KR102508346B1 - Novel Toxoplasma gondii-GRA8 recombinant peptides and composition for preventing or treating cancer comprising the same - Google Patents
Novel Toxoplasma gondii-GRA8 recombinant peptides and composition for preventing or treating cancer comprising the same Download PDFInfo
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Abstract
본 발명은 신규 톡소포자충 GRA8 유래 재조합 펩타이드, 이를 유효성분으로 포함하는 암 질환 예방 또는 치료용 약학적 조성물 및 건강기능식품에 관한 것이다. 본 발명에 따른 톡소포자충 GRA8 유래 재조합 펩타이드는 ATRAM(acidity-triggered rational membrane)에 GRA8의 특정 미토콘드리아 표적화 서열과 ATP5A1/SIRT3 서열을 접합시킨 새로운 재조합 펩타이드로서 기존 GRA8 유래 펩타이드(rGRA8)에 비해 저해 농도 50(Inhibitory concentration 50)가 최대 200배(인비트로) 또는 500배(인비보)에 가까이 상당히 향상된 효능을 보였다. 또한, 상기 펩타이드 처리는 암 질환의 마우스 모델에서 주목할 만한 뚜렷한 치료적 효과를 보이므로, 이를 암 질환 예방 또는 치료용 약학적 조성물 또는 건강기능식품으로 유용하게 사용될 수 있다.The present invention relates to a novel Toxoplasma gondii GRA8-derived recombinant peptide, a pharmaceutical composition for preventing or treating cancer disease, and a health functional food containing the same as an active ingredient. The recombinant peptide derived from Toxoplasma gondii GRA8 according to the present invention is a new recombinant peptide obtained by conjugating the specific mitochondrial targeting sequence of GRA8 and the ATP5A1/SIRT3 sequence to ATRAM (acidity-triggered rational membrane). (Inhibitory concentration 50) showed significantly improved efficacy close to 200 times (in vitro) or 500 times (in vivo). In addition, since the peptide treatment shows a remarkable therapeutic effect in mouse models of cancer disease, it can be usefully used as a pharmaceutical composition or health functional food for preventing or treating cancer disease.
Description
본 발명은 신규 톡소포자충(Toxoplasma gondii) GRA8 유래 재조합 펩타이드 및 이를 포함하는 암 예방 또는 치료용 조성물 및 건강기능식품에 관한 것이다.The present invention relates to a novel Toxoplasma gondii GA8-derived recombinant peptide, a composition for preventing or treating cancer, and a health functional food containing the recombinant peptide.
암은 전세계적으로 주요 사망 원인이며, 미국에서는 심장병 다음으로 두 번째 원인 사망 원인이다. 신진 대사는 암의 발달 및 예방에 중요하다. 암 세포는 신진 대사적으로 활성화되어 있으며 미토콘드리아 SIRT3 를 통한 성장, 증식 및 생존을 위해 ATP가 필요하다. 따라서, 신진 대사 조절제가 암에서 어떻게 특정하게 변화하는지 이해하는 것은 효과적인 암 치료법 개발에 도움이 된다. 미토콘드리아 표적 치료는 종양 사망율을 예방, 완화 또는 역전시키는 유망한 전략으로 간주된다. "대사 소생술"로 알려진 이 조절은 약리학적 또는 영양제를 통해 미토콘드리아 활성을 향상시킬 수 있다. 그러나, 종양 내에서 톡소포자충 GRA8-매개 미토콘드리아 그리고 대사 변화는 설명되지 않은 채 남아 있다.Cancer is the leading cause of death worldwide and the second leading cause of death in the United States after heart disease. Metabolism is important for the development and prevention of cancer. Cancer cells are metabolically active and require ATP for growth, proliferation and survival via mitochondrial SIRT3. Thus, understanding how metabolic modulators specifically change in cancer is helpful in developing effective cancer therapies. Mitochondrial-targeted therapy is considered a promising strategy to prevent, alleviate or reverse tumor mortality. Known as “metabolic resuscitation,” this modulation can enhance mitochondrial activity either pharmacologically or through nutritional agents. However, Toxoplasma gondii GRA8-mediated mitochondrial and metabolic changes within tumors remain unexplained.
본 발명자들은 이전에 단백질 카이네이즈인 Cα (PKCα)-포스포릴화된 톡소포자충 GRA8이 미토콘드리아로 이동되어 미토콘드리아 SIRT3과 상호 작용하는 것을 발견한 바 있다[특허문헌 1]. SIRT3는 ATP5A1와 함께 탈아세틸화되고 그 활성을 조절하여 생체 내 멸균 활성에 기여한다. 탈아세틸화된 ATP5A1은 다양한 미토콘드리아 과정에 관여하고, SIRT3는 전반적인 변화를 종양 성장에 중요한 미토콘드리아의 활성에서 조정할 수 있다. SIRT3는 종양 유전자 또는 종양 억제자로 기능할 수 있으며, 주요 조절자 및 이들의 경로를 표적화함으로써 세포 사멸에 영향을 미친다. 따라서, 미토콘드리아 대사를 조절하는 GRA8 중재 세포 메커니즘은 종양에 대한 치료 접근법에서 이용될 수 있으며, 패혈증에 대해 사용된 전략과 유사하다.The present inventors have previously found that protein kinase Cα (PKCα)-phosphorylated Toxoplasma gondii GRA8 moves into mitochondria and interacts with mitochondrial SIRT3 [Patent Document 1]. SIRT3 is deacetylated along with ATP5A1 and contributes to bactericidal activity in vivo by regulating its activity. Deacetylated ATP5A1 is involved in various mitochondrial processes, and SIRT3 can coordinate global changes in mitochondrial activity that are important for tumor growth. SIRT3 can function as an oncogene or tumor suppressor, influencing cell death by targeting key regulators and their pathways. Thus, GRA8-mediated cellular mechanisms that regulate mitochondrial metabolism can be exploited in therapeutic approaches to tumors, similar to strategies used for sepsis.
한편, 펩타이드 및 단백질은 치료제로서 큰 잠재력을 갖는다. 현재, 소형분자 약물은 제약 시장의 대부분을 차지한다; 이러한 일반적인 소형분자 약물들과 비교하면, 펩타이드 및 단백질은 매우 선택적이며, 그들은 목표와 여러 접촉점을 가지고 있기 때문이다. 또한 증가된 선택성은 부작용과 독성을 감소시킬 수 있다. 펩타이드는 광범위한 분자를 대상으로 하여 디자인될 수 있으며, 이들은 종양학, 면역학, 감염성 질병 그리고 내분비학 분야 응용에서 거의 무한한 가능성을 제공한다. On the other hand, peptides and proteins have great potential as therapeutic agents. Currently, small molecule drugs occupy the majority of the pharmaceutical market; Compared to these common small molecule drugs, peptides and proteins are highly selective because they have multiple contact points with the target. In addition, increased selectivity may reduce side effects and toxicity. Peptides can be designed to target a wide range of molecules, and they offer virtually unlimited possibilities for applications in oncology, immunology, infectious diseases and endocrinology.
이에, 본 발명자들은 미토콘드리아 표적화와 ATP5A1/SIRT3 결합을 위한 최소의 펩타이드 부분을 바탕으로 하여, 이전에 치료적 접근에서 중요한 세포-침투 펩타이드 개발에 연구 노력한 결과, 시험관 내 및 생체 내에서 종양 세포를 표적으로 하는 새로운 톡소포자충 GRA8 유래 재조합 펩타이드(ATRAM-접합된 다기능 GRA8 펩타이드, rATRAM-G8-M/AS)를 설계하고, 상기 재조합 펩타이드가 미토콘드리아 활성화에 의한 암세포의 세포 사멸을 유도하고, 항암 활성이 우수함을 확인함으로써, 본 발명을 완성하게 되었다.Accordingly, the present inventors, based on the minimal peptide portion for mitochondrial targeting and ATP5A1/SIRT3 binding, have previously made research efforts to develop important cell-penetrating peptides in therapeutic approaches, targeting tumor cells in vitro and in vivo A new Toxoplasma gondii GA8-derived recombinant peptide (ATRAM-conjugated multifunctional GRA8 peptide, rATRAM-G8-M/AS) was designed, and the recombinant peptide induces apoptosis of cancer cells by activating mitochondria and has excellent anticancer activity By confirming, the present invention was completed.
따라서, 본 발명은 신규 톡소포자충 GRA8 유래 재조합 펩타이드를 제공하는데 그 목적이 있다.Accordingly, an object of the present invention is to provide a novel recombinant peptide derived from Toxoplasma gondii GRA8.
또한, 본 발명은 신규 톡소포자충 GRA8 유래 재조합 펩타이드를 유효성분으로 포함하는 포함하는 암 예방 또는 치료용 약학적 조성물을 제공하는데 또 다른 목적이 있다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer containing a novel Toxoplasma gondii GRA8-derived recombinant peptide as an active ingredient.
또한, 본 발명은 신규 톡소포자충 GRA8 유래 재조합 펩타이드를 포함하는 암 예방 또는 개선용 건강기능식품을 제공하는데 또 다른 목적이 있다.Another object of the present invention is to provide a health functional food for preventing or improving cancer containing a novel Toxoplasma gondii GRA8-derived recombinant peptide.
본 발명은 톡소포자충(Toxoplasma gondii) GRA8 유래 재조합 펩타이드에 관한 것이다.The present invention relates to a recombinant peptide derived from Toxoplasma gondii GRA8.
본 발명에서 "톡소포자충 GRA8 유래 재조합 펩타이드"는 (GLAGLAGLLGLEGLLGLPLGLLEGLWLGLELEGN)2-GG-ATATTTTTTR-GG-YRFTVQTTQN의 아미노산 서열을 갖는 ATRAM-접합된 다기능 GRA8 펩타이드로서, 서열번호 1로 나타내며, 본원 내에서는 rATRAM-G8-M/AS로 명명된다. 또한, 상기 펩타이드의 변형체(mutant)가 본 발명의 범위 내에 포함된다. 상기 단백질의 변형체란 재조합 펩타이드의 아미노산 서열과 하나 이상의 아미노산 잔기가 결실, 삽입, 비보전적 또는 보전적 치환 또는 이들의 조합에 의하여 상이한 서열을 가지는 단백질을 의미한다. 분자의 활성을 전체적으로 변경시키지 않는 단백질 및 단편에서의 아미노산 교환은 당해 분야에 공지되어 있다(H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979).In the present invention, the "recombinant peptide derived from Toxoplasma gondii GLA8" is (GLAGLAGLLGLEGLLGLPLGLLEGLWLGLELEGN) having an amino acid sequence of 2 -GG-ATATTTTTTTR-GG-YRFTVQTTQN As an ATRAM-conjugated multifunctional GRA8 peptide, represented by SEQ ID NO: 1, it is named rATRAM-G8-M/AS within the present application. Mutants of the above peptides are also included within the scope of the present invention. The variant of the protein refers to a protein having a sequence different from the amino acid sequence of the recombinant peptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof of one or more amino acid residues. Amino acid exchanges in proteins and fragments that do not entirely alter the activity of the molecule are known in the art (H. Neurath, RLHill, The Proteins, Academic Press, New York, 1979).
본 발명에서 용어 "GRA8(dense granule antigen 8; GenBank accession no. XP-002369526.1)"이란, 톡소포자충(Toxoplasma gondii) 유래 항원 단백질로, 269개의 서열번호 2의 아미노산 서열로 구성되어 있다. GRA8은 숙주세포 내에서 기생충 생존을 가능하게 하는 급성 특이성 항원(acute phase-specific antigen)이며, 톡소플라즈마증(toxoplasmosis)에 대한 유망한 백신 후보 물질로 보고된 바 있다(J.G. Costa et.al., Microb. Pathog., 100:229-236, 2016).In the present invention, the term "GRA8 (dense granule antigen 8; GenBank accession no. XP-002369526.1)" is an antigen protein derived from Toxoplasma gondii, and is composed of 269 amino acid sequences of SEQ ID NO: 2. GRA8 is an acute phase-specific antigen that enables parasite survival in host cells and has been reported as a promising vaccine candidate against toxoplasmosis (J.G. Costa et.al., Microb Pathog., 100:229-236, 2016).
본 발명에 있어서, 상기 '톡소포자충(Toxoplasma gondii)' 및 'GRA8(dense granule antigen 8; GenBank accession no. XP-002369526.1(서열번호 2))'은 전술한 바와 같다.In the present invention, 'Toxoplasma gondii' and 'GRA8 (dense granule antigen 8; GenBank accession no. XP-002369526.1 (SEQ ID NO: 2))' are as described above.
또 다른 하나의 양태로서, 본 발명은 상기 톡소포자충 GRA8 유래 재조합 펩타이드를 암호화하는 폴리뉴클레오타이드(유전자)를 제공한다.As another aspect, the present invention provides a polynucleotide (gene) encoding the recombinant peptide derived from Toxoplasma gondii GRA8.
상기 폴리뉴클레오타이드는 서열번호 3의 염기서열을 포함한다.The polynucleotide includes the nucleotide sequence of SEQ ID NO: 3.
