH6 870-910 and GST-MARH6 870-900, but does not bind to GST-MARH6 870-891 and GST-MARH6 892-910. This indicates that the FRR domain (MARH6 870-900) sequence is responsible for binding to NADPH. Through this, it was confirmed that NADPH and RING overlap with FRR. In addition, as shown in Figure 14B, it was confirmed through GST pull-down analysis that the FRR-RING ha interaction significantly increased in the presence of NADPH, but not in NADH.
도 14C 의 시험관내 유비퀴틴화 분석 결과 역시, MARH63f-His6UBE2J2DTM/His6UBE2G2 매개 폴리유비퀴틸화가 NADPH의 존재에 의해 현저하게 향상되었지만 NADH의 존재에 의해 향상되지 않았다는 결과를 나타내었다. 이러한 결과는 NADPH 의 존재가 MARH6 의 활성을 높일 수 있음을 보여주는 결과이다. The in vitro ubiquitination assay in FIG. 14C also showed that MARH63f-His6UBE2J2DTM/His6UBE2G2 mediated polyubiquitylation was significantly enhanced by the presence of NADPH but not by the presence of NADH. These results show that the presence of NADPH can increase the activity of MARH6.
실시예 8. MARH6의 페로토시스 효과기 조절 확인 Example 8. Confirmation of ferrotosis effector regulation of MARH6
MARH6이 세포 사멸에 대응하기 위해 특정 페로토시스 조절자의 metabolic turnover를 조절하는지에 대한 실험을 수행하였다. 야생형 및 MARH6 KO HeLa 또는 A549 세포에서 이들의 분해 패턴을 모니터링하여 이전에 알려진 페로토시스 관련 단백질의 하위 집합을 조사하였다. 보다 구체적으로 MARH6가 페로토시스 조절 단백질인 ACSL4(acyl-CoA synthetase long-chain family member 4), ALOX5(arachidonate 5- lipoxygenase 5) 및 TfR1(transferrin receptor 1), p53 분해에 관여하는지 확인하기 위해 CHX 추적 어세이를 수행하였다. MARH6 유전자가 부재한 상황에서 ACSL4, ALOX5, TFR1, p53 단백질의 분해 속도를 함께 확인하고, 그 결과를 도 15 및 도 16에 나타내었다. An experiment was performed to see whether MARH6 regulates the metabolic turnover of specific ferrotosis regulators in response to apoptosis. A subset of previously known ferrotosis-related proteins was investigated by monitoring their degradation patterns in wild-type and MARH6 KO HeLa or A549 cells. More specifically, a CHX tracking assay was performed to confirm that MARH6 is involved in the degradation of acyl-CoA synthetase long-chain family member 4 (ACSL4), arachidonate 5-lipoxygenase 5 (ALOX5), transferrin receptor 1 (TfR1), and p53, which are proteins that regulate fertility. In the absence of the MARH6 gene, the degradation rates of ACSL4, ALOX5, TFR1, and p53 proteins were also confirmed, and the results are shown in FIGS. 15 and 16 .
도 15에 나타낸 바와 같이, ACSL4, ALOX5 및 TfR1 같은 주요 pro-ferroptosis 효과기들이 MARH6 KO 과 같은 MARH6의 부재하에서는 현저하게 또는 식별가능할 정도로 안정화되어 단백질 분해 속도가 현저하게 감소하는 것을 확인하였다. As shown in FIG. 15, major pro-ferroptosis effectors, such as ACSL4, ALOX5, and TfR1, were significantly or identifiablely stabilized in the absence of MARH6, such as MARH6 KO, and it was confirmed that the rate of protein degradation was remarkably reduced.
또한 도 16에 나타낸 바와 같이, MARH6 결실은 pro-ferroptosis 단백질을 효과적으로 안정화시켰으며, 현저하거나 검출가능할 정도로 핵심 항-페로토시스 단백질인 SLC7A11 (cystine/glutamate antiporter xCT), GPX4, 및 NRF2의 수준을 하향 조절한다는 것을 또한 확인하였다. 이들 중에서 SLC7A11 하향 조절이 가장 현저하게 나타났다.In addition, as shown in FIG. 16, MARH6 deletion effectively stabilized the pro-ferroptosis protein and markedly or detectably downregulated the levels of key anti-ferrotosis proteins, SLC7A11 (cystine/glutamate antiporter xCT), GPX4, and NRF2. It was also confirmed. Among them, SLC7A11 downregulation was most prominent.
