JP2004089053A - Method for screening aids drug - Google Patents
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Abstract
Description
【0001】
【発明の属する技術分野】
本発明は、宿主細胞由来のトポイソメラーゼIの一部のアミノ酸に結合する化合物を選択することからなる、レトロウイルスに対する治療薬のスクリーニング方法、該スクリーニング方法によって確認された化合物、該化合物を活性成分として含有する医薬組成物、及び、該医薬組成物を投与することからなる後天性免疫不全症の治療方法等に関する。
【0002】
【従来の技術】
AIDS(Acquired immunodeficiency syndrome)はレトロウイルス属に属する1本領RNAウイルスであるHIV−1(Human immunodeficiency virus type 1)の感染によって惹起される致死的疾患である。HIV‐1は主に、宿主CD4陽性細胞に感染し宿主免疫不全状態を招く。
【0003】
HIV−1感染者は世界で3千万人を教え、アフリカ、南アジア、中国において爆発的な感染者増加を認める。HIV−1感染後、治療を行わなければAIDSの発症が必発である。感染者が増加している環境においては感染予防に対するワクチンが必要であるが、HIV−1のゲノムはウイルス遺伝子の組換えや突然変異(ミューテーション)が容易に生じてしまうことから、現在有効なワクチンは開発されていない。
【0004】
【発明が解決しようとする課題】
HIV‐1感染者に対して逆転写酵素阻害剤とプロテアーゼ阻害剤治療が行われているが、長期加療におけるHIV−1の中和抵抗性変異株(エスケープミュータント)の出現が問題となっており、新たな抗ウイルス剤の開発が切に望まれている。
【0005】
【課題を解決するための手段】
発明者らの研究室では1990年初頭より一貫してHIV−1の研究を継続しており、平成10年からは科学技術振興事業団のCREST「脳を守る」プロジェクトの一員として、ウイルス脳症の治療法の確立を目指して研究を遂行している。
最近、発明者らはHIV‐1の宿主域は、HIV‐1粒子内に取り込まれる宿主由来のトポイソメラーゼIによって調節されていること、さらにトポイソメラーゼI構成アミノ酸のうち236番目のグルタミン酸、及び237番目のアスパラギンがこの調節の責任アミノ酸であることを見出した。
【0006】
即ち、本発明は、第一の態様として、宿主細胞由来のトポイソメラーゼIの一部のアミノ酸、特に、第236番目のグルタミン酸及び第237番目のアスパラギンの少なくとも一つ、好ましくは、これら2つのアミノ酸の双方に結合する化合物を選択することからなる、レトロウイルスに対する治療薬のスクリーニング方法に係る。
レトロウイルスには、RNA腫瘍ウイルス、レンチウイルス、スプマウイルスの3つの亜科がある。レンチウイルスの代表的な例としては、ヒト免疫不全ウイルス(HIV−1又はHIV−2)等の免疫不全ウイルスを挙げることができる。
本発明のスクリーニング方法の一例において、RNAウイルス粒子は当業者に公知の適当な培養細胞にプロウイルスDNAを含むベクターを導入するステップを含む。かかる培養細胞の例として、当業者に公知の任意のヒト由来細胞株、及びその他の哺乳動物細胞由来の細胞株を挙げることが出来る。
【0007】
阻害効果を測定する方法自体は当業者に公知の任意の手段で行うことが出来る。このような手段の例として、例えば、上記の培養細胞から得られた上清で感染させた細胞が発現するベータガラクトシダーゼを定量することにより、ウイルス感染価を測定する方法を挙げることができる。このようなベータガラクトシダーゼを発現し得る細胞の例として、MAGIC5細胞(Mochizuki N, Otsuka N, Matsuo K, Shiino T, Kojima A, Kurata T, Sakai K, Yamamoto N, Isomura S, Dhole TN, Takebe Y, Matsuda M, Tatsumi M. An infectious DNA clone of HIV type 1 subtype C.AIDS Res Hum Retroviruses 1999 Sep 20;15(14):1321−4)がある。
尚、本発明のスクリーニング方法において使用する試薬類の種類、反応温度及び時間などの各種測定条件は当業者が適宜選択することが出来る。
【0008】
本発明は第二の態様として、宿主細胞由来のトポイソメラーゼIの一部のアミノ酸に結合し、レトロウイルスの複製過程、より具体的には、逆転写反応を阻害する化合物に係る。このような効果を有する化合物は本発明のスクリーニング方法によって確認し、同定することが可能である。