또한, 상기 염기서열의 상동체가 본 발명의 범위 내에 포함된다. 구체적으로는, 상기 폴리뉴클레오타이드는 서열번호 1의 염기서열로 이루어진 군으로부터 선택되는 염기서열과 각각 70% 이상, 더욱 바람직하게는 80% 이상, 더 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 염기서열을 포함할 수 있다. 폴리뉴클레오타이드에 대한 "서열 상동성의 %"는 두 개의 최적으로 배열된 서열과 비교 영역을 비교함으로써 확인되며, 비교 영역에서의 폴리뉴클레오티드 서열의 일부는 두 서열의 최적 배열에 대한 참고 서열(추가 또는 삭제를 포함하지 않음)에 비해 추가 또는 삭제(즉, 갭)를 포함할 수 있다.In addition, homologues of the above nucleotide sequences are included within the scope of the present invention. Specifically, the polynucleotide is 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more of a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1. It may include a nucleotide sequence having more than % sequence homology. The "% of sequence homology" for a polynucleotide is determined by comparing two optimally aligned sequences with a region of comparison, wherein a portion of the polynucleotide sequence in the region of comparison is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. may include additions or deletions (i.e., gaps) compared to (not including).
"코돈 최적화"는 코딩된 단백질이 유기체에서 보다 효율적으로 발현되도록 특정 유기체에서 우선적으로 사용되는 것으로 단백질을 코딩하는 폴리뉴클레오타이드의 코돈을 변화시키는 것을 의미한다. 대부분의 아미노산이 "동의어" 또는 "동의" 코돈이라고 하는, 몇몇 코돈에 의해 표시된다는 점에서, 유전자 코드가 축퇴적이지만, 특정 유기체에 의한 코돈 용법은 임의적이지 않고 특정 코돈 트리플렛에 편향적이다. 이러한 코돈 용법 편향성은 소정 유전자, 공통 기능 또는 조상 기원의 유전자, 고도로 발현되는 단백질 대 낮은 복제수 단백질, 및 유기체 게놈의 집합적 단백질 코딩 영역과 관련하여 더 높을 수 있다. 본 발명의 상기 서열번호 3의 염기서열은 유전자가 대장균 내에서 발현될 수 있도록 대장균의 코돈에 최적화된 서열이다."Codon optimization" means changing the codons of a polynucleotide encoding a protein to those preferentially used in a particular organism so that the encoded protein is more efficiently expressed in the organism. While the genetic code is degenerate, in that most amino acids are represented by several codons, called "synonymous" or "synonymous" codons, codon usage by particular organisms is not arbitrary and is biased toward particular codon triplets. This codon usage bias can be higher with respect to certain genes, genes of common function or ancestral origin, highly expressed versus low copy number proteins, and collective protein coding regions of an organism's genome. The nucleotide sequence of SEQ ID NO: 3 of the present invention is a sequence optimized for the codon of E. coli so that the gene can be expressed in E. coli.
또 다른 하나의 양태로서, 본 발명은 상기 톡소포자충 GRA8 유래 재조합 펩타이드를 암호화하는 폴리뉴클레오타이드를 포함하는 재조합 발현 벡터를 제공한다.As another aspect, the present invention provides a recombinant expression vector containing a polynucleotide encoding the recombinant peptide derived from Toxoplasma gondii GRA8.
본 발명은 또한, 상기 SV82 폴리펩타이드 코딩 유전자를 포함하는 재조합 벡터 및 상기 재조합 벡터로 형질전환된 대장균을 제공한다.The present invention also provides a recombinant vector containing the gene encoding the SV82 polypeptide and E. coli transformed with the recombinant vector.
용어 "재조합"은 세포가 이종의 핵산을 복제하거나, 상기 핵산을 발현하거나 또는 펩타이드, 이종의 펩타이드 또는 이종의 핵산에 의해 암호된 단백질을 발현하는 세포를 지칭하는 것이다. 재조합 세포는 상기 세포의 천연 형태에서는 발견되지 않는 유전자 또는 유전자 절편을, 센스 또는 안티센스 형태 중 하나로 발현할 수 있다. 또한, 재조합 세포는 천연 상태의 세포에서 발견되는 유전자를 발현할 수 있으며, 그러나 상기 유전자는 변형된 것으로서 인위적인 수단에 의해 세포 내 재도입된 것이다.The term "recombinant" refers to a cell that replicates, expresses a heterologous nucleic acid, or expresses a peptide, a protein encoded by a heterologous peptide or a heterologous nucleic acid. Recombinant cells can express genes or gene segments not found in the cell's native form, either in sense or antisense form. Recombinant cells can also express genes found in cells in their natural state, but the genes have been reintroduced into the cell by artificial means as modified.
본 발명에서, 상기 톡소포자충 GRA8 유래 재조합 펩타이드를 코딩하는 유전자는 재조합 발현 벡터 내로 삽입될 수 있다. 용어 "재조합 발현 벡터"는 세균 플라스미드, 파아지, 효모 플라스미드, 식물 세포 바이러스, 포유동물 세포 바이러스, 또는 다른 벡터를 의미한다. 대체로, 임의의 플라스미드 및 벡터는 숙주 내에서 복제 및 안정화할 수 있다면 사용될 수 있다. 상기 발현 벡터의 중요한 특성은 복제 원점, 프로모터, 마커 유전자 및 번역 조절 요소(translation control element)를 가지는 것이다.In the present invention, the gene encoding the recombinant peptide derived from Toxoplasma gondii GRA8 can be inserted into a recombinant expression vector. The term “recombinant expression vector” refers to a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In general, any plasmid and vector may be used provided that it is capable of replicating and stabilizing in the host. An important characteristic of the expression vector is that it has an origin of replication, a promoter, a marker gene and a translation control element.
톡소포자충 GRA8 유래 재조합 펩타이드를 코딩하는 유전자 서열 및 적당한 전사/번역 조절 신호를 포함하는 발현 벡터는 당업자에 주지된 방법에 의해 구축될 수 있다. 상기 방법은 시험관 내 재조합 DNA 기술, DNA 합성 기술 및 생체 내 재조합 기술 등을 포함한다. 상기 DNA 서열은 mRNA 합성을 이끌기 위해 발현 벡터 내의 적당한 프로모터에 효과적으로 연결될 수 있다. 또한 발현 벡터는 번역 개시 부위로서 리보좀 결합 부위 및 전사 터미네이터를 포함할 수 있다.An expression vector containing a gene sequence encoding a recombinant peptide derived from Toxoplasma gondii GRA8 and suitable transcriptional/translational control signals can be constructed by methods well known to those skilled in the art. The method includes in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like. The DNA sequence can be effectively linked to a suitable promoter in an expression vector to direct mRNA synthesis. In addition, the expression vector may include a ribosome binding site and a transcription terminator as a translation initiation site.
본 발명의 일 구현 예에 따른 재조합 벡터는, pRSFDuet-1 벡터에 합성한 톡소포자충 GRA8 유래 재조합 펩타이드를 코딩하는 유전자(서열번호 2)를 5' 말단(BamHI 제한효소 사이트)과 3' 말단 (XhoI 제한효소 사이트)에 인프레임(in frame)으로 융합하여 제조하였고, 상기 유전자를 T7 프로모터와 lac 오퍼레이터에 의하여 효과적으로 발현시켜 톡소포자충 GRA8 유래 재조합 펩타이드를 제조하는 것을 특징으로 하는 벡터이다.In the recombinant vector according to an embodiment of the present invention, the gene (SEQ ID NO: 2) encoding the recombinant peptide derived from Toxoplasma gondii GRA8 synthesized in the pRSFDuet-1 vector is 5' end (BamHI restriction enzyme site) and 3' end (XhoI restriction enzyme site), and the gene is effectively expressed by the T7 promoter and the lac operator to prepare a recombinant peptide derived from Toxoplasma gondii GRA8.
본 발명의 벡터를 원핵세포에 안정되면서 연속적으로 클로닝 및 발현시킬 수 있는 숙주세포는 당업계에 공지된 어떠한 숙주세포도 이용할 수 있으며, 예컨대, E. coli Rosetta, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, 바실러스 서브틸리스, 바실러스 츄린겐시스와 같은 바실러스 속 균주, 그리고 살모넬라 티피무리움, 세라티아 마르세슨스 및 다양한 슈도모나스 종과 같은 장내균과 균주 등이 있다.Any host cell known in the art can be used as a host cell capable of continuously cloning and expressing the vector of the present invention while being stable in a prokaryotic cell, such as E. coli Rosetta, E. coli JM109, E. coli BL21 , E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus strains such as Bacillus thuringiensis, and Salmonella typhimurium, Serratia Enterobacteriaceae and strains such as Marcessons and various Pseudomonas species;
또한, 본 발명의 벡터를 진핵 세포에 형질전환시키는 경우에는 숙주세포로서, 효모(Saccharomyce cerevisiae), 곤충세포, 사람세포 (예컨대, CHO 세포주 (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN 및 MDCK 세포주) 및 식물세포 등이 이용될 수 있다.In addition, when the vector of the present invention is transformed into a eukaryotic cell, as a host cell, yeast (Saccharomyce cerevisiae), insect cell, human cell (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and plant cells, etc. may be used.
본 발명의 일 구현 예에 따른 재조합 벡터로 형질전환된 숙주세포는 대장균일 수 있으나, 이에 제한되지 않는다.The host cell transformed with the recombinant vector according to one embodiment of the present invention may be Escherichia coli, but is not limited thereto.
본 발명의 벡터를 숙주세포 내로 운반하는 방법은, 숙주 세포가 원핵 세포인 경우, CaCl2 방법, 하나한 방법(Hanahan, D., 1983 J. Mol. Biol. 166, 557-580) 및 전기천공 방법 등에 의해 실시될 수 있다. 또한, 숙주세포가 진핵세포인 경우에는, 미세주입법, 칼슘포스페이트 침전법, 전기천공법, 리포좀-매개 형질감염법, DEAE-덱스트란 처리법, 및 유전자 밤바드먼트 등에 의해 벡터를 숙주세포 내로 주입할 수 있다.Methods for delivering the vectors of the present invention into host cells, when the host cells are prokaryotic cells, include the CaCl 2 method, the Hanhan method (Hanahan, D., 1983 J. Mol. Biol. 166, 557-580), and electroporation. It can be implemented by methods and the like. In addition, when the host cell is a eukaryotic cell, the vector can be injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment. can
본 발명의 일 실시예에 있어서, 톡소포자충 GRA8 염기서열 중 미토콘드리아 SIRT3 단백질과 결합하는 표적 펩타이드(G8-M/AS)를 수득하였다. 이를 암세포 표적 ATRAM 펩타이드와 결합한 염기서열을 벡터에 삽입하여 발현벡터를 수득한 다음, 이를 대장균에 도입하여 rATRAM-G8-M/AS을 생산하는 형질전환체를 수득하였으며, 상기 형질전환체를 배양하여 재조합 rATRAM-G8-M/AS을 대량 생산하고 정제하였다. 또한, 상기 정제된 재조합 rATRAM-G8-M/AS을 대상으로 면역 블롯(immunoblotting, IB) 분석을 수행한 결과, 상기 rATRAM-G8-M/AS은 암세포 표적능이 우수하였고 (도 5), 암세포내 미토콘드리아 활성 (Oxphos V 활성도, 미토콘드리아 mass 및 ATP 생성)을 유도함을 확인하였다(도 4). 이에 따라, rATRAM-G8-M/AS이 미토콘드리아 생합성 및 활성에 영향을 미치므로, 미토콘드리아 기능 장애에 의해 유발되는 암 치료에 이용될 수 있음을 유추할 수 있었다.In one embodiment of the present invention, a target peptide (G8-M/AS) binding to mitochondrial SIRT3 protein in the GRA8 nucleotide sequence of Toxoplasma gondii was obtained. An expression vector was obtained by inserting the nucleotide sequence combined with the cancer cell-targeting ATRAM peptide into a vector, and then introducing it into E. coli to obtain a transformant producing rATRAM-G8-M/AS, and culturing the transformant Recombinant rATRAM-G8-M/AS was mass-produced and purified. In addition, as a result of performing immunoblotting (IB) analysis on the purified recombinant rATRAM-G8-M/AS, the rATRAM-G8-M/AS had excellent cancer cell targeting ability (FIG. 5), and It was confirmed that mitochondrial activity (Oxphos V activity, mitochondrial mass and ATP production) was induced (FIG. 4). Accordingly, it could be inferred that since rATRAM-G8-M/AS affects mitochondrial biosynthesis and activity, it can be used to treat cancer caused by mitochondrial dysfunction.
또한, 본 발명에 따른 rATRAM-G8-M/AS 유도 미토콘드리아 활성화에 의한 암 세포주의 세포 사멸에 대해, 기존 GRA8 유래 펩타이드(rGRA8)에 비해 저해 농도 50(Inhibitory concentration 50)가 약 200배 개선된 효과를 확인하였다(도 4). 또한, rATRAM-G8-M/AS는 암 세포주에서 항종양 활성에 대해 기존 GRA8 유래 펩타이드(rGRA8)에 비해 저해 농도 50(Inhibitory concentration 50)가 약 500배 개선된 효과를 나타내었다.In addition, the inhibitory concentration 50 improved about 200 times compared to the existing GRA8-derived peptide (rGRA8) on cell death of cancer cell lines by rATRAM-G8-M/AS-induced mitochondrial activation according to the present invention was confirmed (FIG. 4). In addition, rATRAM-G8-M/AS exhibited about 500-fold improved effect of inhibitory concentration 50 compared to the existing GRA8-derived peptide (rGRA8) for antitumor activity in cancer cell lines.
