Enzymes that help retroviruses integrate DNA carrying viral genetic information into host DNA
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Integrase helpsRetrovirusAn enzyme that integrates DNA carrying viral genetic information into the host's DNA.It is usually carried by the virus itself and does not exist inhost cell, so it can be used asAntiviral drugsA suitable target for.
Researchers may have found a new way to deal with HIV, that is, to inhibit an HIV enzyme called "integrase". Researchers are testing a new drug, which can inhibit "integrase". Through experiments on monkeys, they found that this drug can control the spread of AIDS.
HIV-1 integrase isRetrovirusThe essential enzyme for replication, which catalyzes the integration of viral DNA and host chromosome DNA, and has no analogues in human cells, has become an attractive and reasonable target for the treatment of AIDS.
A large number of HIV-1 integrase inhibitors have been identified, some of which show selective inhibition of HIV-1 integrase and blocking of viral replication activities. The two most influential types of inhibitors are catechol containing polyhydroxy aromatic compounds and recently reported aryl groupsβ-Diketoacids.
There are different kinds of compounds of new anti HIV drugs, including benzimidazole derivatives, nucleosides, polypeptides, hydroxyl substituted aromatic compounds and diketoacids.
Inhibitor research
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Research and development of inhibitors
HIV type 1 (HIV-1) integrase inhibitor developed by Merck scientists began to enter phase I clinical research in the United States and Belgium in March 2003, targeting volunteers who have not been infected with HIV.Phase I clinical study is the initial stage of human experiment, which aims to evaluate the safety, tolerance andPharmacokinetics(the level of drug metabolism in the blood).According to Jeffrey Chodakewitz, MD, Vice President of Clinical Research Department of Infectious Diseases and Vaccines of Merck, up to now, a small number of healthy volunteers (less than 70 people) have participated in the research, and their overall drug tolerance is good.He noted that the existing preclinical data support two doses per day. If all the studies can be carried out smoothly, the determination of the appropriate dosage still needs to pass the clinical study.
A research article published in Science on July 8, 2004 pointed out that Merck reported that when monkeys were infected with HIV at the initial stage, taking this drug could significantly protect monkeys. Therefore, this drug is also called "integrase" inhibitor, and it also has effects on the seriously ill.Merck is also studying several "integrase" inhibitors, and they use several volunteers to test them to determine whether these drugs are safe and whether they can inhibit the filtration virus.
A High throughput Screening Method for Novel Integrase Inhibitors
With the continuous upgrading of resistance to HIV-1, the research and development of anti HIV drugs is far behind. The discovery of new targets of anti HIV-1 drugs and the research and development of innovative drugs have become urgent.HIV-1 integrase catalyzes the integration of viral cDNA and host cell genome, and is an essential key enzyme in HIV replication cycle.There is no functional analogue of integrase in human body.Therefore, integrase is an ideal target for the development of anti AIDS drugs.
in recent years,School of Life Science and Bioengineering, Beijing University of TechnologyThe Laboratory of Molecular Design and Protein Function led by Professor Wang Cunxin, supported by two National Natural Science Foundation of China and five Beijing municipal projects, and supported by the "211 Project" of Beijing University of Technology, is committed to drug design and screening with HIV-1 integrase and gp 41 protein as the main targets, and has achieved remarkable results.In March 2008, the research group published a report in Acta Pharmacologica Sinica, 29 (3): 397-404).In this work, the magnetic bead capture DNA technology was introduced to replace the traditional DNA coating microplate method to modify and process DNA. A high-throughput integrase activity detection method was established through integrase catalyzed DNA strand transfer reaction. Based on this method, HIV inhibitors targeting integrase were screened, and a molecular level integrase inhibitor screening platform was established.This screening method discards the shortcomings of the traditional autoradiography method, such as radioactive contamination and difficulty in high-throughput screening, and improves the time-consuming and labor-intensive shortcomings of the traditional enzyme-linked immunosorbent assay (ELISA) coating, sealing micropores, etc.The method has high sensitivity, good specificity, convenience and rapidity, and can be used for high-throughput screening.Therefore, the workload of drug screening can be greatly reduced.This method can be applied to scientific research institutions and companies engaged in drug research and development to create higher social value.
The title of this research is: "A new high-throughput format assay for HIV-1 integrated strand transfer reaction using magnetic beads".
Protein background
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Human immunodeficiency virus (HIV) includes HIV-1 and HIV-2, of which HIV-2 is mainly distributed in West Africa, while HIV-1 is widely distributed around the world, which is the pathogen causing the AIDS epidemic worldwide.The integration process in HIV replication cycle is the process of integrating HIV-1 DNA into host DNA, and is also an indispensable process in HIV replication cycle.HIV-1 integrase (IN) participates in the whole integration process and catalyzes the whole integration reaction. It is an essential enzyme in the replication process of HIV-1, and is also an indispensable enzyme for stable viral infection.Therefore, inhibiting the activity of this enzyme will effectively fight against HIV-1, so many AIDS researchers take IN as a target for developing new drugs.However, due to the difficulty in dissolving HIV-1 IN and other reasons, the understanding of this enzyme is limited. Therefore, obtaining soluble IN is to study its activity and provides important help for screening anti HIV drugs.