상기와 같은 결과를 통해, 페로토시스 유도자가 NADPH 결핍 및 연속적인 MARH6 불활성화를 통해 pro-페로토시스 단백질을 안정화시킬 수 있다는 가능성을 확인하였다. Through the above results, it was confirmed that the ferrotosis inducer can stabilize the pro-ferrotosis protein through NADPH depletion and continuous MARH6 inactivation.
생리학적 조건 하에서 MARH6의 페로토시스 인자 조절 효과를 확인하기 위하여, 에라스틴을 처리하거나 하지 않은 모체로부터 분리한 Marh6-/- 배아 및 야생형의 간 조직에서 p53, SLC7A11, SM 및 4-HNE(lipid peroxidation marker 4-hydroxynonenal) 의 수준을 측정하였다. 또한, 야생형 및 MARH6 KO A549 세포 유래 이종이식 종양에서도 동일 인자들의 발현양 변화를 확인하였으며, 그 결과를 도 17에 나타내었다. To confirm the effect of MARH6 on regulating ferrotosis factors under physiological conditions, the levels of p53, SLC7A11, SM and 4-HNE (lipid peroxidation marker 4-hydroxynonenal) were measured in liver tissues of Marh6 −/− embryos and wild-type cells isolated from mothers treated with or without elastin. In addition, changes in the expression level of the same factors were confirmed in wild-type and MARH6 KO A549 cell-derived xenograft tumors, and the results are shown in FIG. 17 .
도 17에 나타낸 바와 같이, pro- 페로토시스 인자인 p53, SM 및 4-HNE 수준은 Marh6-/- 마우스에서 야생형과 비교하여 증가한 반면, SLC7A11 수준은 크게 감소하였다. 이러한 결과는 야생형 및 MARH6 KO A549 세포 유래 이종이식 종양에서도 유사하게 나타났다. As shown in FIG. 17, the levels of pro- ferrotesis factors p53, SM and 4-HNE were increased in Marh6 -/- mice compared to the wild type, whereas the level of SLC7A11 was greatly decreased. These results were similar in wild-type and MARH6 KO A549 cell-derived xenograft tumors.
상기와 같은 결과를 종합하면, MARH6가 NADPH 를 감지하여 페로토시스 조절에 관여하는 단백질이고, NADPH 는 MARH6의 FRR 도메인에 직접적으로 결합하여 링 도메인의 활성을 높여주고, MARH6가 pro-ferroptosis 단백질인 ACSL4, ALOX5, TFR1, p53 를 분해하여 페로토시스를 억제할 수 있음을 확인하였다. Summarizing the above results, it was confirmed that MARH6 is a protein involved in ferrotosis regulation by sensing NADPH, NADPH directly binds to the FRR domain of MARH6 to increase the activity of the ring domain, and MARH6 inhibits ferrotosis by degrading pro-ferroptosis proteins ACSL4, ALOX5, TFR1, and p53.
실시예 9. MARH6의 페로토시스 조절을 통한 항암 활성 확인 Example 9. Confirmation of anticancer activity through ferrotosis regulation of MARH6
MARH6가 페로토시스 조절인자가 될 수 있음을 확인하였으므로, MARH6가 암세포에서의 페로토시스 조절을 통해 종양의 성장을 조절할 수 있는지 확인하기 위한 실험을 수행하였다. Since it was confirmed that MARH6 can be a ferrotosis regulator, an experiment was performed to determine whether MARH6 can regulate tumor growth through ferrotosis control in cancer cells.
먼저 MARH6의 제거가 종양의 형성에 영향을 미치는지 확인하기 위하여 야생형 및 MARH6 KO A549 폐암세포를 이용한 이종이식 실험을 수행하였다. 이종 이식 초반에는 Fer-1 을 주사하여 페로토시스에 의한 영향을 최소화하였고, 이후에는 Fer-1 를 중단하여 페로토시스에 의한 영향을 확인하였다. 매 4일 간격으로 종양의 부피를 측정하였고, 야생형 암과 MARH6 유전자가 제거된 암의 생장 속도를 확인한 결과를 도 18에 나타내었다. First, xenograft experiments using wild-type and MARH6 KO A549 lung cancer cells were performed to confirm whether the removal of MARH6 had an effect on tumor formation. At the beginning of the xenotransplantation, Fer-1 was injected to minimize the effect of ferrotosis, and then Fer-1 was discontinued to confirm the effect of ferrotosis. The volume of the tumor was measured every 4 days, and the results of confirming the growth rates of the wild-type cancer and the cancer from which the MARH6 gene was removed are shown in FIG. 18 .