또 다른 하나의 양태로서, 본 발명은 신규 톡소포자충 GRA8 유래 재조합 펩타이드를 유효성분으로 포함하는, 암 질환 예방 또는 치료용 약학적 조성물을 제공한다.In another aspect, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a novel Toxoplasma gondii GRA8-derived recombinant peptide as an active ingredient.
상기 암 질환은 뇌암, 식도암, 위암, 간암, 췌장암, 신장암, 소장암, 대장암, 직장암, 폐암, 유방암, 자궁암, 난소암 및 전립선암으로 이루어진 군으로부터 선택된 것일 수 있다. The cancer disease may be selected from the group consisting of 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, and prostate cancer.
본 발명에서 용어 "대장암(colorectal cancer)"이란 대장에 생긴 암세포로 이루어진 악성종양을 말한다. 구체적으로, 대장암은 결장과 직장에 발생하는 암을 합쳐서 일컫는다.In the present invention, the term "colorectal cancer" refers to a malignant tumor composed of cancer cells in the large intestine. Specifically, colorectal cancer refers to cancers that occur in the colon and rectum.
본 발명에서 용어 "예방"이란, 본 발명의 약학적 조성물의 투여로 대장암의 발병을 억제 또는 지연시키는 모든 행위를 의미하며, "치료"란, 본 발명의 약학적 조성물의 투여로 인해 이미 유발된 대장암의 증세가 호전되거나 이롭게 되는 모든 행위를 의미한다.In the present invention, the term "prevention" refers to any action that inhibits or delays the onset of colon cancer by administration of the pharmaceutical composition of the present invention, and "treatment" refers to any activity that has already occurred due to administration of the pharmaceutical composition of the present invention. It means any action that improves or benefits the symptoms of colorectal cancer.
특히, 본 발명에서 제공하는 rATRAM-G8-M/AS는 암, 특히 대장암의 예방 또는 치료 효과를 갖는 약학적 조성물의 유효성분으로 사용될 수 있음을 알 수 있었다.In particular, it was found that the rATRAM-G8-M/AS provided in the present invention can be used as an active ingredient of a pharmaceutical composition having an effect of preventing or treating cancer, particularly colorectal cancer.
상기 본 발명의 약학적 조성물은 약학적으로 허용가능한 담체를 추가로 포함할 수 있다. 본 발명의 용어 "약학적으로 허용가능한"이란 상기 조성물에 노출되는 세포나 인간에게 독성이 없는 특성을 나타내는 것을 의미한다.The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" of the present invention means exhibiting characteristics that are not toxic to cells or humans exposed to the composition.
상기 담체는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제, 기제, 부형제, 윤활제 등 당업계에 공지된 것이라면 제한없이 사용할 수 있다. The carrier may be used without limitation as long as it is known in the art such as a buffer, a preservative, a pain reliever, a solubilizer, an isotonic agent, a stabilizer, a base, an excipient, a lubricant, and the like.
또한, 본 발명의 약학적 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 본 발명의 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시 벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다.In addition, the pharmaceutical composition of the present invention is formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. can be used Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
상기 약학적 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다.The administration route of the pharmaceutical composition may be administered through any general route as long as it can reach the target tissue.
본 발명의 용어 "투여"란, 어떠한 적절한 방법으로 환자에게 소정의 물질을 도입하는 것을 의미하며, 인체 또는 수의용으로 제형화되어 다양한 경로로 투여된다. 본 발명의 재조합 펩타이드는 비경구 경로, 예컨대 혈관내, 정맥내, 동맥내, 근육내 또는 피하 등의 경로로 투여될 수 있고, 경구, 비강, 직장, 경피 또는 에어로졸을 통한 흡입 경로로 투여될 수도 있으며, 볼루스(bolus)로 투여하거나 또는 서서히 주입할 수도 있으나, 바람직하게는 근육내 또는 피하 주사로 투여되는 것이 바람직하다.The term "administration" of the present invention means introducing a predetermined substance into a patient by any suitable method, and is formulated for human or veterinary use and administered through various routes. The recombinant peptide of the present invention may be administered by a parenteral route, such as intravascular, intravenous, intraarterial, intramuscular, or subcutaneous routes, and may be administered by oral, nasal, rectal, transdermal or aerosol inhalation routes. It may be administered as a bolus or by slow infusion, but it is preferably administered by intramuscular or subcutaneous injection.
한편, 본 발명의 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명의 용어 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미하며, 유효용량 수준은 환자의 건강상태, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dose level is the patient's health condition, It may be determined according to severity, drug activity, drug sensitivity, administration method, administration time, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field.
본 발명의 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 다른 항암제와는 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with other conventional anticancer agents. And it can be single or multiple administrations. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, and can be easily determined by those skilled in the art.
상기 종래의 다른 항암제는, 이에 제한되지는 않으나, 세툭시맙(cetuximab), 엘로티닙(erlotinib), 게피티닙(Gefitinib), 트라스투주맙(trastuzumab), 라파티닙(lapatinib), 파클리탁셀, 타목시펜, 및 시스플라틴으로 이루어진 군에서 선택된 어느 하나 또는 그 이상일 수 있다.Other conventional anticancer agents include, but are not limited to, cetuximab, erlotinib, gefitinib, trastuzumab, lapatinib, paclitaxel, tamoxifen, and It may be any one or more selected from the group consisting of cisplatin.
본 발명의 약학적 조성물은 rATRAM-G8-M/AS와 함께 암 예방 및 치료 효과를 갖는 공지의 유효성분을 1종 이상 포함할 수 있다.The pharmaceutical composition of the present invention may contain one or more known active ingredients having cancer prevention and treatment effects together with rATRAM-G8-M/AS.
또 다른 하나의 양태로서, 본 발명은 암의 발병이 예상되거나 또는 상기 암이 발병된 대상체에 상기 약학적 조성물을 투여하는 단계를 포함하는, 암을 예방 또는 치료하는 방법을 제공한다.As another aspect, the present invention provides a method for preventing or treating cancer, comprising administering the pharmaceutical composition to a subject who is expected to develop cancer or has the cancer.
본 발명의 용어 "대상체"란, 상기 암이 발병하였거나 발병할 수 있는 살아있는 유기체를 의미하는데, 인간을 포함한 원숭이, 소, 말, 양, 돼지, 닭, 칠면조, 메추라기, 고양이, 개, 마우스, 쥐, 토끼 또는 기니아 피그를 포함한 모든 포유동물을 의미한다. 본 발명의 약학적 조성물을 대상체에게 투여함으로써 상기 암을 효과적으로 예방 또는 치료할 수 있다. 본 발명의 약학적 조성물은 기존의 치료제와 병행하여 투여될 수 있다.The term "subject" of the present invention refers to a living organism that has or may develop the cancer, including humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, and rats. , any mammal, including rabbits or guinea pigs. By administering the pharmaceutical composition of the present invention to a subject, the cancer can be effectively prevented or treated. The pharmaceutical composition of the present invention may be administered in parallel with existing therapeutic agents.
구체적으로, 본 발명에 따른 조성물에 함유된 상기 rATRAM-G8-M/AS의 유효량은 환자의 나이, 성별, 체중에 따라 달라질 수 있으며, 일반적으로는 본 발명의 상기 rATRAM-G8-M/AS는 고형분을 기준으로 1일 체중 kg 당 0.001 내지 50 mg, 바람직하게는 체중 kg 당 0.1 내지 15 mg을 매일 또는 격일 투여하거나 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나, 투여 경로, 질병의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.Specifically, the effective amount of the rATRAM-G8-M/AS contained in the composition according to the present invention may vary depending on the patient's age, sex, and weight. In general, the rATRAM-G8-M/AS of the present invention Based on the solid content, 0.001 to 50 mg/kg of body weight per day, preferably 0.1 to 15 mg/kg of body weight per day may be administered daily or every other day or divided into 1 to 3 times a day. However, since it may increase or decrease according to the route of administration, severity of disease, sex, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.
또 다른 하나의 양태로서, 본 발명은 상기 rATRAM-G8-M/AS를 포함하는 암 질환 예방 또는 개선용 건강기능식품을 제공한다.As another aspect, the present invention provides a health functional food for preventing or improving cancer disease comprising the rATRAM-G8-M/AS.
본 발명에서 용어 "개선"은 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다.In the present invention, the term "improvement" refers to any action that at least reduces a parameter related to the condition being treated, eg, the severity of a symptom.
본 발명의 조성물을 식품으로 사용하는 경우, 상기 "rATRAM-G8-M/AS"을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용할 수 있고, 통상의 방법에 따라 적절하게 사용할 수 있다. 상기 조성물은 유효성분 이외에 식품학적으로 허용가능한 식품보조첨가제를 포함할 수 있으며, 유효성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다.When the composition of the present invention is used as a food, the "rATRAM-G8-M/AS" 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. In addition to the active ingredient, the composition may include food additives acceptable in food science, and the mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment).
본 발명에서 사용되는 용어 "식품보조첨가제"란 식품에 보조적으로 첨가될 수 있는 구성요소를 의미하며, 각 제형의 건강기능식품을 제조하는데 첨가되는 것으로서 당업자가 적절히 선택하여 사용할 수 있다. 식품보조첨가제의 예로는 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 충진제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등이 포함되지만, 상기 예들에 의해 본 발명의 식품보조첨가제의 종류가 제한되는 것은 아니다.The term "food supplement additive" used in the present invention refers to a component that can be added to food supplementally, and can be appropriately selected and used by those skilled in the art as being added to prepare a health functional food of each formulation. Examples of food additives include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners , pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc. are included, but the types of food additives of the present invention are not limited by the above examples.
본 발명에서 사용되는 용어 "건강기능식품"이란 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 정제, 캅셀, 분말, 과립, 액상 및 환 등의 형태로 제조 및 가공한 식품을 말한다. 여기서 '기능성'이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 건강기능식품은 당업계에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조 시에는 당업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한 상기 건강기능식품의 제형 또한 건강기능식품으로 인정되는 제형이면 제한 없이 제조될 수 있다. 본 발명의 식품용 조성물은 다양한 형태의 제형으로 제조될 수 있으며, 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어나, 본 발명의 건강기능식품은 류마티스 관절염의 효과를 증진시키기 위한 보조제로 섭취가 가능하다.The term "health functional food" used in the present invention refers to food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functionalities for the human body. Here, 'functionality' means obtaining useful effects for health purposes, such as adjusting nutrients for the structure and function of the human body or physiological functions. The health functional food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation. In addition, the formulation of the health functional food may also be manufactured without limitation as long as the formulation is recognized as a health functional food. The composition for food of the present invention can be prepared in various types of formulations, and unlike general drugs, it has the advantage of not having side effects that can occur when taking drugs for a long time by using food as a raw material, and has excellent portability. Of the health functional foods can be taken as supplements to enhance the effect of rheumatoid arthritis.
본 발명에 따른 톡소포자충 GRA8 유래 재조합 펩타이드는 ATRAM(acidity-triggered rational membrane)에 GRA8의 특정 미토콘드리아 표적화 서열과 ATP5A1/SIRT3 서열을 접합시킨 새로운 재조합 펩타이드로서 암 특히 대장암에 대한 예방 또는 우수한 치료 효과를 보이므로, 이를 암 질환 예방 또는 치료용 약학적 조성물 또는 건강기능식품으로 유용하게 사용될 수 있다.The recombinant peptide derived from Toxoplasma gondii GRA8 according to the present invention is a new recombinant peptide obtained by conjugating a specific mitochondrial targeting sequence of GRA8 and an ATP5A1/SIRT3 sequence to ATRAM (acidity-triggered rational membrane), and has excellent preventive or therapeutic effects on cancer, particularly colorectal cancer. Since it is visible, it can be usefully used as a pharmaceutical composition or health functional food for preventing or treating cancer.
도 1은 rGRA8 처리가 미토콘드리아 경로에 의해 HCT116 세포 사멸을 증가시킴을 확인한 것이다[(A 및 B). 다양한 암세포를 지시된 시간동안 WT rGRA8 (20 μg/ml) 및 이것의 돌연변이와 함께 배양한 다음, MTT 분석 (위쪽)으로 세포 생존력을 측정하고 αATP5A1, αSIRT3 또는 αActin (아래쪽) 면역블롯팅을 하였다. (C) HCT116 세포를 2일 동안 폴리브렌 (8 μg/mL) (오른쪽)으로 렌티바이러스-shRNA-NS 또는 렌티바이러스-shRNA-PKCα 또는 SIRT3 (MOI = 100)로 형질도입 하였다. 세포를 지시된 시간동안 WT rGRA8과 함께 배양하고 αLys-AC 또는 αHis와 함께 IP를 진행하였으며 αATP5A1, αATP5C1, αSIRT3, αPGC-1, αPKCα 또는 αActin로 IB를 수행하였다. 데이터는 유사한 결과를 가진 5개의 독립적인 실험을 나타낸다. 유의미한 차이는 rVector-처리된 것과 비교하였다 (** P<0.01; *** P<0.001)].