【0009】
本発明は更に、第三の態様として、上記化合物を活性成分として含有する医薬組成物に係る。この医薬組成物はレトロウイルスが病因となる様々な疾患の予防及び治療に使用することが出来る。このような疾患の代表的な例として、後天性免疫不全症候群(AIDS)を挙げることが出来る。本発明の医薬組成物には、上記活性成分の他に、当業者に公知の任意の様々な補助剤等を、適宜含有させることが出来る。活性成分である本発明化合物及びこのような各種補助剤の種類及び含有量、並びに該医薬組成物の形態は、当該組成物の使用目的、患者の病状・性別・年齢・体重等に応じて、当業者が適宜選択することが出来る。医薬組成物の形態としては、例えば、錠剤、粉末剤等の各種固形剤、水溶液、懸濁液及び乳濁液等の液剤、更には、エアゾール等を挙げることが出来る。
本発明は更に、第四の態様として、上記医薬組成物をヒト及び哺乳動物などの患者に投与することからなる、上記各種疾患の予防及び治療等の各種処置方法に係る。投与方法は医薬組成物の種類、並びに患者の病状・性別・年齢・体重等に応じて、当業者に公知の任意の手段で行うことが出来る。例えば、経口投与、静注及びインハレーション等を挙げることが出来る。本発明医薬組成物の一回の投与量及び投与間隔等は、患者の病状・性別・年齢・体重等に応じて、当業者が適宜選択することが出来る。
【0010】
以下、実施例に沿って本発明を説明するが、本発明の技術的範囲はこれら実施例に何等拘束されるものではない。
【0011】
【実施例】
HIV ‐1感染価のモニターによる HIV ‐ 1 複製におけるトポイソメラーゼIの役割の解析
1.サルトポイソメラーゼIのクローニングとシークエンス、およびヒトトポイソメラーゼIとの比較
以下の手順に従い、アフリカミドリザル腎臓細胞株Vero細胞からcDNAライブラリーを作成し、PCRによってサルトポイソメラーゼIをクローニングし、シークエンスを行った。
1、ポリA RNAをVero細胞より抽出した。
2、SuperScript RNaseH ̄ reverse transcriptase (Invitrogen Life Technologies, Carlsbad, California)によりcDNAを合成した。
3、プライマーtopoF (5’ ̄ATGAGTGGGGACCACCTCCACAACGA ̄3’)及び topoR (5’ ̄CTAAAACTCATAGTCTTCATCAGCCA ̄3’)によりpolymerase chain reaction (PCR)にてサルtopoisomerase I cDNAを合成した。
4、約2.3kbの産物をpSTblue ̄1 cloning vector (Novagen, Madison, Wisconsin)へクローニングした。
5、ベクターのインサートをa model 377A DNA sequencer (Perkin Elmer)を使用してシークエンスした。
6、得られたサルトポイソメラーゼIのシークエンスを元にSMARTTM RACE cDNA Amplificationキット(BD Biosciences Clontech CA USA)を使用して5’及び3’のシークエンスを得た。
得られたサルトポイソメラーゼIのcDNAおよびアミノ酸シークエンスは、それぞれ、配列番号1及び配列番号2で示した。尚、該配列は、GenBank the accession number AB089321 に登録されている(公開は平成15年2月3日予定)。又、ヒトトポイソメラーゼIの配列はGenBank the accession number J03250で登録されている。
その結果、サルトポイソメラーゼIはヒトと比較して、核酸塩基配列で47箇所異なっていた。その結果から予測したアミノ酸配列では8箇所異なっており、98.4%の相似性を有していた(図1)。
【0012】
2.発現ベクターの作製
サルcDNAをプライマー5’ ̄AGTGAATTCATGAGTGGGGACCACCTCCAC ̄3’ 及び 5’ ̄ACGTGCGGCCGCCTAAAACTCATAGTCTTC ̄3’(Eco RI及びNot Iサイトを下線で示す。)でPCRにて増幅し、産物をさらにEco RI及びNot Iにて消化し、pcDNA4/HisMax ̄C (Invitrogen)のEco RI およびNot Iサイトへ挿入した。
さらにヒトトポイソメラーゼIのうちサルと異なるアミノ酸部分についてそれぞれサル型に変換した変異トポイソメラーゼIを発現するべクターも作成した。具体的には、ヒトトポイソメラーゼIの第236番目のグルタミン酸をアスパラギン酸に、第237番目のアスパラギンをセリンに変換したミュータントトポイソメラーゼI(HuE236D/N237S)を作成した。
