Discovered in 1980, HTLV-1 (Human T-cell Leukemia Virus-1), was the first identified human retrovirus and is shown to be associated with a variety of diseases including: adult T-cell leukemia lymphoma (ATLL), tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM), chronic arthropathy, uveitis, infective dermatitis, and polymyositis. The mechanism by which the virus causes disease is still unknown. HTLV- 1 infection has been reported in many regions of the world but is most prevalent in Southern Japan, the Caribbean basin, Central and West Africa, the Southeastern United States, Melanesia, parts of South Africa, the Middle East and India. Approximately 30 million people are infected by HTLV-1 worldwide, although only 3-5% of the infected individuals evolve Adult T-cell Leukemia (ATL) during their life and the prognosis for those infected is still poor. The retroviral proteases (PRs) are essential for viral replication because they process viral Gag and Gag-(Pro)-Pol polyproteins during maturation, much like the PR from Human Immunodeficiency Virus-1 (HIV-1). Various antiviral inhibitors are in clinical use and one of the most significant classes is HIV-1 PR inhibitors, which have used for antiretroviral therapy in the treatment of AIDS. HTLV-1 PR and HIV-1 PR are homodimeric aspartic proteases with 125 and 99 residues, respectively. Even though substrate specificities of these two enzymes are different, HTLV-1 PR shares 28% similarity with HIV-1 PR overall and the substrate binding sites have 45% similarity. In addition to the 125-residue full length HTLV-1 PR, constructs with various C- terminal deletions (giving proteases with lengths of 116, 121, or 122 amino acids) were made in order to elucidate the effect of the residues in the C-terminal region. It was suggested that five amino acids in the C-terminal region are not necessary for the enzymatic activity in Hayakawa et al. 1992. In 2004 Herger et al. had suggested that 10 amino acids at the C-terminal region are not necessary for catalytic activity. A recent paper suggested that C-terminal residues are essential; and that catalytic activity lowers upon truncation, with even the last 5 amino acids necessary for full catalytic activity (1). The mutation L40I has been made to prevent autoproteolysis and the W98V mutation was made to make the active site of HTLV-1 PR similar to HIV-1 PR. We have characterized C-terminal amino acids of HTLV-1 PR as not being essential for full catalytic activity. We have discovered potential new inhibitors by in silico screening of 116-HTLV-1 PR. These small molecules were tested kinetically for various constructs including the 116, 121 and 122-amino acid forms of HTLV-1 PR. Inhibitors with the best inhibition constants were used in HTLV-1 infected cells and one of the inhibitors seems to inhibit gag processing.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18129号 / 医博第3849号 / 新制||医||1001(附属図書館) / 30987 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 髙折 晃史, 教授 五十嵐 樹彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No description available.

Discovered in 1980, HTLV-1 (Human T-cell Leukemia Virus-1), was the first identified human retrovirus and is shown to be associated with a variety of diseases including: adult T-cell leukemia lymphoma (ATLL), tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM), chronic arthropathy, uveitis, infective dermatitis, and polymyositis. The mechanism by which the virus causes disease is still unknown. HTLV- 1 infection has been reported in many regions of the world but is most prevalent in Southern Japan, the Caribbean basin, Central and West Africa, the Southeastern United States, Melanesia, parts of South Africa, the Middle East and India. Approximately 30 million people are infected by HTLV-1 worldwide, although only 3-5% of the infected individuals evolve Adult T-cell Leukemia (ATL) during their life and the prognosis for those infected is still poor. The retroviral proteases (PRs) are essential for viral replication because they process viral Gag and Gag-(Pro)-Pol polyproteins during maturation, much like the PR from Human Immunodeficiency Virus-1 (HIV-1). Various antiviral inhibitors are in clinical use and one of the most significant classes is HIV-1 PR inhibitors, which have used for antiretroviral therapy in the treatment of AIDS. HTLV-1 PR and HIV-1 PR are homodimeric aspartic proteases with 125 and 99 residues, respectively. Even though substrate specificities of these two enzymes are different, HTLV-1 PR shares 28% similarity with HIV-1 PR overall and the substrate binding sites have 45% similarity. In addition to the 125-residue full length HTLV-1 PR, constructs with various C- terminal deletions (giving proteases with lengths of 116, 121, or 122 amino acids) were made in order to elucidate the effect of the residues in the C-terminal region. It was suggested that five amino acids in the C-terminal region are not necessary for the enzymatic activity in Hayakawa et al. 1992. In 2004 Herger et al. had suggested that 10 amino acids at the C-terminal region are not necessary for catalytic activity. A recent paper suggested that C-terminal residues are essential; and that catalytic activity lowers upon truncation, with even the last 5 amino acids necessary for full catalytic activity (1). The mutation L40I has been made to prevent autoproteolysis and the W98V mutation was made to make the active site of HTLV-1 PR similar to HIV-1 PR. We have characterized C-terminal amino acids of HTLV-1 PR as not being essential for full catalytic activity. We have discovered potential new inhibitors by in silico screening of 116-HTLV-1 PR. These small molecules were tested kinetically for various constructs including the 116, 121 and 122-amino acid forms of HTLV-1 PR. Inhibitors with the best inhibition constants were used in HTLV-1 infected cells and one of the inhibitors seems to inhibit gag processing.