도 2는 ATRAM-접합된 다기능 GRA8 펩타이드(rATRAM-G8-M/AS) 기반 단백질의 설계 및 발현을 나타낸 것이다[(A) 293T 세포의 세포 내 분획은 GST-GRA8 WT를 발현하거나 WT rGRA8을 (1 μg/ml) BMDM과 함께 배양하고 여러 Tat-GRA8 펩타이드로 6시간 동안 (10 μM) 처리하였다. 미토콘드리아 및 세포질 분획을 분류하고 IB를 이용해 GST의 발현에 대해 분석하였다. 분획의 순도는 VDAC (미토콘드리아 단백질) 및 액틴 (세포질 단백질)에 대한 블롯팅에 의해 확인되었다. (B) 293T 세포에 포유류 GST-GRA8 구조를 V5-ATP5A1 또는 Flag-SIRT3 와 함께 12시간 동안 형질감염 후 6시간 동안 여러 개의 Tat-GRA8 펩타이드 (10 μM)로 처리하였다. 293T 세포를 GST 풀다운에 사용하였고, 이어서 αV5 또는 αFlag로 IB를 수행하였다. WCL는 αGST, αV5, αFlag 또는 αActin과 함께 IB에 사용되었다. (C) ATRAM-GRA8-M/AS 및 그 돌연변이의 설계 도식. (D) 박테리아로 정제된 6xHis-rATRAM-GRA8-M/AS 및 이것의 돌연변이 체를 쿠마시 블루 염색 (왼쪽) 또는 αHis (오른쪽)로 면역블롯팅(IB)에 의해 분석하였다. (E) BMDM을 지시된 시간 동안 rATRAM-GRA8-M/AS 및 이것의 돌연변이 (20 μg / ml)와 함께 배양한 다음, MTT 분석으로 세포 생존율을 측정하였다. 데이터는 유사한 결과 (A, B, D 및 E)를 가진 4 개의 독립적인 실험을 나타낸다.].
도 3은 확인된 GRA8의 최소 영역이 ATP5A1 및 SIRT3과 직접 상호작용함을 나타낸 것이다[포유 동물 GST-ATP5A1 또는 GST-SIRT3를 Flag-GRA8와 함께 구성하여 형질 감염 12시간 후에 6시간 동안 293T에 여러 가지 Tat-GRA8 펩타이드 (10 μM)를 처리하였다. 293T 세포를 GST 풀다운에 사용하였고, 이어서 항Flag로 갖는 IB를 수행하였다. 세포 전체 용해물은 αGST, αFlag 또는 항Actin과 함께 IB에 사용되다. 데이터는 유사한 결과를 가진 4개의 독립적인 실험을 나타낸다.].
도 4는 rATRAM-GRA8-M/AS-유도 미토콘드리아 활성화에 의한 HCT116 세포 사멸을 나타낸 것이다[(A 및 B) HCT116 세포를 지시된 시간 동안 WT rGRA8 또는 rATRAM-GRA8-M/AS 및 그의 돌연변이와 함께 배양한 다음, MTT 분석으로 세포 생존력을 측정하였다. (C-E) HCT116 세포를 2일 동안 폴리브렌 (8 μg/mL) (오른쪽)으로 lentivirus-shRNA-NS 또는 lentivirus-shRNA-PKCα 또는 SIRT3 (MOI = 100)로 형질 도입 하였다. 세포를 1일 동안 rATRAM-GRA8-M/ AS로 자극하고, OXPHOS V 효소 활성을 적용하였다, (C) 미토 트래커 형광 신호를 유세포 분석에 의해 확인하였다. 막대 그래프는 미토콘드리아 질량 MFI를 보여준다 (D) 또는 BMDM의 mtDNA 함량을 정량적 실시간 PCR에 의해 측정하였다. mtDNA 함량은 핵 DNA 로 표준화하였다. (E) 유의미한 차이는 대조군 처리와 비교하였다 (** P <0.01; *** P <0.001). 데이터는 유사한 결과를 가진 5개의 독립적인 실험을 나타낸다].
도 5는 rATRAM-GRA8-M/AS의 약동학 및 약물 분포를 나타낸 것이다[(A) 종양-보유 마우스에서 rATRAM-GRA8-M/AS에 대한 약동학 및 약동학적 분포. 방법 부분에서 설명된 바와 같이, HCT116 세포를 BALB/c 마우스의 측면에 피하 주사하였다. 마우스에 복강주사를 통해 1 또는 2회 투여되었고 5일째에 죽였다. His 발현은 마우스로부터의 다양한 기관에서 면역 블롯팅 (IB)에 의해 평가되었다. 전체 세포 용해물을 항Actin과 함께 IB에 사용하였다. 데이터는 유사한 결과를 가진 4개의 독립적인 실험을 나타낸다. (B) 또는 rGRA8 WT (위)로 처리된 이종 이식편 모델의 개략도. 누드 마우스의 측면에 HCT116 세포를 피하 주사하였다. 캘리퍼를 사용하여 종양의 길이 및 폭을 측정하고, 30일 동안 3일마다 종양 부피를 계산하였다. 각 그룹으로부터의 각각의 마우스로부터의 개별 종양 부피를 평균하고 접종 후 수일에 대해 그래프를 그렸다. 통계적 유의성은 Tukey의 사후 검정을 통한 양방향 분산 (ANOVA) 분석에 의해 결정되었다; rVector와 비교하여 * P <0.05, *** P <0.001. 각 그룹은 10마리의 마우스를 포함하였다. 데이터는 유사한 결과를 가진 두 개의 독립적 인 실험을 나타낸다.
도 6은 rATRAM-GRA8-M/AS가 HCT116 이종 이식에서 항종양 활성을 보여주는 것이다[(A 및 B) 또는 rATRAM-GRA8-M/AS 또는 그의 돌연변이(상단)로 처리된 이종이식 모델의 모식도. 누드 마우스의 측면에 HCT116 세포를 피하 주사하였다. 캘리퍼를 사용하여 종양의 길이 및 폭을 측정하고, 30일 동안 3일마다 종양 부피를 계산하였다. 투여 후 15일에, 마우스를 안락사시키고, GRA8과의 상호작용, ATP5A1의 아세틸화 수준 또는 OXPHOS의 발현을 분석하였다 (A, 아래). 15일에 rATRAM-GRA8-M/AS 또는 돌연변이로 처리된 마우스로부터의 종양의 대표적인 이미지 (오른쪽). (C-F) HCT116 세포를 렌티 바이러스-shRNA-NS 또는 렌티 바이러스-shRNA-PKCα, SIRT3, 또는 폴리브렌 (C-E) 또는 ATP5A1 WT 또는 돌연변이가 발현되는 HCT116 세포를 누드 마우스의 측면에 피하주사 하였다. 유의미한 차이 (* P <0.05; ** P <0.01; *** P <0.001)는 rVector와 비교하였다. 각 그룹은 10 마리의 마우스를 포함하였다. 데이터는 유사한 결과를 가진 두 개의 독립적인 실험을 나타낸다.],
도 7은 HCT 116에서의 녹다운 효과 및 생체 내 rATRAM-GRA8-M/AS의 면역원성을 나타낸 것이다 [(A) HCT116 세포를 2일 동안 폴리브렌 (8㎍/mL)과 함께 렌티 바이러스-shRNA-NS 또는 렌티 바이러스-shRNA-PKCα, SIRT3 또는 ATP5A1로 형질 도입하였다. 항PKCα, 항ATP5A1, 항SIRT3 또는 항Actin으로 면역 블롯팅을 수행하였다. 데이터는 유사한 결과를 가진 5개의 독립적인 실험을 보여준다. (B) rVector (상단)로 처리된 이종 이식편 모델의 개략도. HCT116-발현된 ATP5A1 WT 및 돌연변이 세포를 누드 마우스의 측면에 피하 주사하였다. 유의적 차이는 rVector와 비교하였다. 각 그룹은 10마리의 마우스를 포함하였다. 데이터는 유사한 결과를 가진 두 개의 독립적인 실험을 보여준다. (C) BALB/C 마우스에서의 체액성 면역 반응. 마우스를 3일마다 각각의 경로 (정맥주사 또는 복강주사)에서 면역시키고 40일에 채혈하였다. rATRAM-GRA8-M/AS 또는 오발부민 (OVA)에 특이적인 IgG의 수준을 ELISA를 사용하여 측정하였다. 값은 450nm에서의 흡광도(OD)에 대한 평균 및 표준 편차를 나타낸다. 각 그룹은 5마리의 마우스를 포함하였다. (D) 종양 보유 마우스에서 rATRAM-GRA8-M/AS에 대한 사이토카인 반응. 방법 부분에 설명된 바와 같이, HCT116 세포를 BALB/c 마우스의 측면에 피하 주사하였다. 혈청 사이토카인 수준은 투여 후 30일에 측정되었다. 각 그룹은 10마리의 마우스를 포함하였다.
도 8은 본 발명에 따른 재조합 발현벡터 제작에 사용된 pRSFDuet-1 벡터의 개열지도를 나타낸 것이다.
도 9는 본 발명에 따른 재조합 발현벡터 pRSFDuet-1-ATRAM-G8-M/AS 의 개열지도를 나타낸 것이다.do 1 confirms that rGRA8 treatment increases HCT116 cell death by the mitochondrial pathway [(A and B). Various cancer cells were incubated with WT rGRA8 (20 μg/ml) and its mutants for the indicated times, then cell viability was measured by MTT assay (top) and αATP5A1, αSIRT3 or αActin (bottom) immunoblotting. (C) HCT116 cells were transduced with lentiviral-shRNA-NS or lentiviral-shRNA-PKCα or SIRT3 (MOI = 100) with polybrene (8 μg/mL) (right) for 2 days. Cells were incubated with WT rGRA8 for the indicated times, followed by IP with αLys-AC or αHis, and IB with αATP5A1, αATP5C1, αSIRT3, αPGC-1, αPKCα or αActin. Data represent 5 independent experiments with similar results. Significant differences compared to rVector-treated (** P<0.01; *** P<0.001)].
Figure 2 shows the design and expression of an ATRAM-conjugated multifunctional GRA8 peptide (rATRAM-G8-M/AS) based protein [(A) intracellular fraction of 293T cells expressing GST-GRA8 WT or WT rGRA8 ( 1 μg/ml) BMDM and treated with various Tat-GRA8 peptides for 6 hours (10 μM). Mitochondrial and cytosolic fractions were sorted and analyzed for expression of GST using IB. The purity of the fractions was confirmed by blotting for VDAC (mitochondrial protein) and actin (cytoplasmic protein). (B) 293T cells were transfected with mammalian GST-GRA8 constructs together with V5-ATP5A1 or Flag-SIRT3 for 12 hours and then treated with several Tat-GRA8 peptides (10 μM) for 6 hours. 293T cells were used for GST pulldown followed by IB with αV5 or αFlag. WCL was used for IB together with αGST, αV5, αFlag or αActin. (C) Schematic of ATRAM-GRA8-M/AS and its mutants. (D) Bacterially purified 6xHis-rATRAM-GRA8-M/AS and its mutants were analyzed by immunoblotting (IB) for Coomassie blue staining (left) or αHis (right). (E) BMDMs were incubated with rATRAM-GRA8-M/AS and its mutants (20 μg/ml) for the indicated times, then cell viability was measured by MTT assay. Data represent 4 independent experiments with similar results (A, B, D and E)].
Figure 3 shows that a minimal region of identified GRA8 directly interacts with ATP5A1 and SIRT3 [mammalian GST-ATP5A1 or GST-SIRT3 was constructed together with Flag-GRA8 to 293T for 6 h after 12 h of transfection. Eggplant Tat-GRA8 peptide (10 μM) was treated. 293T cells were used for GST pulldown followed by IB with anti-Flag. Whole cell lysates were used for IB with αGST, αFlag or anti-Actin. Data represent 4 independent experiments with similar results].
Figure 4 shows HCT116 cell death by rATRAM-GRA8-M/AS-induced mitochondrial activation [(A and B) HCT116 cells were treated with WT rGRA8 or rATRAM-GRA8-M/AS and mutants thereof for the indicated times. After culturing, cell viability was measured by MTT assay. (CE) HCT116 cells were transduced with lentivirus-shRNA-NS or lentivirus-shRNA-PKCα or SIRT3 (MOI = 100) with polybrene (8 μg/mL) (right) for 2 days. Cells were stimulated with rATRAM-GRA8-M/AS for 1 day, and OXPHOS V enzymatic activity was applied. (C) Mito Tracker fluorescence signal was confirmed by flow cytometry. Bar graphs show mitochondrial mass MFI (D) or mtDNA content of BMDM was measured by quantitative real-time PCR. mtDNA content was normalized to nuclear DNA. (E) Significant difference compared to control treatment (** P <0.01; *** P <0.001). Data represent 5 independent experiments with similar results].