【0013】
3.発現べクタ−による感染性ウイルス粒子の作製
HIV‐1は1本鎖直鎖状RNAウイルスで染色体上のブロウイルスとしては9,709bpの長さを有する。HIV−1のブロウイルスDNAを含むべクターpNL43(1986年にAdachi等が発表)を哺乳類細胞へ導入するとHIV‐1蛋白群が転写、翻訳され、感染性を有する粒子を培養細胞上清に得ることが出来る。
上述のpNL432を1 マイクログラム、野生株ヒト、サル及び上記で作成したミュータントトポイソメラーゼI発現べクタ−を1マイクログラム、同時にサル由来のCOSI細胞へ哺乳細胞遺伝子導入試薬Fugene 6(Rosche)を用いて導入し、2m1の培養液で培養した。
【0014】
4.ウイルス感染価の定量
Hachiya A, Aizawa ̄Matsuoka S, Tanaka M, Takahashi Y, Ida S, Gatanaga H, Hirabayashi Y, Kojima A, Tatsumi M, Oka S.Rapid and simple phenotypic assay for drug susceptibility of human immunodeficiency virus type 1 using CCR5 ̄expressing HeLa/CD4(+) cell clone 1 ̄10 (MAGIC ̄5). Antimicrob Agents Chemother 2001 Feb;45(2):495 ̄501に記載の方法に従い、遺伝子導入から48時間後、上清のHIV‐1抗原(P24)を定量した。
具体的には、以下の方法で実施した。
1.96穴ディッシュにMAGIC5細胞を1穴あたり1万個でまく。
2.サンプルの希釈配列を作成し、それぞれ37℃5%CO2の条件で48時間培養する。
3.ウイルスを含む上精を除き1%formaldehyde,0.2%glutaraldehydeを含むPBSにて常温で固定した。
4.X ̄Gal(5 ̄bromo ̄4 ̄chloro ̄3 ̄indolyl ̄b ̄D ̄galactopyranosideによって感染細胞を染色した。
5.顕微鏡下に数えた。
【0015】
ヒトトポイソメラーゼI野生株(HuWT)、サルトポイソメラーゼI野生株(SimWT)、及び236、237番目のグルタミン酸、アスパラギン(236GIu、237Asn)をサル型にしたミュータントトポイソメラーゼI(HuE236D/N237S)を発現したサル細胞から得られたウイルス感染価を比べた結果を図2に示す。
サルトポイソメラーゼIとは対照的にヒトトポイソメラーゼIを発現することにより、サル細胞由来HIV‐1ウイルスの感染価は回復できた。さらにヒトトポイソメラーゼIのうち236GIu、237Asnをサル型にしたミュータントトポイソメラーゼI(HuE236D/N237S)を発現した細胞由来のHIV‐1 ウイルスはサルトポイソメラーゼI野生株(SimWT)を発現した細胞またはべクタ−(pcDNA)を導入した細胞由来のHN−1と同様、ウイルス感染価を回復することはできなかった。
【0016】
以上の結果が示すように、本発明者等はサル細胞由来のHIV‐1 ウイルス感染価がヒト細胞由来HIV‐1の感染価に比べ約7から9倍減少していることから、ヒトウイルス産生細胞の細胞宿主因子の中にサル細胞の宿主因子では代償できない因子が含まれていると考えられる。
ヒトとサルのトポイソメラーゼIをサル由来細胞に発現し、その細胞でウイルスを産生させた結果、サルトポイソメラーゼIはサル細胞由来HIV‐1の感染価を回復させることができないがヒトトポイソメラーゼIを発現することにより、サル細胞由来HIV‐1ウイルスの感染価は回復したこと、ヒトトポイソメラーゼIのミュータントを用いて、ウイルス感染価の回復には236GIu、237Asnが不可欠であることが明らかになった。
トポイソメラーゼIはHIV‐1の複製過程においてcDNAの産生に関与していることも既に明らかにしており、合わせて考えると236GIu、237Asnは逆転写においてウイルス因子と相互作用していると部位であると予想される。
【0017】
【発明の効果】
上記の結果に基づき、発明によって、トポイソメラーゼIの236GIu、237Asnに結合する化合物(薬物)のスクリーニングをが提供される。見出された薬剤群はトポイソメラーゼIとウイルス因子の関係を阻害することが予測され、培養細胞のHIV‐1複製系における作用と細胞毒性を調べ実用性を確認する。
宿主因子とウイルス因子との相互作用を標的としたAIDS治療の場合、ウイルス因子を対象とする場合に比べエスケープミュータントの出現効率が極めて低く細胞毒性の低い特異的抗ウイルス剤開発につながることが期待できる。
