METHOD FOR CONTROLLING THE FIDELITY AND PROCESSIVITY OF...

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage

Reexamination Certificate

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C435S006120, C435S339000

Reexamination Certificate

active

06727059

ABSTRACT:

The present invention in particular relates to the treatment of pathological conditions induced by retroid viruses, in particular HIV 1, using nucleotide analogs which modulate the fidelity and processivity of the reverse transcriptase of this virus.
The viruses responsible for the acquired immuno-deficiency syndrome (AIDS) have been the subject of many studies since 1981, the date at which the disease was identified for the first time. AIDS is a public health problem for many countries in the world. Thus, in the USA for example, the number of declared AIDS cases exceeds 100 000 and the number of individuals infected has been estimated at more than one million. The propagation of the disease is accentuated by the number of chronic carriers of the virus responsible for AIDS who remain asymptomatic for many years, if not throughout their entire life, and are therefore unidentified sources of contagion. This disease can be transmitted sexually and via the blood.
AIDS is a disease which affects the host's immune system, thus favoring the appearance of opportunistic infections or pathological conditions against which a healthy immune system would have protected the host. When AIDS is declared death generally occurs two to three years after diagnosis, subsequent to a collapse of the patient's immune defenses and to multiple opportunistic infections.
It is very difficult to classify the AIDS viruses, given the extreme genetic and antigenic variability which they show; conventionally, it is accepted that two types of virus responsible for human AIDS exist: HIV-1 and HIV-2 (Fauci, 1998). The HIV-1 virus is the agent responsible for AIDS in Central Africa, in Europe, in the USA and in many other countries, while HIV-2 is predominant in the west of Africa. Precise comparative analysis of various viral genomes has shown that the HIV-2 virus is closer to the simian viruses of the macaque (SIVmac) and of the sooty mangabey (SIVsm) than to the human HIV-1 virus and its homolog in chimpanzees (SIVcpz). Besides the three types of simian virus, feline viruses and viruses in other mammals exist.
The AIDS viruses are part of the group of retroid viruses which have the major common characteristic of using, during the viral and infective cycle, a very particular enzyme, reverse transcriptase (RT). The characteristic of this enzyme is that it catalyzes the synthesis of a double-stranded DNA from a single-stranded RNA matrix. The diverse members of the retroid group comprise retroviruses (oncogenic and non oncogenic), hepadnaviruses (hepatitis viruses) and caulimoviruses (viruses of higher plants). Three subgroups are conventionally identified in the retrovirus group: RNA oncoviruses, lentiviruses (HIV) and spumaviruses.
Many efforts have been made to attempt to understand and identify the molecular bases of each step of the viral cycle of HIV in order to develop curative or preventative therapies for AIDS. The CD4 receptor is the molecule which has a predominant role during infection with HIV; this receptor is located at the surface of several cells of the immune system: helper T4 lymphocytes, monocytes and macrophages. This molecule interacts with high affinity with viral glycoproteins (gp120 for HIV-1 and gp140 for HIV-2) embedded in the external viral envelope. The virus enters the target cell carrying the CD4 receptor only in the presence of a cofactor (for example CCR5, CCR3 and CCR2b) which allows the viral envelope to fuse with the cell membrane (Alkhatib et al., 1996; Deng et al., 1996, Choe et al., 1996). When the virus infects the cell, its genomic RNA is released. The reverse transcriptase (RT) catalyzes proviral DNA synthesis in the cytoplasm within the hour following infection. After retrotranscription, the resulting double-stranded viral DNA molecule penetrates into the nucleus of the infected cell. Integrase P32 cleaves the genomic DNA of the host cell, thus allowing the open nuclear DNA to receive the proviral DNA (Kim et al., 1989; Sato et al., 1992). There do not appear to be, a priori, preferential regions of integration in the chromosomes of the target cells. Integration is followed by a long latency period and then the integrated provirus is expressed (transcription and translation of its genes). It is transcribed into RNA which will be used as a genome for new virions and also as messenger for the synthesis of viral proteins.
For multiple reasons, the mechanism of replication of HIV poses many problems for the production of an effective therapy. Specifically, by virtue of its integration into the cellular genome, the proviral DNA behaves like a genetic element of the host. In addition, the HIV virus is disseminated throughout the entire body, in the T lymphocytes, monocytes and macrophages and also in the central nervous system. Finally, the HIV virus possesses very great antigenic variability, of which there are two main causes: the low fidelity of the viral reverse transcriptase (Gojobori et al. (1985); Jolly et al. (1986)), which has no mechanism for error correction, and the high recombination rate of the viral genome linked to the diploid nature of this same genome (Pathak et al., 1990 a and b; Hu and Temin, 1990b).
The diverse curative therapies currently used clinically consist, mostly, of meeting the virus head on, either by blocking the activity of the reverse transcriptase or by inhibiting the activity of viral enzymes essential for infection or replication (proteases, integrases). Diverse conventional antiviral agents have been identified by systematic assessment (screening) of molecules improved by successive chemical substitutions after demonstration of an antiviral effect. More recently, the construction, based on crystallographic molecular models, of viral inhibitors, aimed at strict modification of their target, has been explored. These medicinal products are classified according to their site of action and their chemical structure. The most commonly used in clinical trials in multitherapy are nucleotide and non-nucleotide reverse transcriptase inhibitors.
The nucleotide analogs developed to date interfere with infection by HIV and its replication via specific incorporations which inhibit the reverse transcriptase. Specifically, when the reverse transcriptase incorporates into the DNA chain, during synthesis, chemical compounds analogous to nucleotides blocked in 3′, the molecule thus neosynthesized terminates with a site incapable of accepting the addition of another nucleotide. The truncated DNA has lost part of the genetic information and almost certainly its infectivity.
Among these agents, mention should be made of 3′-azido-3′-deoxythimidine (AZT), 2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxycitidine (ddC), 2′,3′-dideoxy-3′-thia-cytidine (3TC), 2′,3′-didehydro-2′,3′-dideoxythymidine (D4T) and 2′-deoxy-5-fluoro-3′-thiacytidine (FTC). The clinical use of AZT makes it possible to decrease the frequency and seriousness of opportunistic infections and to reduce mortality in patients, but its not insignificant toxicity prohibits it being used continuously. Even though some analogs prove to be less toxic, such as dideoxyinosine (ddI), while at the same time being as active as AZT, the effectiveness of these agents remains limited in that the absorption of these antiviral agents causes side effects. In addition, due to its high mutation rate, the HIV virus rapidly becomes resistant to AZT and to the other nucleotide analogs during therapy. The emergence of resistants makes it necessary to increase the therapeutic doses administered to patients. It has thus been demonstrated that, after a certain amount of time, AZT may inhibit some cellular DNA polymerases at concentrations lower than those which inhibit HIV-1 reverse transcriptase, which explains the toxicity of these nucleotide compounds.
Another group of reverse transcriptase-specific inhibitors has been discovered more recently using a very different approach. Whereas research on nucleotide analogs follows a

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