Figure 5 shows the pharmacokinetics and drug distribution of rATRAM-GRA8-M/AS [(A) Pharmacokinetics and pharmacokinetic distribution of rATRAM-GRA8-M/AS in tumor-bearing mice. As described in the Methods section, HCT116 cells were injected subcutaneously into the flanks of BALB/c mice. Mice were administered once or twice via intraperitoneal injection and sacrificed on
Figure 6 shows the antitumor activity of rATRAM-GRA8-M/AS in HCT116 xenografts [(A and B) or schematic of xenograft models treated with rATRAM-GRA8-M/AS or mutants thereof (top). HCT116 cells were subcutaneously injected into the flanks of nude mice. Tumor length and width were measured using calipers, and tumor volume was calculated every 3 days for 30 days. On
Figure 7 shows the knockdown effect in HCT 116 and immunogenicity of rATRAM-GRA8-M/AS in vivo [(A) HCT116 cells were incubated with polybrene (8 μg/mL) for 2 days and lentivirus-shRNA- NS or lentivirus-shRNA-PKCα, SIRT3 or ATP5A1 were transduced. Immunoblotting was performed with anti-PKCα, anti-ATP5A1, anti-SIRT3 or anti-Actin. Data show 5 independent experiments with similar results. (B) Schematic diagram of a xenograft model treated with rVector (top). HCT116-expressed ATP5A1 WT and mutant cells were injected subcutaneously into the flanks of nude mice. Significant differences compared to rVector. Each group contained 10 mice. Data show two independent experiments with similar results. (C) Humoral immune response in BALB/C mice. Mice were immunized by each route (intravenous or intraperitoneal) every 3 days and bled on
8 shows a cleavage map of the pRSFDuet-1 vector used to construct the recombinant expression vector according to the present invention.
9 shows a cleavage map of the recombinant expression vector pRSFDuet-1-ATRAM-G8-M/AS according to the present invention.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시하나, 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명이 범주 및 기술사상 범위 내에서 다양한 변경 및 수정이 가능함은 이 기술분야의 통상의 기술자에게 있어서 명백한 것이며, 이러한 변형 및 수정이 첨부된 특허청구범위에 속하는 것도 당연한 것이다.Hereinafter, preferred embodiments are presented to aid understanding of the present invention, but the following examples are only illustrative of the present invention, and various changes and modifications are possible within the scope and spirit of the present invention. It is obvious to those skilled in the art, and it goes without saying that these variations and modifications fall within the scope of the appended claims.
[재료와 방법][Materials and methods]
모든 동물 실험 절차는 기관 동물 관리 및 사용위원회 (프로토콜 2016-0221 및 2017-0218)와 한양대학교 기관 검토위원회(HYI-17-227-1)에 의해 검토되고 승인되었다. 모든 동물 실험은 한국 식품 의약품 안전청 지침에 따라 수행되었다.All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (Protocols 2016-0221 and 2017-0218) and the Institutional Review Board of Hanyang University (HYI-17-227-1). All animal experiments were performed in accordance with the guidelines of the Korea Food and Drug Administration.
마우스 및 세포 배양Mice and cell culture
야생형 C57BL/6 및 BALB/c 마우스는 오리엔트 바이오 (경기도)에서 구입하였다. 골수-유래 대식세포 (BMDM)를 C57BL/6 마우스로부터 분리하고, M-CSF (R & D Systems, 416-ML)를 포함한 DMEM에서 3-5 일 동안 배양하였다 (Toxoplasma gondii GRA7-Targeted ASC and PLD1 Promote Antibacterial Host Defense via PKCalpha. PLoS Pathog. 2017; 13:e1006126. 참조). HEK293T 세포 (ATCC-11268; American Type Culture Collection), HCT116 (ATCC-CCL247), HT-29 (ATCC-HTB-38), HepG2 (ATCC-HB-8065), Hep3B (ATCC-HB-8064), MCF7 (ATCC-HTB-22) 및 MDAMB-231 (ATCC-HTB-26)은 10% FBS (Invitrogen), 피루브산 나트륨, 비필수 아미노산, 페니실린 G (100IU/ml) 및 스트렙토마이신 (100 μg/ml)를 함유하는 DMEM (Invitrogen)에서 유지되었으며, 일시적인 형질감염은 제조사의 지시에 따라 리포펙타민3000(Lipofectamine3000; Invitrogen) 또는 인산 칼슘 (Clontech)을 사용하여 수행되었다. 400-800 ㎍/ml의 G418을 갖는 표준 선택 프로토콜을 사용하여 HCT116 안정 세포주를 생성하였다.Wild-type C57BL/6 and BALB/c mice were purchased from Orient Bio (Gyeonggi-do). Bone marrow-derived macrophages (BMDM) were isolated from C57BL/6 mice and cultured in DMEM containing M-CSF (R & D Systems, 416-ML) for 3-5 days (Toxoplasma gondii GRA7-Targeted ASC and PLD1 See Promote Antibacterial Host Defense via PKCalpha. PLoS Pathog. 2017; 13:e1006126.). HEK293T cells (ATCC-11268; American Type Culture Collection), HCT116 (ATCC-CCL247), HT-29 (ATCC-HTB-38), HepG2 (ATCC-HB-8065), Hep3B (ATCC-HB-8064), MCF7 (ATCC-HTB-22) and MDAMB-231 (ATCC-HTB-26) were formulated with 10% FBS (Invitrogen), sodium pyruvate, non-essential amino acids, penicillin G (100 IU/ml) and streptomycin (100 μg/ml). containing DMEM (Invitrogen), and transient transfection was performed using Lipofectamine 3000 (Invitrogen) or calcium phosphate (Clontech) according to the manufacturer's instructions. The HCT116 stable cell line was generated using a standard selection protocol with 400-800 μg/ml of G418.
재조합 ATRAM-GRA8 mito-ATP5A1/SIRT3 단백질Recombinant ATRAM-GRA8 mito-ATP5A1/SIRT3 protein
Novagen의 표준 프로토콜에 따라서, 재조합 정제된 단백질은 ATRAM 아미노산 잔기 (GLAGLAGLLGLEGLLGLPLGLLEGLWLGLELEGN), 미토콘드리아 표적화 서열 (ATATTTTTTR) 및 ATP5A1/SIRT3 결합 서열 (YRFTVQTTQN)을 수득하기 위해 Novagen사의 N-말단(N-terminal)이 6×His 표지(tag)된 pRSFDuet-1 벡터(vector)[도 8]에 삽입, 복제(clone)하여 사용하였다. 이때, 상기 벡터 제조사인 Novagen사가 추천한 표준 프로토콜(standard protocol)에 따라 단백질 발현 숙주로서 E. coli BL-21(DE-3)pLysS를 사용하였으며, 상기 E.coli 균주에서 단백질 발현을 유도하고, 수집(harvest)하여 정제하여 사용하였다. rGRA8을 투과성 셀룰로스 막으로 투석하고 Limulus amebocyte lysate assay (BioWhittaker)로 리포폴리 사카라이드 오염을 테스트하고 여기에 설명된 실험에 사용된 rGRA8 단백질 농도는 < 20 pg/ml를 함유하였다.According to Novagen's standard protocol, the recombinantly purified protein was modified at the N-terminus of Novagen to obtain the ATRAM amino acid residue (GLAGLAGLLLGLEGLLGLPLGLLEGLWLGLELEGN), the mitochondrial targeting sequence (ATATTTTTTR) and the ATP5A1/SIRT3 binding sequence (YRFTVQTTQN). ×His-tagged pRSFDuet-1 vector (vector) [Fig. 8] was inserted and cloned and used. At this time, E. coli BL-21 (DE-3) pLysS was used as a protein expression host according to the standard protocol recommended by Novagen, the vector manufacturer, and protein expression was induced in the E. coli strain, It was harvested, purified, and used. rGRA8 was dialyzed against a permeable cellulose membrane and tested for lipopolysaccharide contamination by Limulus amebocyte lysate assay (BioWhittaker) and the rGRA8 protein concentration used in the experiments described herein contained <20 pg/ml.
시약 및 항체reagents and antibodies
인간 PKCα (RHS4531-EG5578) 및 SIRT3 (RHS4531-EG23410) 표적 shRNA 플라스미드 DNA은 Open Biosystems에서 구입하였다. ATP5C1 (PA5-29975)에 대한 특이적 항체는 Invitrogen에서 구입하였다. ATP5A1 (51), SIRT3 (14.45), VDAC (B-6), SDHA (B-1), UQCRC2 (G-10), COX IV (D-20), PGC-1β (E-9), Tfam (H-203), PKCα (C-20), Actin (I-19), V5 (H-9), Flag (D-8), His (AD1.1.10), Lys-AC (AKL5C1) 및 GST (B-14)에 특정한 항체는 Santa Cruz Biotechnology에서 구입하였다.Human PKCα (RHS4531-EG5578) and SIRT3 (RHS4531-EG23410) targeting shRNA plasmid DNAs were purchased from Open Biosystems. A specific antibody against ATP5C1 (PA5-29975) was purchased from Invitrogen. ATP5A1 (51), SIRT3 (14.45), VDAC (B-6), SDHA (B-1), UQCRC2 (G-10), COX IV (D-20), PGC-1β (E-9), Tfam ( H-203), PKCα (C-20), Actin (I-19), V5 (H-9), Flag (D-8), His (AD1.1.10), Lys-AC (AKL5C1) and GST (B Antibodies specific to -14) were purchased from Santa Cruz Biotechnology.
플라스미드 제조Plasmid manufacturing
GRA8 (GST 또는 Flag-GRA8), ATP5A1 (GST 또는 V5-ATP5A1) 및 SIRT3 (GST 또는 Flag-SIRT3) 플라스미드의 전장을 암호화하는 플라스미드는 비특허문헌 1에 기술되었다. 본 발명에 사용된 플라스미드는 다음 표 1에 나타내었다.Plasmids encoding the full lengths of GRA8 (GST or Flag-GRA8), ATP5A1 (GST or V5-ATP5A1) and SIRT3 (GST or Flag-SIRT3) plasmids are described in
[표 1][Table 1]
펩타이드peptide
Tat-접합 GRA8 펩타이드는 상업적으로 합성하고 세포에서의 비정상적인 반응을 피하기 위해 아세테이트 염 형태로 Peptron (한국)에서 정제되었다. 본 발명에서 사용된 펩타이드의 아미노산 서열은 하기 표 2 및 3에 기재되어 있다. Limulus amebocyte lysate assay (BioWhittaker)에 의해 측정된 내독소 함량은 실험에 사용된 펩타이드의 농도에서 3-5 pg/ml 미만을 함유하였다.The Tat-conjugated GRA8 peptide was commercially synthesized and purified by Peptron (Korea) in the form of its acetate salt to avoid abnormal reactions in cells. The amino acid sequences of the peptides used in the present invention are listed in Tables 2 and 3 below. The endotoxin content measured by Limulus amebocyte lysate assay (BioWhittaker) contained less than 3-5 pg/ml at the concentration of the peptide used in the experiment.
[표 2] [Table 2]
[표 3][Table 3]
GST 풀다운, 면역 블롯 및 면역침강 분석GST pull-down, immunoblot and immunoprecipitation assays
GST 풀다운, 면역침강 및 면역 블롯 분석은 C.S. Yang et al., Nat. Commun., 6:6115 (2015) 및 H.J. Koh et al., PLoS Pathog., 13(1):e1006126 (2017)에 설명되어 있다. GST 풀다운의 경우, 세포를 얻어 complete protease inhibitor cocktail (Roche)이 보충된 NP-40 완충액에 용해시켰다. 원심 분리 후, 상층액을 단백질 A/G bead로 4 ℃에서 2시간 동안 예비-정제하였다. 예비-정제된 용해물을 glutathione-conjugated Sepharose beads (Amersham Biosciences)의 50% 슬러리와 혼합하고, 결합반응을 4 ℃에서 4시간 동안 배양하였다. 침전물을 용해 완충액으로 여러 번 세척하였다. 글루타티온 비드에 결합된 단백질을 5분 동안 가열함으로써 SDS 로딩 완충액으로 녹였다. GST pull-down, immunoprecipitation and immunoblot analysis were performed by C.S. Yang et al., Nat. Commun., 6:6115 (2015) and H.J. Koh et al., PLoS Pathog., 13(1):e1006126 (2017). For GST pull-down, cells were harvested and lysed in NP-40 buffer supplemented with complete protease inhibitor cocktail (Roche). After centrifugation, the supernatant was pre-purified with protein A/G beads at 4°C for 2 hours. The pre-purified lysate was mixed with a 50% slurry of glutathione-conjugated Sepharose beads (Amersham Biosciences), and the conjugation reaction was incubated at 4°C for 4 hours. The precipitate was washed several times with lysis buffer. Proteins bound to glutathione beads were dissolved in SDS loading buffer by heating for 5 minutes.
면역침강을 위해, 세포를 얻은 후 컴플리트 프로테아제 저해제 칵테일(complete protease inhibitor cocktail; Roche)이 보충된 NP-40 완충액에 용해시켰다. 4 ℃에서 1 시간 동안 단백질 A/G 아가로스 비드로 예비-정제한 후, 전 세포 용해물을 지시된 항체와의 면역 침전을 위해 사용하였다. 일반적으로, 1-4 μg의 상업용 항체를 1 ml의 세포 용해물에 첨가하고 4 ℃에서 8 내지 12 시간 동안 배양하였다. 단백질 A/G 아가로스 비드를 6시간 동안 첨가한 후, 면역 침전물을 용해 완충액으로 여러 번 세척하고 5분 동안 가열시킴으로써 SDS 로딩 완충액으로 녹였다. For immunoprecipitation, cells were harvested and lysed in NP-40 buffer supplemented with complete protease inhibitor cocktail (Roche). After pre-purification with protein A/G agarose beads for 1 hour at 4°C, whole cell lysates were used for immunoprecipitation with the indicated antibodies. Typically, 1-4 μg of commercial antibody was added to 1 ml of cell lysate and incubated at 4° C. for 8-12 hours. After adding protein A/G agarose beads for 6 hours, the immunoprecipitates were washed several times with lysis buffer and lysed with SDS loading buffer by heating for 5 minutes.