宿主因子を標的としたAIDS治療の場合、ウイルス因子を対象とする場合に比べエスケープミュータントの出現効率が極めて低いと予測できる。宿主因子自体ではなく、宿主因子とウイルス因子の相互作用を阻害する薬物が選択出来れば、さらに細胞毒性の低い特異的抗ウイルス剤開発につながると考えられる。
ゲノムRNAの切断と再結合はHIV−1の高い変異効率の原因の一つでもあり、その原因を阻害する薬剤は宿主免疫によるウイルス排除機構を大幅に伸長させる効果も望まれる。
【0018】
【配列表】
【図面の簡単な説明】
【図1】サルとヒトのトポイソメラーゼIのアミノ酸配列を比較した結果を示す。
【図2】ヒトトポイソメラーゼI野生株(HuWT)、サルトポイソメラーゼI野生株(SimWT)、及び236、237番目のグルタミン酸、アスパラギン(236GIu、237Asn)をサル型にしたミュータントトポイソメラーゼI(HuE236D/N237S)を発現したサル細胞から得られたウイルス感染価を比べた結果を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention provides a screening method for a therapeutic agent against a retrovirus, comprising selecting a compound that binds to some amino acids of topoisomerase I derived from a host cell, a compound confirmed by the screening method, and using the compound as an active ingredient. The present invention relates to a pharmaceutical composition, a method for treating acquired immunodeficiency, which comprises administering the pharmaceutical composition.
[0002]
[Prior art]
AIDS (Acquired immunodeficiency syndrome) is a lethal disease caused by infection of HIV-1 is 1 stride RNA virus belonging to the retrovirus genus (H uman i mmunodeficiency v irus type 1). HIV-1 mainly infects host CD4-positive cells and causes a host immunodeficiency state.
[0003]
HIV-1 infected teaches 30 million people worldwide and has seen explosive increases in Africa, South Asia and China. After HIV-1 infection, the development of AIDS is inevitable without treatment. In an environment where the number of infected people is increasing, a vaccine for infection prevention is necessary, but the HIV-1 genome is currently effective because the recombination and mutation (mutation) of viral genes can easily occur. No vaccine has been developed.
[0004]
[Problems to be solved by the invention]
HIV-1 infected patients are treated with reverse transcriptase inhibitors and protease inhibitors, but the emergence of HIV-1 neutralization resistant mutants (escape mutants) in long-term treatment has become a problem. The development of new antiviral agents is urgently desired.