도 18에 나타낸 바와 같이, 이종이식실험 결과는 종양의 크기가 야생형 세포와 비교하여 MARH6 KO 세포에서 현저하게 감소하였음을 나타내었다. 도 18B 에서 확인할 수 있는 바와 같이, Fer-1 이 주사된 40일 시점까지는 종양 크기의 차이가 없었으나, 페로토시스 억제제인 Fer-1 의 주사를 중단한 40일 시점부터 MARH6 KO A549 세포를 이식한 마우스에서는 종양의 크기가 현저하게 저해되었음을 확인하였다. As shown in Figure 18, the xenograft test results showed that the tumor size was significantly reduced in MARH6 KO cells compared to wild-type cells. As can be seen in FIG. 18B, there was no difference in tumor size until 40 days after Fer-1 was injected. However, it was confirmed that tumor size was significantly inhibited in mice transplanted with MARH6 KO A549 cells from 40 days after injection of Fer-1, a ferrotosis inhibitor, was stopped.
이러한 결과는 암세포의 MARH6 억제를 통해, 암세포의 페로토시스를 유도하여 암의 생장 속도를 억제할 수 있음을 보여주는 결과이다. These results show that the growth rate of cancer can be inhibited by inducing ferrotosis of cancer cells through inhibition of MARH6 in cancer cells.
실시예 10. MARH6의 페로토시스 조절에 따른 생존률 확인 Example 10. Confirmation of survival rate according to ferrotosis regulation of MARH6
MARH6-/- 마우스는 배아 발달 단계에 페로토시스를 겪고, 성장 지연 및/또는 출생 후 높은 치사율을 보이는 것을 확인하였다. 한편 친유성 항산화제인 비타민 E 를 식이 첨가하면 Gpx4-/- 마우스에서 페로토시스 표현형이 억제됨이 알려져 있다. It was confirmed that MARH6 -/- mice undergo ferrotosis during embryonic development, exhibit growth retardation and/or high mortality after birth. On the other hand, it is known that the addition of vitamin E, a lipophilic antioxidant, to the diet inhibits the fertosis phenotype in Gpx4 -/- mice.
본 발명의 MARH6-/- 에 의하여 페로토시스가 유도된 마우스에서도 비타민 E 를 식이에 첨가하면 페로토시스 표현형이 억제되고 생존율이 증가될 수 있는지 확인하였다. MARH6+/- 와 MARH6+/- 를 교배하고 임신중인 모체에 19일차까지 정상 수준 (45mg/kg food) 및 고용량 (567mg/kg food) 비타민 E 식이를 공급하였다. 그 후 야생형과 MARH6-/- 마우스 및 야생형 마우스의 생존율을 확인하였다. 실험 모식도 및 생존율 측정 결과를 도 19에 나타내었다. In mice whose ferrotosis was induced by MARH6 -/- of the present invention, it was confirmed whether the ferrotosis phenotype could be suppressed and the survival rate increased if vitamin E was added to the diet. MARH6 +/- and MARH6 +/- were mated, and pregnant mothers were fed normal (45 mg/kg food) and high-dose (567 mg/kg food) vitamin E diets until the 19th day. Then, the survival rates of wild-type and MARH6 -/- mice and wild-type mice were confirmed. A schematic diagram of the experiment and the results of measuring the survival rate are shown in FIG. 19 .