면역 블롯팅을 위해, 폴리펩타이드는 SDS-폴리아크릴아마이드 겔에 의해 전기영동(PAGE)하고 PVDF 막 (Bio-Rad)으로 옮겨서 분리하였다. 면역 검출은 특정 항체로 진행되었다. 항체 결합은 화학 발광 (ECL; Millipore)에 의해 시각화하고 Vilber 화학 발광 분석기 (Fusion SL 3; Vilber Lourmat)에 의해 검출되었다.For immunoblotting, polypeptides were separated by electrophoresis (PAGE) on SDS-polyacrylamide gels and transferred to PVDF membranes (Bio-Rad). Immunodetection was performed with specific antibodies. Antibody binding was visualized by chemiluminescence (ECL; Millipore) and detected by a Vilber chemiluminescence analyzer (
세포 분획cell fraction
사이토졸 및 미토콘드리아는 미토콘드리아 분획 키트 (Active Motif, 40015)를 사용하거나 Toxoplasma gondii GRA8 induces ATP5A1-SIRT3- mediated mitochondrial metabolic resuscitation: a potential therapy for sepsis. Exp Mol Med. 2018; 50:e464.에 기술된 바와 같이 세포로부터 분리되었다. 세포 내 분획 화 단백질을 2% SDS를 함유하는 완충액에 용해시키고 SDS-PAGE를 위해 2x reducing sample buffer으로 가열시켰다.Cytosol and mitochondria were isolated using a mitochondrial fractionation kit (Active Motif, 40015) or Toxoplasma gondii GRA8 induces ATP5A1-SIRT3- mediated mitochondrial metabolic resuscitation: a potential therapy for sepsis. Exp Mol Med. 2018; Cells were isolated as described in 50:e464. Intracellular fractionated proteins were dissolved in buffer containing 2% SDS and heated with 2x reducing sample buffer for SDS-PAGE.
MTT 분석MTT assay
비특허문헌 1에 설명한 것과 같이, 비처리 군에 대한 세포 생존율은 MTT 분석에 의해 측정되었다. 명시된 시간 동안 배양한 후, 배지에 5 mg/ml의 MTT (3-(4,5-디메틸 티아졸-2-일)-2,5-디페닐테트라졸륨브로마이드) 용액을 첨가하고, 세포를 추가로 4시간 배양하였다. 이어서, 모든 배지를 제거하고 동일한 부피의 디메틸 설폭사이드 (DMSO) 용액을 15 분 동안 첨가하여 포르마잔을 용해시켰다. UV/VIS 분광 광도계를 사용하여, 플레이트의 각각의 웰을 540 nm에서 측정하여 상대 세포 생존력을 측정하였다.As described in
미토콘드리아 DNA 정량Mitochondrial DNA quantification
미토콘드리아 DNA(mtDNA)의 카피 수(copy number)를 정량화하기 위해, The AMPK-PPARGC1A pathway is required for antimicrobial host defense through activation of autophagy. Autophagy. 2014; 10:785-802..에 설명되어 있는 방법에 따라 미토콘드리아 DNA(mitochondrial DNA, mtDNA) 대비 핵 DNA(nuclear DNA, nDNA) 비율(ratio)로 측정하였다. 피루베이트 키나제 (Pklr)를 mtDNA에 대한 nDNA 및 NADH 탈수소 효소 서브유닛 1 (mt-Nd1)에 대한 마커로서 사용하였다. 제조사의 지시 (QuantiFast SYBR 녹색 PCR 마스터 믹스; Qiagen, 204052)에 따라 실시간 PCR 반응을 수행하고, QuantStudio™ 3 (ABI)에서 온도 사이클링을 수행하였다. mtDNA 함량을 핵산 DNA 함량으로 표준화하였다.To quantify the copy number of mitochondrial DNA (mtDNA), The AMPK-PPARGC1A pathway is required for antimicrobial host defense through activation of autophagy. Autophagy. 2014; According to the method described in 10:785-802.., the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nuclear DNA, nDNA) was measured. Pyruvate kinase (Pklr) was used as a marker for nDNA and NADH dehydrogenase subunit 1 (mt-Nd1) for mtDNA. Real-time PCR reactions were performed according to the manufacturer's instructions (QuantiFast SYBR Green PCR Master Mix; Qiagen, 204052) and temperature cycling was performed in QuantStudio™ 3 (ABI). mtDNA content was normalized to nucleic acid DNA content.
미토콘드리아 막 전위 측정Measurement of mitochondrial membrane potential
C.S. Yang et.al., Autophagy, 10(5):[0198] 785-802 (2014)에 설명된 바와 같이, 온전한 세포의 미토콘드리아 막 전위 (ΔΨm)를 변형과 함께 측정하였다. TMRE (테트라 메틸로다민, 에틸 에스테르; 200 nM, 분자 프로브-Invitrogen, T669)를 세포 현탁액에 첨가하였다. 세포를 어두운 곳에서 37℃에서 30분 동안 배양하였다. ΔΨm은 유세포 분석에 의해 측정되었고, 데이터는 FlowJo 소프트웨어를 사용하여 분석되었다. FLRE 채널 (582 nm)을 사용하여 TMRE 형광을 측정하였다.C.S. As described in Yang et.al., Autophagy, 10(5):[0198] 785-802 (2014), mitochondrial membrane potential (ΔΨm) of intact cells was measured with modification. TMRE (tetra methylrhodamine, ethyl ester; 200 nM, Molecular Probes-Invitrogen, T669) was added to the cell suspension. Cells were incubated for 30 minutes at 37°C in the dark. ΔΨm was measured by flow cytometry and data were analyzed using FlowJo software. TMRE fluorescence was measured using the FLRE channel (582 nm).
미토콘드리아 복합체 V(Complex V) 활성 분석Mitochondrial complex V activity assay
제조사의 지시에 따라 Abcam (ab109907, MA, Cambridge)에서 MitoTox Complex V OXPHOS Activity Microplate Assay 키트를 사용하여 Complex V의 활성을 측정하였다. Complex V의 활성은 30℃에서 1시간 동안 340nm에서의 흡광도 변화를 모니터링함으로써 측정되었다. 올리고마이신 (Sigma, O4876)을 분석을 위한 양성 대조군으로 사용하였다.Complex V activity was measured using the MitoTox Complex V OXPHOS Activity Microplate Assay kit from Abcam (ab109907, Cambridge, MA) according to the manufacturer's instructions. The activity of Complex V was measured by monitoring the change in absorbance at 340 nm for 1 hour at 30°C. Oligomycin (Sigma, O4876) was used as a positive control for the assay.
마우스 모델 이종 이식mouse model xenograft
4-6 주령의 암컷 무흉선 누드 마우스 (Central Lab. Animal, Korea)를 종양 이종 이식 실험에 사용하였다 (Bacillus calmette-guerin cell wall cytoskeleton enhances colon cancer radiosensitivity through autophagy. Autophagy. 2010; 6:46-60.). 간략하게, 동물에게 0.1 mL 세포 배지에 1 x 106 HCT116 세포를 피하주사하고 종양 부피 측정으로 ~ 7-10일 동안 관찰하였다. 종양 크기가 200 또는 600 mm3의 평균 부피에 도달했을 때 치료를 시작하였다. 피부 캘리퍼로 종양 부피를 3일마다 측정하고 종양 길이 × 종양 폭2 × 0.5로 계산하고 mm3으로 나타내었다. 모든 동물은 특정 병원체가없는 환경에서 유지되었다.4-6 week old female athymic nude mice (Central Lab. Animal, Korea) were used for tumor xenograft experiments (Bacillus calmette-guerin cell wall cytoskeleton enhances colon cancer radiosensitivity through autophagy. Autophagy. 2010; 6:46-60 .). Briefly, animals were subcutaneously injected with 1 x 10 6 HCT116 cells in 0.1 mL cell medium and observed for ~7-10 days by tumor volume measurement. Treatment was started when the tumor size reached an average volume of 200 or 600 mm 3 . Tumor volume was measured every 3 days with skin calipers and calculated as tumor length x tumor width 2 x 0.5 and expressed in mm 3 . All animals were maintained in a specific pathogen-free environment.
통계 분석statistical analysis
모든 데이터는 다중 비교를 위해 Bonferroni adjustment 또는 ANOVA와 함께 Student 's t-test를 사용하여 분석되었으며 평균 ± SD로 표시된다. SPSS (Version 12.0) 통계 소프트웨어 프로그램 (SPSS)을 사용하여 통계 분석을 수행하였다. 차이는 p <0.05에서 유의미한 것으로 간주되었다.All data were analyzed using Student's t-test with Bonferroni adjustment or ANOVA for multiple comparisons and are presented as mean ± SD. Statistical analysis was performed using the SPSS (Version 12.0) statistical software program (SPSS). Differences were considered significant at p < 0.05.
[결과][result]
rGRA8-PKCα-SIRT3-ATP5A1 경로를 통해 결장 세포 사멸을 유도한다Induces colon apoptosis through the rGRA8-PKCα-SIRT3-ATP5A1 pathway
PKCα-포스포릴화된 GRA8은 미토콘드리아 SIRT3과 결합하고; SIRT3은 ATP5A1과 상호 작용하여 아세틸화 상태 및 활성을 조절한다. 탈아세틸화된 ATP5A1은 다양한 미토콘드리아 과정에 관여하며, SIRT3는 종양 성장에 중요한 미토콘드리아 활성에서 전반적인 변화를 조정할 수 있다. 우리는 rGRA8 및 그 돌연변이가 다양한 암 세포주에서 세포 사멸을 유발하는지 여부를 결정하였다. 도 1의 A 및 B는 WT rGRA8이 시간에 따라 인간 결장암 세포주(HCT116 및 HT-29)에서 세포 사멸을 유도하였지만, rGRA8 돌연변이는 그렇게 않는 것을 보여준다. 대식세포에서 처리와 유사하게(비특허문헌 1), WT rGRA8에 의한 HCT116 세포의 처리는 ATP5A1, SIRT3 및 ATP5C1에 대한 rGRA8의 결합을 현저하게 증가시킬뿐 만 아니라 ATP5A1 탈아세틸화를 증가시켰다 (도 1의 C). 따라서, WT rGRA8은 미토콘드리아 대사 소생 경로를 통해 인간 결장암 세포에서 세포 사멸을 유도한다.PKCα-phosphorylated GRA8 binds to mitochondrial SIRT3; SIRT3 interacts with ATP5A1 to regulate its acetylation status and activity. Deacetylated ATP5A1 is involved in various mitochondrial processes, and SIRT3 can mediate global changes in mitochondrial activity that are important for tumor growth. We determined whether rGRA8 and its mutations induce apoptosis in various cancer cell lines. 1A and B show that WT rGRA8 induced apoptosis in human colon cancer cell lines (HCT116 and HT-29) over time, but rGRA8 mutants did not. Similar to treatment in macrophages (Non-Patent Document 1), treatment of HCT116 cells with WT rGRA8 markedly increased the binding of rGRA8 to ATP5A1, SIRT3 and ATP5C1, as well as increased ATP5A1 deacetylation (Fig. 1 C). Thus, WT rGRA8 induces apoptosis in human colon cancer cells through the mitochondrial metabolic resuscitation pathway.