[0005]
[Means for Solving the Problems]
The laboratory of the inventors has been consistently conducting research on HIV-1 since the beginning of 1990. Since 1998, as a member of the Japan Science and Technology Corporation (JST) 's CREST “Protecting the Brain” project, he has been working on viral encephalopathy. He is conducting research with the aim of establishing a cure.
Recently, the inventors have determined that the host range of HIV-1 is regulated by topoisomerase I from the host, which is incorporated into HIV-1 particles, and furthermore, glutamic acid at position 236 of the amino acids constituting topoisomerase I and position 237 at position 237 Asparagine was found to be the amino acid responsible for this regulation.
[0006]
That is, the present invention provides, as a first aspect, some amino acids of topoisomerase I derived from a host cell, in particular, at least one of 236th glutamic acid and 237th asparagine, preferably at least one of these two amino acids. The present invention relates to a method for screening a therapeutic agent for a retrovirus, comprising selecting a compound that binds to both.
There are three subfamilies of retroviruses: RNA oncoviruses, lentiviruses, and spumaviruses. Representative examples of lentiviruses include immunodeficiency viruses such as human immunodeficiency virus (HIV-1 or HIV-2).
In one example of the screening method of the present invention, the RNA virus particle includes a step of introducing a vector containing proviral DNA into appropriate cultured cells known to those skilled in the art. Examples of such cultured cells include any human-derived cell line known to those skilled in the art, and other mammalian cell-derived cell lines.
[0007]
The method itself for measuring the inhibitory effect can be performed by any means known to those skilled in the art. As an example of such a means, for example, a method of measuring a virus infectivity by quantifying beta-galactosidase expressed by a cell infected with a supernatant obtained from the above cultured cells can be mentioned. Examples of cells capable of expressing such a beta-galactosidase include MAGIC5 cells (Mochizuki N, Otsuka N, Matsuo K, Shino T, Kojima A, Kurata T, Sakai K, Yamamoto N, Yamamoto N, Yamamoto N Matsuda M, Tatsumi M. An infectious DNA clone of HIV type 1 subtype C. AIDS Res Hum Retroviruses 1999 Sep 20; 15 (14): 1321-4.
In addition, those skilled in the art can appropriately select various measurement conditions such as the types of reagents, reaction temperature and time used in the screening method of the present invention.
[0008]
As a second aspect, the present invention relates to a compound that binds to some amino acids of topoisomerase I derived from a host cell and inhibits the retrovirus replication process, more specifically, the reverse transcription reaction. Compounds having such effects can be confirmed and identified by the screening method of the present invention.
[0009]
The present invention further relates, as a third aspect, to a pharmaceutical composition containing the above compound as an active ingredient. This pharmaceutical composition can be used for prevention and treatment of various diseases caused by retrovirus. A representative example of such a disease is acquired immunodeficiency syndrome (AIDS). The pharmaceutical composition of the present invention may appropriately contain, in addition to the above active ingredients, any of various adjuvants known to those skilled in the art. The type and content of the compound of the present invention as an active ingredient and such various adjuvants, and the form of the pharmaceutical composition are determined according to the purpose of use of the composition, the condition, gender, age, body weight, etc. of the patient. A person skilled in the art can appropriately select. Examples of the form of the pharmaceutical composition include various solid preparations such as tablets and powders, liquid preparations such as aqueous solutions, suspensions and emulsions, and aerosols.
The present invention further relates, as a fourth aspect, to various treatment methods such as prevention and treatment of the above-mentioned various diseases, which comprise administering the above-mentioned pharmaceutical composition to patients such as humans and mammals. The administration method can be performed by any means known to those skilled in the art according to the type of the pharmaceutical composition and the condition, gender, age, weight, etc. of the patient. For example, oral administration, intravenous injection, inhalation and the like can be mentioned. A person skilled in the art can appropriately select a single dose, a dosing interval, and the like of the pharmaceutical composition of the present invention according to the patient's condition, gender, age, body weight, and the like.
[0010]
Hereinafter, the present invention will be described with reference to examples, but the technical scope of the present invention is not limited to these examples.