도 19에 나타낸 바와 같이, 비타민 E를 풍부하게 공급한 모체에서는 MARH6 유전자를 가지고 있지 않은 새끼 쥐가 태어날 수 있음 (12%) 을 확인하였다. 반면 정상 수준의 비타민 E 를 공급한 모체에서는 페로토시스 스트레스로 인하여 MARH6-/- 유전자형의 새끼가 거의 태어나지 못함을 확인하였다. 이러한 결과는 MARH6 가 배아의 발달 및 출생 과정에서 페로토시스 조절에 중요한 역할을 할 수 있음을 나타낸다. As shown in FIG. 19, it was confirmed that baby mice without the MARH6 gene could be born (12%) in mothers who were abundantly supplied with vitamin E. On the other hand, it was confirmed that in mothers supplied with normal levels of vitamin E, offspring of the MARH6 -/- genotype were hardly born due to ferrotosis stress. These results indicate that MARH6 may play an important role in regulating fertility during embryonic development and birth.
Claims (9)
A pharmaceutical composition for preventing or treating cancer comprising an inhibitor of MARCH6 activity or expression.
The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the MARH6 activity or expression inhibitor is at least one selected from the group consisting of an antisense nucleotide, a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a ribozyme, a compound that specifically binds to the MARH6 protein, a peptide, a peptide mimetic, a substrate analog, an aptamer, and an antibody that complementarily bind to the mRNA of the MARH6 gene.
The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the MARCH6 activity or expression inhibitor is for inducing ferrotosis.
The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the MARH6 activity or expression inhibitor inhibits the binding of MARH6 and NADPH or the binding of MARH6 ring domain and ferroptosis regulatory region (FRR) domain.
SLC7A11 (cystine/glutamate antiporter xCT), GPX4, 및 NRF2 로 이루어진 군에서 선택 1종 이상의 항-페로토시스 인자들의 발현을 하향 조절하는 것인, 암 예방 또는 치료용 약학적 조성물.
The method of claim 1, wherein the MARH6 activity or expression inhibitor is selected from the group consisting of ACSL4, ALOX5, TFR1 and p53 - inhibits degradation of pro-ferroptosis proteins; or
SLC7A11 (cystine / glutamate antiporter xCT), GPX4, and one or more selected from the group consisting of NRF2 - to down-regulate the expression of ferrotosis factors, a pharmaceutical composition for preventing or treating cancer.
The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is at least one selected from the group consisting of breast cancer, colon cancer, rectal cancer, lung cancer, colon cancer, thyroid cancer, oral cancer, pharynx cancer, laryngeal cancer, cervical cancer, brain cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, uterine cancer, stomach cancer, bone cancer, and blood cancer.
An anti-cancer adjuvant composition comprising a MARCH6 activity or expression inhibitor.
A health functional food composition for preventing or improving cancer containing a MARCH6 activity or expression inhibitor.
2) 후보물질 처리 후 MARCH6 의 활성 또는 발현의 변화를 측정하는 단계; 및
3) 상기 2) 단계의 MARCH6 활성 또는 발현 수준이 감소되면, 상기 후보물질을 항암제로 판정하는 단계; 를 포함하는 페로토시스 유도 항암제의 스크리닝 방법. 1) processing a candidate substance into a sample;
2) measuring the change in activity or expression of MARCH6 after treatment with the candidate substance; and
3) determining the candidate substance as an anticancer drug when the activity or expression level of MARCH6 in step 2) is reduced; A screening method for ferrotosis-inducing anticancer agents comprising a.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020220006597A KR20230110972A (en) | 2022-01-17 | 2022-01-17 | Anticancer composition comprising MARCH6 inhibitor |
| PCT/KR2022/019509 WO2023136471A1 (en) | 2022-01-17 | 2022-12-02 | Anticancer composition including marh6 (marchf6) inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020220006597A KR20230110972A (en) | 2022-01-17 | 2022-01-17 | Anticancer composition comprising MARCH6 inhibitor |
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| KR20230110972A true KR20230110972A (en) | 2023-07-25 |
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| KR1020220006597A KR20230110972A (en) | 2022-01-17 | 2022-01-17 | Anticancer composition comprising MARCH6 inhibitor |
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| KR (1) | KR20230110972A (en) |
| WO (1) | WO2023136471A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080090775A1 (en) * | 2005-10-31 | 2008-04-17 | Chong Huang | Antagonist of TEB4 and Methods of Use |
| KR101135173B1 (en) * | 2010-01-19 | 2012-04-16 | 한국생명공학연구원 | Composition comprising expression or activity inhibitors of SH3RF2 for the prevention or treatment of cancer |
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- 2022-01-17 KR KR1020220006597A patent/KR20230110972A/en not_active Application Discontinuation
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