종양을 표적을 위해 설계된 ATRAM-접합 GRA8 펩타이드ATRAM-conjugated GRA8 peptide designed to target tumors
결합 연구는 GRA8의 C-말단 40개의 아미노산 서열 (aa 183-222) 또는 28개의 아미노산 서열 (aa 242-269)이 각각 미토콘드리아 표적화 또는 ATP5A1/SIRT3 상호 작용에 충분하다는 것을 보여주었다 (비특허문헌 1). 따라서, 본 발명자들은 단백질 분해를 피하면서 세포 내 전달을 위해 retro-inverso 펩타이드로서 알려진 HIV-1 Tat 단백질 형질 도입 도메인을 GRA8의 9-10 번째 아미노산 서열에 추가하였으며 Tat-GRA8 펩타이드로 나타낸다 (표 1 및 2). 이들 펩타이드는 미토콘드리아 표적화 및 ATP5A1/SIRT3 상호 작용을 위해 잠재적인 GRA8의 최소 잔기를 시험하였다. GST-GRA8에 의한 형질감염 12시간 후 또는 rGRA8에 의한 처리에서, 293T세포 또는 골수 유래 대식세포를 Tat 단독 또는 다른 Tat-GRA8 펩타이드와 함께 배양한 후 세포이하의 분획화하여 GRA8의 미토콘드리아 위치를 결정하였다. 이는 Tat-GRA8 (aa 213-222) 펩타이드가 배양 6시간 이내에 미토콘드리아 표적을 효율적으로 차단할 수 있는 반면, Tat 펩타이드만으로는 불가능하다는 것을 보여주었다 (도 2의 A). 또한, Tat-GRA8 (aa260-269) 펩타이드는 293T 세포에서 GST-풀다운 분석에 의해 결정된 바와 같이 배양 6시간 이내에 GRA8-ATP5A1/SIRT3 상호 작용을 효과적으로 차단할 수 있었다 (도 2의 B 및 도 3). 따라서, 이들 데이터는 미토콘드리아 표적화 및 ATP5A1/SIRT3 결합에 대한 GRA8의 최소 영역을 보여준다. Binding studies showed that the C-
ATRAM은 산성 조건에서 암 세포와 강한 상호 작용을 하여 우수한 CTP를 강조하였다. 따라서, 종양을 표적으로 하고 미토콘드리아 대사를 활성화시키기 위해 ATRAM 접합된 다기능 GRA8 펩타이드를 설계하였다 (도 2의 C). 암 세포에서 ATRAM-접합된 다기능의 GRA8 펩타이드의 역할을 조사하기 위해, 본 발명자들은 박테리아 정제된 His가 태그된 rATRAM-GRA8-M/AS 및 그의 돌연변이를 생성하였다. 정제된 rATRAM-GRA8-M/AS (10 kDa)는 SDS-PAGE 및 면역 블롯팅에 의해 확인되었다 (도 2의 D). rATRAM-GRA8-M/AS 및 그 돌연변이 대해 대식세포에서 유의한 세포 독성 차이가 벡터 대조군과 비교하여 관찰되지 않았다 (도 2의 E). 따라서, ATRAM-접합된 다기능 GRA8 펩타이드는 암세포 미토콘드리아 대사를 조절함으로써 암세포에 특이적일 것으로 예상된다.ATRAM had a strong interaction with cancer cells under acidic conditions, highlighting good CTP. Therefore, an ATRAM-conjugated multifunctional GRA8 peptide was designed to target tumors and activate mitochondrial metabolism (Fig. 2C). To investigate the role of ATRAM-conjugated multifunctional GRA8 peptides in cancer cells, we generated bacterially purified His-tagged rATRAM-GRA8-M/AS and mutants thereof. Purified rATRAM-GRA8-M/AS (10 kDa) was confirmed by SDS-PAGE and immunoblotting (Fig. 2D). No significant difference in cytotoxicity in macrophages was observed for rATRAM-GRA8-M/AS and its mutants compared to the vector control (Fig. 2E). Thus, ATRAM-conjugated multifunctional GRA8 peptides are expected to be cancer cell specific by regulating cancer cell mitochondrial metabolism.
rATRAM-GRA8-M/AS는 HCT116에서 미토콘드리아 활성과 생합성을 보여준다rATRAM-GRA8-M/AS shows mitochondrial activity and biosynthesis in HCT116
본 발명자들은 rATRAM-GRA8-M/AS에 rGRA8 유사 모방 약리학적 및 생물학적 프로파일이 있는지 확인하였다. rGRA8 단독의 활성과 일치하여, rATRAM-GRA8-M/AS는 또한 HCT116 세포에서 용량-의존적 방식으로 세포 사멸을 유도하였다. 놀랍게도, rATRAM-GRA8-M/AS의 IC50은 0.1 μg/ml였으며, 이는 rGRA8 단독에 대해 발견된 20 μg/ml의 양 보다 IC50에서 200배 개선된 것이다 (도 4의 A). 또한, rATRAMGRA8-M/AS와 HCT116 세포에서 그의 돌연변이 또는 벡터 대조군 사이에 세포 독성의 유의한 차이가 관찰되지 않았다 (도 4의 B). 이 발견은 미토콘드리아 표적화 및 ATP5A1/SIRT3에 대한 GRA8 영역이 암세포 사멸에 필수적이라는 것을 보여준다. 이후, 본 발명자들은 rATRAMGRA8-M/AS의 미토콘드리아 대사 파라미터를 조사하였다. 도 4의 C 및 도 4의 D에서, rATRAM-GRA8-M/AS는 HCT116 세포에서 SIRT3 및 PKCα를 통한 Complex V OXPHOS 활성, 미토콘드리아 질량 및 미토콘드리아 DNA 함량을 향상시켰다. 따라서, rATRAM-GRA8-M/AS는 미토콘드리아-표적된 대사 소생법에 의해 선택적이고 강력한 대사-조절제로서 작용한다.We confirmed that rATRAM-GRA8-M/AS has rGRA8-like pharmacological and biological profiles. Consistent with the activity of rGRA8 alone, rATRAM-GRA8-M/AS also induced apoptosis in HCT116 cells in a dose-dependent manner. Surprisingly, the IC 50 of rATRAM-GRA8-M/AS was 0.1 μg/ml, a 200-fold improvement in IC 50 over the amount of 20 μg/ml found for rGRA8 alone (FIG. 4A). In addition, no significant difference in cytotoxicity was observed between rATRAMGRA8-M/AS and its mutant or vector control in HCT116 cells (FIG. 4B). This finding shows that the GRA8 domain for mitochondrial targeting and ATP5A1/SIRT3 is essential for cancer cell death. Then, the present inventors investigated mitochondrial metabolic parameters of rATRAMGRA8-M/AS. 4C and 4D , rATRAM-GRA8-M/AS enhanced Complex V OXPHOS activity, mitochondrial mass, and mitochondrial DNA content through SIRT3 and PKCα in HCT116 cells. Thus, rATRAM-GRA8-M/AS acts as a selective and potent metabolism-modulator by mitochondria-targeted metabolic resuscitation.
HCT116 이종 이식에서 rATRAM-GRA8-M/AS의 항종양 활성Antitumor activity of rATRAM-GRA8-M/AS in HCT116 xenografts
생체 내에서의 치료 가능성을 확인하기 전에 rATRAMGRA8-M/AS의 약동학 및 약전분포를 조사하였다. rATRAM-GRA8-M/AS 단백질은 면역 블롯팅에 의해 검출된 바와 같이 여러 기관의 종양 세포에 위치하였다. 이 위치는 최대 2일 동안 유지되었고 3일째까지 점차 사라졌다 (도 5의 A).The pharmacokinetics and pharmacopoietic distribution of rATRAMGRA8-M/AS were investigated before confirming its therapeutic potential in vivo. rATRAM-GRA8-M/AS protein was localized to tumor cells in several organs as detected by immunoblotting. This position was maintained for up to 2 days and gradually disappeared by the 3rd day (Fig. 5A).
HCT116 세포를 함유하는 이종 이식 마우스에서 rATRAM-GRA8-M/AS의 항종양 활성을 확인하였다. 마우스에서 rATRAM-GRA8-M/AS (20 μg/kg, 복강 내 주사) 처리는 처음 성장이 시작되는 종양 (도 6의 A) 및 활발히 성장하는 종양의 (도 6의 B) 성장을 억제하였다. 또한, 잠재적인 임상 적용 가능성을 평가하기 위해 미토콘드리아 관련 대상 단백질-결합 프로필 및 OXPHOS 활동을 결정하여 rATRAM-GRA8-M/AS를 약리학적으로 생체 조건에서 활성 여부를 확인하였다. rATRAM-GRA8-M/AS로의 처리는 rGRA8의 ATP5A1, SIRT3 및 ATP5C1에 대한 결합을 현저하게 증가시켰다. 또한, ATP5A1 탈아세틸화 수준 및 미토콘드리아 생발생 단백질 수준은 종양 용해물에서 증가하였다 (도 6의 A, 하단). 놀랍게도, rATRAM-GRA8-M/AS는 20 μg/kg의 IC50을 가졌으며, 이는 생체 내에서 rGRA8 단독에 대해 10 mg/kg의 것에 비해 IC50에서 500배 개선된 것 (도 6의 A 및 도 5의 B)이다. Antitumor activity of rATRAM-GRA8-M/AS was confirmed in xenograft mice containing HCT116 cells. In mice, treatment with rATRAM-GRA8-M/AS (20 μg/kg, intraperitoneal injection) inhibited the growth of initially growing tumors (FIG. 6A) and actively growing tumors (FIG. 6B). In addition, to evaluate the potential clinical application, the pharmacological activity of rATRAM-GRA8-M/AS in vivo was confirmed by determining the mitochondrial-related target protein-binding profile and OXPHOS activity. Treatment with rATRAM-GRA8-M/AS markedly increased the binding of rGRA8 to ATP5A1, SIRT3 and ATP5C1. In addition, the levels of ATP5A1 deacetylation and mitochondrial biogenesis were increased in tumor lysates (Fig. 6A, bottom). Surprisingly, rATRAM-GRA8-M/AS had an IC 50 of 20 μg/kg, a 500-fold improvement in IC 50 compared to that of 10 mg/kg for rGRA8 alone in vivo (Fig. 6A and 5 B).
rATRAM-GRA8-M/AS에 의해 유발된 항종양 활성에서 PKCα, SIRT3 및 ATP5A1의 역할을 명확히 하기 위해, 특정 lentiviral shRNA를 사용하여 PKCα-, SIRT3- 및 ATP5A1-결핍 HCT116 세포를 생성하였다. 그 다음 rATRAM-GRA8-M/AS로 처리된 이종 이식 마우스에서 shPKCα, shSIRT3 및 shATP5A1-HCT116 세포의 종양 억제 효과를 평가하였다. HCT116 결장암 이종이식 모델에서 rATRAM-GRA8-M/AS에 의한 종양 크기 감소 및 종양 성장 지연은 PKCα, SIRT3, ATP5A1 (도 6의 C-E 및 도 7의 A) 및 ATPA1의 K506 및 K531의 아세틸화 상태에 의존하였다 (도 6의 F 및 도 7의 B). To clarify the roles of PKCα, SIRT3 and ATP5A1 in the antitumor activity induced by rATRAM-GRA8-M/AS, PKCα-, SIRT3- and ATP5A1-deficient HCT116 cells were generated using specific lentiviral shRNAs. Then, the tumor suppression effect of shPKCα, shSIRT3 and shATP5A1-HCT116 cells was evaluated in xenograft mice treated with rATRAM-GRA8-M/AS. In the HCT116 colon cancer xenograft model, the tumor size reduction and tumor growth delay by rATRAM-GRA8-M/AS were dependent on the acetylation status of PKCα, SIRT3, ATP5A1 (Fig. 6 C-E and Fig. 7 A) and ATPA1 K506 and K531. depended (FIG. 6F and FIG. 7B).
면역원성은 안전성 및 효능과 밀접한 관련이 있기 때문에 치료용 단백질을 개발하는데 중요한 요소 중 하나이다. 따라서 면역화 된 마우스에서 rATRAM-GRA8-M/AS에 대한 항체의 생산을 모니터링함으로써 rATRAM-GRA8-M/AS의 면역원성을 조사 하였다. rATRAM-GRA8-M/AS를 3회 연속 주사한 후, 효소결합면역 흡착분석법을 사용하여 IgG 항체의 수준을 분석하였다. Ovalbumin을 양성 대조군으로 사용하였다. rATRAM-GRA8-M/AS에 특이적인 항체의 수준은 무시할 수 있는 반면, Ovalbumin은 항체 수준의 현저한 증가를 일으켰다 (도 7의 C). 또한, rATRAM-GRA8-M/AS는 종양이 있는 마우스에서 염증성 사이토카인의 무시할만한 생산을 유도하였다. BALB/c 마우스에서 혈청의 TNF-α, IL-6 및 IL-1β의 수준은 음성 대조군의 수준과 거의 같았으며 (도 7의 D), rATRAM-GRA8-M/AS는 무시할 수 있는 면역원성과 면역 반응을 유발함을 보였다. 따라서, rATRAM-GRA8-M/AS는 생체적합성 생체 물질이고, rATRAM-GRA8-M/AS-매개 미토콘드리아 대사 소생술은 항종양 활성에 중요하다.Immunogenicity is one of the important factors in developing therapeutic proteins because it is closely related to safety and efficacy. Therefore, the immunogenicity of rATRAM-GRA8-M/AS was investigated by monitoring the production of antibodies against rATRAM-GRA8-M/AS in immunized mice. After three consecutive injections of rATRAM-GRA8-M/AS, the levels of IgG antibodies were analyzed using enzyme-linked immunosorbent assay. Ovalbumin was used as a positive control. While the level of antibodies specific to rATRAM-GRA8-M/AS was negligible, Ovalbumin caused a marked increase in antibody levels (Fig. 7C). In addition, rATRAM-GRA8-M/AS induced negligible production of inflammatory cytokines in tumor bearing mice. The levels of TNF-α, IL-6 and IL-1β in the serum of BALB/c mice were almost the same as those of the negative control group (Fig. 7D), and rATRAM-GRA8-M/AS had negligible immunogenicity and immunity. It has been shown to elicit a reaction. Thus, rATRAM-GRA8-M/AS is a biocompatible biomaterial, and rATRAM-GRA8-M/AS-mediated mitochondrial metabolic regeneration is important for its antitumor activity.