[0011]
【Example】
Analysis of the Role of Topoisomerase I in HIV - 1 Replication by Monitoring HIV- 1 Infectivity Cloning and sequencing of saltopoisomerase I and comparison with human topoisomerase I According to the following procedure, a cDNA library was prepared from African green monkey kidney cell line Vero cells, and sartopoisomerase I was cloned by PCR and sequenced.
1. Poly A RNA was extracted from Vero cells.
2, SuperScript RNaseH ¯ reverse transcriptase ( Invitrogen Life Technologies, Carlsbad, California) and cDNA was synthesized by.
3. A cDNA obtained by synthesizing a monkey topoisomerase I cDNA by polymerase chain reaction (PCR) using primers topoF (5 ′ @ ATGAGTGGGGACCACCTCCCACAACGA ̄3 ′) and topoR (5′ ̄CTAAAACTCATAGTCTTCATCAGCCCA ̄3 ′).
4. The product of about 2.3 kb was cloned into pSTblue @ 1 cloning vector (Novagen, Madison, Wis.).
5. The vector inserts were sequenced using a model 377A DNA sequencer (Perkin Elmer).
6. Using the SMART ™ RACE cDNA Amplification kit (BD Biosciences Clontech CA USA), 5 ′ and 3 ′ sequences were obtained based on the obtained sequence of astopoisomerase I.
The cDNA and amino acid sequence of the obtained saltopoisomerase I are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The sequence is registered in GenBank the accession number AB089321 (disclosed on February 3, 2003). The sequence of human topoisomerase I is registered in GenBank the accession number J03250.
As a result, as compared with human, saltopoisomerase I had 47 nucleotide differences in nucleic acid base sequence. The amino acid sequences predicted from the results differ from each other by 8 positions, and have 98.4% similarity (FIG. 1).
[0012]
2. Preparation of Expression Vector The monkey cDNA was amplified by PCR with primers 5′ΔAGT GAATTC ATGAGTGGGGACCACCCTCCAC 3 ′ and 5′ΔACGT GCGGCCGCCTAAACTCATATACTCTTC 3 ′ (Eco RI and Not I sites are underlined), and the product was further amplified by PCR. It was digested with EcoRI and NotI, and inserted into EcoRI and NotI sites of pcDNA4 / HisMaxΔC (Invitrogen).
Further, a vector expressing a mutant topoisomerase I in which amino acids different from those of monkeys in human topoisomerase I were converted into monkey types was also prepared. Specifically, mutant topoisomerase I (HuE236D / N237S) was prepared by converting 236th glutamic acid of human topoisomerase I to aspartic acid and 237th asparagine to serine.
[0013]
3. Preparation of Infectious Virus Particles by Expression Vector HIV-1 is a single-stranded linear RNA virus and has a length of 9,709 bp as a chromosome brovirus. When vector pNL43 containing HIV-1 brovirus DNA (published by Adachi et al. In 1986) is introduced into mammalian cells, the HIV-1 protein group is transcribed and translated, and infectious particles are obtained in the culture cell supernatant. I can do it.
One microgram of the above-described pNL432, one microgram of the wild-type human and monkey and the mutant topoisomerase I expression vector prepared above, and simultaneously using the mammalian gene transfection reagent Fugene 6 (Rosche) into monkey-derived COSI cells. The cells were introduced and cultured in a 2 ml culture solution.
[0014]
4. Quantification of virus infectious titer Hachiya A, Aizawa @ Matsuoka S, Tanaka M, Takahashi Y, Ida S, Gatayama H, Hirabayashi Y, Kojima O., Tatsuma A. Rapid and simple phenotypic assay for drug susceptibility of human immunodeficiency virus type 1 usage CCR5 (expressing HeLa / CD4 @ 10C1 + G1 @ c1 + c1 + c1 + c1 + c1 + c1 + c1c1 + c1c1 + c1c1 + c1c1 + c1). According to the method described in Antimicrob Agents Chemother 2001 Feb; 45 (2): 495 ̄501, 48 hours after gene transfer, HIV-1 antigen (P24) in the supernatant was quantified.