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19628号 / 医科博第66号 / 新制||医科||5(附属図書館) / 32664 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 髙折 晃史, 教授 浅野 雅秀, 教授 小柳 義夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Le virus humain T lymphotrope de type 1 (HTLV-1) est l’agent étiologique de la leucémie/lymphome T de l’adulte (ATLL) qui se développe après plusieurs décennies et pour laquelle il n’existe à ce jour pas de traitement efficace. Parmi les protéines virales de HTLV-1, Tax et HBZ jouent un rôle déterminant dans le développement de l’ATLL. Si Tax participe au processus leucémogène dès les étapes précoces, HBZ jouerait plutôt un rôle dans le maintien du phénotype tumoral dans les étapes tardives. Dans ce contexte, là nous nous sommes intéressés à la régulation de l’expression des protéines anti-apoptotiques Bfl-1 et Bcl-xL, par les protéines virales Tax et HBZ. Nous avons montré que Tax induit l’expression des protéines anti-apoptotiques Bfl-1 et Bcl-xL de la famille Bcl-2 via la voie NF-κB, alors que HBZ n’a aucun effet sur leur expression. De plus, Tax coopère avec les facteurs de transcription c-Jun et JunD de la voie AP-1 pour augmenter l’expression de ces gènes anti-apoptotiques. En revanche, HBZ module uniquement la trans-activation de bfl-1 induite par Tax. L’ensemble de nos résultats indique donc que Tax joue un rôle prépondérant dans l’activation de l’expression de Bfl-1 et de Bcl-xL et suggère que Bfl-1 et Bcl-xL sont exprimées au cours des étapes précoces et tardives du développement de l’ATLL. Par une stratégie d’ARN interférence, nous avons ensuite montré que Bfl-1 et/ou Bcl-xL sont impliquées dans la survie de lignées cellulaires T infectées par HTLV-1, suggérant que Bfl-1 et Bcl-xL représentent des cibles thérapeutiques potentielles pour traiter l’ATLL. Actuellement, il existe des petites molécules ciblant les membres anti-apoptotiques de la famille Bcl-2, mais aucune ne cible spécifiquement Bfl-1. En collaboration avec la société IMAXIO, nous avons identifié par deux cribles à haut débit 83 molécules capables d’inhiber l’activité anti-apoptotique de Bfl-1. L’une de ces molécules induit spécifiquement la mort de lignées cellulaires T infectées par HTLV-1 pour lesquelles Bfl-1 représente un gène de survie. Ainsi, ce travail doit permettre à terme de développer de futurs médicaments dirigés contre Bfl-1 et de proposer une nouvelle stratégie thérapeutique ciblée contre l’ATLL / Human T lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL) that develops after several decades and for which there is no effective treatment. Among the viral proteins of HTLV-1, Tax and HBZ play a major role in the development of ATLL. If Tax participates in the initiation of leukemogenesis from the early stages, HBZ rather plays a role in maintaining the tumor phenotype in the late stages. The aims of our study were to better understand the regulation of Bfl-1 and Bcl-xL anti-apoptotic protein expression by Tax and HBZ viral proteins, as well as their role in the survival of HTLV-1-infected T-cells to propose new therapeutic strategies. We showed that Tax induces Bfl-1 and Bcl-xL expression via the NF-κB pathway, whereas HBZ has no effect on their expression. Tax also cooperates with c-Jun and JunD transcription factors of AP-1 family to increase the expression of these anti-apoptotic genes. By contrast, HBZ modulates the Tax-induced bfl-1 trans-activation. Altogether, our data indicate that Tax plays a key role in activating Bfl-1 and Bcl-xL expression and suggests that Bfl-1 and Bcl-xL are potentially expressed during the early and the late stages of ATLL development. Using short hairpin RNA strategy, we then showed that Bfl-1 and/or Bcl-xL are involved in HTLV-1-infected T-cell line survival, indicating that Bfl-1 and Bcl-xL represent potential therapeutic targets in the case of ATLL. One approach currently being developed in anti-cancer drug discovery is to search for small inhibitory compounds targeting anti-apoptotic proteins of the Bcl-2 family. But so far, no drug specifically targeting Bfl-1 is available. In collaboration with the IMAXIO Company, we have identified 83 molecules able to inhibit Bfl-1 anti-apoptotic activity using two high-throughput screening. One of these molecules specifically induced the death of HTLV-1-infected T-cell for which Bfl-1 represents a survival gene. This work provides new insight for long-term development of future drugs directed against Bfl-1 and should allow us to propose new therapeutic strategy for ATLL treatment