<110> Industry-University Cooperation Foundation Hanyang University ERICA Campus <120> Novel Toxoplasma gondii-GRA8 recombinant peptides and composition for preventing or treating cancer comprising the same <130> P19U22C1799 <150> KR 10-2019-0174088 <151> 2019-12-24 <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 92 <212> PRT <213> Artificial Sequence <220> <223> rATRAM-G8-M/AS <400> 1 Gly Leu Ala Gly Leu Ala Gly Leu Leu Gly Leu Glu Gly Leu Leu Gly 1 5 10 15 Leu Pro Leu Gly Leu Leu Glu Gly Leu Trp Leu Gly Leu Glu Leu Glu 20 25 30 Gly Asn Gly Leu Ala Gly Leu Ala Gly Leu Leu Gly Leu Glu Gly Leu 35 40 45 Leu Gly Leu Pro Leu Gly Leu Leu Glu Gly Leu Trp Leu Gly Leu Glu 50 55 60 Leu Glu Gly Asn Gly Gly Ala Thr Ala Thr Thr Thr Thr Thr Thr Arg 65 70 75 80 Gly Gly Tyr Arg Phe Thr Val Gln Thr Thr Gln Asn 85 90 <210> 2 <211> 269 <212> PRT <213> Toxoplasma gondii <400> 2 Met Ala Leu Pro Leu Arg Val Ser Ala Thr Val Phe Val Val Phe Ala 1 5 10 15 Val Phe Gly Val Ala Arg Ala Met Asn Gly Pro Leu Ser Tyr His Pro 20 25 30 Ser Ser Tyr Gly Ala Ser Tyr Pro Asn Pro Ser Asn Pro Leu His Gly 35 40 45 Met Pro Lys Pro Glu Asn Pro Val Arg Pro Pro Pro Pro Gly Phe His 50 55 60 Pro Ser Val Ile Pro Asn Pro Pro Tyr Pro Leu Gly Thr Pro Ala Gly 65 70 75 80 Met Pro Gln Pro Glu Val Pro Pro Leu Gln His Pro Pro Pro Thr Gly 85 90 95 Ser Pro Pro Ala Ala Ala Pro Gln Pro Pro Tyr Pro Val Gly Thr Pro 100 105 110 Gly Met Pro Gln Pro Glu Ile Pro Pro Val His Arg Pro Pro Pro Pro 115 120 125 Gly Phe Arg Pro Glu Val Ala Pro Val Pro Pro Tyr Pro Val Gly Thr 130 135 140 Pro Thr Gly Met Pro Gln Pro Glu Ile Pro Ala Val His His Pro Phe 145 150 155 160 Pro Tyr Val Thr Thr Thr Thr Thr Ala Ala Pro Arg Val Leu Val Tyr 165 170 175 Lys Ile Pro Tyr Gly Gly Ala Ala Pro Pro Arg Ala Pro Pro Val Pro 180 185 190 Pro Arg Met Gly Pro Ser Asp Ile Ser Thr His Val Arg Gly Ala Ile 195 200 205 Arg Arg Gln Pro Ala Thr Ala Thr Thr Thr Thr Thr Thr Arg Asn Val 210 215 220 Leu Leu Arg Thr Ala Ile Leu Ala Ala Ala Ala Ala Thr Leu Ile Ala 225 230 235 240 Leu Phe Arg Gln Arg Pro Leu Phe Thr Glu Gly Val Arg Met Phe Pro 245 250 255 Asp Phe Gln Tyr Arg Phe Thr Val Gln Thr Thr Gln Asn 260 265 <210> 3 <211> 276 <212> DNA <213> Artificial Sequence <220> <223> rATRAM-G8-M/AS <400> 3 ggcctggccg gcctggccgg cctgctgggc ctggaaggcc tgccgggcct gcccctgggc 60 ctgctggaag gcctgtggct gggcctggaa ctggaaggaa acggcctggc cggcctggcc 120 ggcctgctgg gcctggaagg cctgccgggc ctgcccctgg gcctgctgga aggcctgtgg 180 ctgggcctgg aactggaagg aaacgggggg gctaccgcca ccacaaccac tactacccgc 240 ggggggagat tcaccgtcca aacgacgcag aattaa 276 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Tat <400> 4 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 5 <211> 51 <212> PRT <213> Artificial Sequence <220> <223> Tat-GRA8-(183-222) <400> 5 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ala Ala Pro Pro Arg 1 5 10 15 Ala Pro Pro Val Pro Pro Arg Met Gly Pro Ser Asp Ile Ser Thr His 20 25 30 Val Arg Gly Ala Ile Arg Arg Gln Pro Ala Thr Ala Thr Thr Thr Thr 35 40 45 Thr Thr Arg 50 <210> 6 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Tat-GRA8-(183-192) <400> 6 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ala Ala Pro Pro Arg 1 5 10 15 Ala Pro Pro Val Pro 20 <210> 7 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Tat-GRA8-(193-202) <400> 7 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Pro Arg Met Gly Pro 1 5 10 15 Ser Asp Ile Ser Thr 20 <210> 8 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Tat-GRA8-(203-212) <400> 8 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly His Val Arg Gly Ala 1 5 10 15 Ile Arg Arg Gln Pro 20 <210> 9 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Tat-GRA8-(213-222) <400> 9 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ala Thr Ala Thr Thr 1 5 10 15 Thr Thr Thr Thr Arg 20 <210> 10 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Tat-GRA8-(242-269) <400> 10 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Phe Arg Gln Arg Pro 1 5 10 15 Leu Phe Thr Glu Gly Val Arg Met Phe Pro Asp Phe Gln Tyr Arg Phe 20 25 30 Thr Val Gln Thr Thr Gln Asn 35 <210> 11 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Tat-GRA8-(242-250) <400> 11 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Phe Arg Gln Arg Pro 1 5 10 15 Leu Phe Thr Glu 20 <210> 12 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Tat-GRA8-(251-259) <400> 12 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly Val Arg Met Phe 1 5 10 15 Pro Asp Phe Gln 20 <210> 13 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Tat-GRA8-(260-269) <400> 13 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Tyr Arg Phe Thr Val 1 5 10 15 Gln Thr Thr Gln Asn 20 <110> Industry-University Cooperation Foundation Hanyang University ERICA Campus <120> Novel Toxoplasma gondii-GRA8 recombinant peptides and composition for preventing or treating cancer comprising the same <130> P19U22C1799 <150> KR 10-2019-0174088 <151> 2019-12-24 <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 92 <212> PRT <213> artificial sequence <220> <223> rATRAM-G8-M/AS <400> 1 Gly Leu Ala Gly Leu Ala Gly Leu Leu Gly Leu Glu Gly Leu Leu Gly 1 5 10 15 Leu Pro Leu Gly Leu Leu Glu Gly Leu Trp Leu Gly Leu Glu Leu Glu 20 25 30 Gly Asn Gly Leu Ala Gly Leu Ala Gly Leu Leu Gly Leu Glu Gly Leu 35 40 45 Leu Gly Leu Pro Leu Gly Leu Leu Glu Gly Leu Trp Leu Gly Leu Glu 50 55 60 Leu Glu Gly Asn Gly Gly Ala Thr Ala Thr Thr Thr Thr Thr Thr Arg 65 70 75 80 Gly Gly Tyr Arg Phe Thr Val Gln Thr Thr Gln Asn 85 90 <210> 2 <211> 269 <212> PRT <213> Toxoplasma gondii <400> 2 Met Ala Leu Pro Leu Arg Val Ser Ala Thr Val Phe Val Val Phe Ala 1 5 10 15 Val Phe Gly Val Ala Arg Ala Met Asn Gly Pro Leu Ser Tyr His Pro 20 25 30 Ser Ser Tyr Gly Ala Ser Tyr Pro Asn Pro Ser Asn Pro Leu His Gly 35 40 45 Met Pro Lys Pro Glu Asn Pro Val Arg Pro Pro Pro Pro Gly Phe His 50 55 60 Pro Ser Val Ile Pro Asn Pro Pro Tyr Pro Leu Gly Thr Pro Ala Gly 65 70 75 80 Met Pro Gln Pro Glu Val Pro Pro Leu Gln His Pro Pro Pro Thr Gly 85 90 95 Ser Pro Pro Ala Ala Ala Pro Gln Pro Pro Tyr Pro Val Gly Thr Pro 100 105 110 Gly Met Pro Gln Pro Glu Ile Pro Pro Val His Arg Pro Pro Pro Pro 115 120 125 Gly Phe Arg Pro Glu Val Ala Pro Val Pro Pro Tyr Pro Val Gly Thr 130 135 140 Pro Thr Gly Met Pro Gln Pro Glu Ile Pro Ala Val His Pro Phe 145 150 155 160 Pro Tyr Val Thr Thr Thr Thr Thr Ala Ala Pro Arg Val Leu Val Tyr 165 170 175 Lys Ile Pro Tyr Gly Gly Ala Ala Pro Pro Arg Ala Pro Pro Val Pro 180 185 190 Pro Arg Met Gly Pro Ser Asp Ile Ser Thr His Val Arg Gly Ala Ile 195 200 205 Arg Arg Gln Pro Ala Thr Ala Thr Thr Thr Thr Thr Thr Arg Asn Val 210 215 220 Leu Leu Arg Thr Ala Ile Leu Ala Ala Ala Ala Ala Thr Leu Ile Ala 225 230 235 240 Leu Phe Arg Gln Arg Pro Leu Phe Thr Glu Gly Val Arg Met Phe Pro 245 250 255 Asp Phe Gln Tyr Arg Phe Thr Val Gln Thr Thr Gln Asn 260 265 <210> 3 <211> 276 <212> DNA <213> artificial sequence <220> <223> rATRAM-G8-M/AS <400> 3 ggcctggccg gcctggccgg cctgctgggc ctggaaggcc tgccgggcct gcccctgggc 60 ctgctggaag gcctgtggct gggcctggaa ctggaaggaa acggcctggc cggcctggcc 120 ggcctgctgg gcctggaagg cctgccgggc ctgcccctgg gcctgctgga aggcctgtgg 180 ctgggcctgg aactggaagg aaacgggggg gctaccgcca ccacaaccac tactacccgc 240 ggggggagat tcaccgtcca aacgacgcag aattaa 276 <210> 4 <211> 10 <212> PRT <213> artificial sequence <220> <223> <400> 4 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 5 <211> 51 <212> PRT <213> artificial sequence <220> <223> Tat-GRA8-(183-222) <400> 5 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ala Ala Pro Pro Arg 1 5 10 15 Ala Pro Pro Val Pro Pro Arg Met Gly Pro Ser Asp Ile Ser Thr His 20 25 30 Val Arg Gly Ala Ile Arg Arg Gln Pro Ala Thr Ala Thr Thr Thr Thr 35 40 45 Thr Thr Arg 50 <210> 6 <211> 21 <212> PRT <213> artificial sequence <220> <223> Tat-GRA8-(183-192) <400> 6 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ala Ala Pro Pro Arg 1 5 10 15 Ala Pro Pro Val Pro 20 <210> 7 <211> 21 <212> PRT <213> artificial sequence <220> <223> Tat-GRA8-(193-202) <400> 7 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Pro Arg Met Gly Pro 1 5 10 15 Ser Asp Ile Ser Thr 20 <210> 8 <211> 21 <212> PRT <213> artificial sequence <220> <223> Tat-GRA8-(203-212) <400> 8 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly His Val Arg Gly Ala 1 5 10 15 Ile Arg Arg Gln Pro 20 <210> 9 <211> 21 <212> PRT <213> artificial sequence <220> <223> Tat-GRA8-(213-222) <400> 9 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ala Thr Ala Thr Thr 1 5 10 15 Thr Thr Thr Thr Arg 20 <210> 10 <211> 39 <212> PRT <213> artificial sequence <220> <223> Tat-GRA8-(242-269) <400> 10 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Phe Arg Gln Arg Pro 1 5 10 15 Leu Phe Thr Glu Gly Val Arg Met Phe Pro Asp Phe Gln Tyr Arg Phe 20 25 30 Thr Val Gln Thr Thr Gln Asn 35 <210> 11 <211> 20 <212> PRT <213> artificial sequence <220> <223> Tat-GRA8-(242-250) <400> 11 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Phe Arg Gln Arg Pro 1 5 10 15 Leu Phe Thr Glu 20 <210> 12 <211> 20 <212> PRT <213> artificial sequence <220> <223> Tat-GRA8-(251-259) <400> 12 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly Val Arg Met Phe 1 5 10 15 Pro Asp Phe Gln 20 <210> 13 <211> 21 <212> PRT <213> artificial sequence <220> <223> Tat-GRA8-(260-269) <400> 13 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Tyr Arg Phe Thr Val 1 5 10 15 Gln Thr Thr Gln Asn 20
Claims (9)
A recombinant peptide derived from dense granule antigen 8 (GRA8) conjugated with acidity-triggered rational membrane (ATRAM) represented by SEQ ID NO: 1.
A gene encoding the peptide of claim 1 and represented by SEQ ID NO: 3.
A recombinant expression vector comprising the gene of claim 2.
A pharmaceutical composition for preventing or treating colon cancer, comprising, as an active ingredient, a recombinant peptide derived from dense granule antigen 8 (GRA8) conjugated with acidity-triggered rational membrane (ATRAM) represented by SEQ ID NO: 1.
A health functional food for preventing or improving colon cancer disease, comprising a recombinant peptide derived from dense granule antigen 8 (GRA8) conjugated with acidity-triggered rational membrane (ATRAM) represented by SEQ ID NO: 1.
A method for preventing or treating colon cancer comprising the step of administering the pharmaceutical composition of claim 4 to a mammal other than a human who is expected to develop colon cancer or has developed colon cancer.
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