Specifically, the following method was used.
1. Spread 10,000 MAGIC5 cells per well in a 96-well dish.
2. A diluted sequence of the sample is prepared, and each is cultured under conditions of 37 ° C. and 5% CO 2 for 48 hours.
3. The cells were fixed at room temperature with PBS containing 1% formaldehyde and 0.2% glutardehyde except for the ejaculate containing the virus.
4. Infected cells were stained with X \ Gal (5-bromo 4-chloro-3 indolyl b-D galactopyranoside).
5. Counted under a microscope.
[0015]
Human topoisomerase I wild strain (HuWT), sartopoisomerase I wild strain (SimWT), and mutant topoisomerase I (HuE236D / N237S) in which glutamic acid at positions 236 and 237 and asparagine ( 236 GIu, 237 Asn) were monkey-type were expressed. FIG. 2 shows the results of comparing virus infectivity titers obtained from monkey cells.
By expressing human topoisomerase I in contrast to saltopoisomerase I, the infectivity of HIV-1 virus derived from monkey cells could be restored. Furthermore, among the human topoisomerase I, HIV-1 virus derived from a cell expressing mutant topoisomerase I (HuE236D / N237S), in which 236GIu and 237Asn are monkey-type, is a cell or vector expressing a wild strain of saltopoisomerase I (SimWT). As with HN-1 derived from cells into which pcDNA) had been introduced, the virus infectivity could not be restored.
[0016]
As shown by the above results, the present inventors show that the infectious titer of HIV-1 virus derived from monkey cells is about 7 to 9 times lower than the infectious titer of HIV-1 derived from human cells. It is considered that the cell host factors of cells include factors that cannot be compensated for by the host factors of monkey cells.
As a result of expressing human and monkey topoisomerase I in monkey-derived cells and producing virus in those cells, sartopoisomerase I cannot restore the infectious titer of monkey cell-derived HIV-1, but expresses human topoisomerase I Thus, it was revealed that the infectivity of the HIV-1 virus derived from monkey cells was restored, and that 236 GIu and 237 Asn are indispensable for the restoration of the infectivity of the virus using a mutant of human topoisomerase I.
It has already been shown that topoisomerase I is involved in the production of cDNA during the replication process of HIV-1. In view of this, 236 GIu and 237 Asn are considered to interact with viral factors in reverse transcription. Expected to be.
[0017]
【The invention's effect】
Based on the above results, the invention provides for the screening of compounds (drugs) that bind to 236 GIu, 237 Asn of topoisomerase I. The discovered drug group is expected to inhibit the relationship between topoisomerase I and viral factors, and the effect and cytotoxicity of the cultured cells on the HIV-1 replication system will be examined to confirm their utility.
AIDS treatment targeting the interaction between host factors and viral factors is expected to lead to the development of specific antiviral agents with extremely low escape mutant appearance efficiency and low cytotoxicity compared to viral factors. it can.
In the case of AIDS treatment targeting a host factor, it can be predicted that the emergence efficiency of escape mutants is extremely low as compared with the case of targeting a viral factor. If a drug that inhibits the interaction between the host factor and the viral factor, rather than the host factor itself, can be selected, it will lead to the development of a specific antiviral agent with even lower cytotoxicity.
Cleavage and recombination of genomic RNA is also one of the causes of high mutation efficiency of HIV-1, and an agent that inhibits the cause is also desired to have an effect of greatly extending the virus elimination mechanism by host immunity.
[0018]
[Sequence list]
[Brief description of the drawings]
1 shows the results of comparing the amino acid sequences of monkey and human topoisomerase I.
FIG. 2: Human topoisomerase I wild strain (HuWT), sartopoisomerase I wild strain (SimWT), and mutated topoisomerase I (HuE236D / N237S) obtained by converting 236 and 237th glutamic acid and asparagine ( 236 GIu, 237 Asn) into monkey type. 4) shows the results of comparing virus infectivity titers obtained from monkey cells expressing).
Claims (21)
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