Technical background
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Soluble expression -- because foreign proteins are easy to be hosted during expressionCellular proteaseDegradation or formation of inclusion bodies, and in vitro renaturation of inclusion bodies is often time-consuming, laborious and uneconomical, so soluble expression of foreign proteins in E. coli or Pichia pastoris has high academic and economic value.
PET-28a -- from the pET series of products produced by Novagen, with the main characteristics of
PET-28a: Kan T7lac His tag (N-terminal tag and optional C-terminal tag) T7 tag (internal tag) protein (thrombin)
Research objectives
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According to Dr. Chodakewitz, Merck plans to start phase I clinical research on a small number of HIV infected volunteers in the second half of 2002.The clinical research project of HIV-1 integrase inhibitor in the trial is expected to involve a wide range of people, including those who have never received antiretroviral treatment, and those who have developed resistance to other treatments, to evaluate the efficacy and tolerance of the drug.If the results of the clinical phase I study are satisfactory, Merck expects to start the clinical phase II study in 2003.
Research direction
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MerckIt has been 15 years since we committed ourselves to developing breakthrough HIV/AIDS therapeutic drugs and vaccines, and we are still working hard so far.In addition to the efforts to develop HIV-l integrase inhibitors, Merck is committed to the research of HIV vaccine. The research direction is not only to protect people from infection, but also to treat HIV patients.Two HIV research vaccines have entered the clinical phase I study.
Viral drugs
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Integrase inhibitors are new drugs for HIV/AIDS treatment, which may become a significant breakthrough in the treatment of antiretroviral drugs.HIV replication requires three enzymes: protease, reverse transcriptase and integrase.Existing drugs can inhibit the first two enzymes, namely protease and reverse transcriptase.However, drugs that inhibit integrase have not been approved.
Dr. Daria Hazuda, Executive Director of Merck's Biochemistry Department and Deputy Leader of HIV-1 Integrase Inhibitor Development Group, said: "According to the preclinical study, the HIV-l Integrase Inhibitor under study may have synergistic effect with reverse transcriptase inhibitor or protease inhibitor.If clinical trials can prove that this drug can be effective in combination with other types of antiretroviral drugs, it will make a valuable supplement to antiretroviral therapy.
Medical mechanism
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In the mechanism of anti AIDS, S - 1360 controls the infection of HIV to cells by inhibiting HIV integrase.Integrase, reverse transcriptase and protease are three enzymes necessary for HIV infected cells to replicate. All existing anti AIDS drugs destroy the function of reverse transcriptase or protease, but they do not commit any offense to integrase.Therefore, the research and development company said that S-1360 has achieved a breakthrough in mechanism, which has expanded the selection of anti AIDS drugs, and may better control the replication of HIV in the human body with the help of other drugs, thus improving the treatment of AIDS.
HIV-1 integrase is an important enzyme in HIV replication and an important target of AIDS drugs.In order to screen inhibitors targeting integrase proteins and construct HIV?1. The recombinant plasmid of integrase was used for soluble expression and functional study in prokaryotic cells.The F185K and C280S mutations were introduced into the integrase cDNA fragment of HIV-1 subtype B standard strain by overlapping PCR technology. The PCR amplified fragment was cloned into the pET-28a (+) expression vector, and the recombinant plasmid was constructed in EColi, SDS-PAGE was used to identify the expression product, affinity chromatography was used to purify the protein, and enzyme-linked immunosorbent assay was used to determine the biological activity of the integrase.Results The recombinant plasmid was highly efficient and stable in soluble expression. The ELISA experiment confirmed that the protein had the activity of integrase 3 'cutting DNA and 5' chain transfer.The study on the soluble expression and activity of HIV-1 integrase protein lays a foundation for the establishment of an anti HIV drug screening platform targeting the integrase
Treatment options
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Although the combination of drugs is highly effective, the need for new treatment options is supported by the spread of multi drug resistant virus strains, insufficient treatment durability, side effects caused by long-term use of existing antiretroviral drugs and other factors.
It is estimated that more than 78% of all patients receiving antiretroviral drugs are resistant to more than one class of drugs.The problem of drug resistance is becoming more and more serious in patients who have not received antiretroviral treatment.Among the patients who have not received drug treatment, the proportion of drug-resistant virus strains has risen from 5% in 1999 to 20% now.In animal experiments, Merck's drugs are effective for the treatment of highly mutated, multi drug resistant HIV-1.
Although there are many drugs for HIV/AIDS treatment, the prevalence of AIDS is still rampant.It is estimated that about 40 million people around the world are infected with HIV, of which more than 95% are in developing countries.AIDS is the infectious disease that causes the most deaths, causing 3 million deaths annually.
Structure function
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Scientists decipher the three-dimensional structure of integrase
British and American researchers reported in the journal Nature on January 31, 2010 that a new study they conducted together simulated the three-dimensional structure of integrase.Integrases can be found in retroviruses, including HIV, and act as "accomplices" when HIV replicates in the human body.This major breakthrough will help scientists solve a problem that has plagued the field of AIDS research for 20 years, so as to find a better way to treat AIDS.
When HIV infects the human body, the integrase is usually used as a tool to "copy and paste" the genetic information of the virus into its DNA, and integrate the viral DNA with the human DNA.Many scientists have tried to decipher the three-dimensional structure of this integrase bound to viral DNA, but the results have failed.Many of the new antiretroviral drugs use the function of inhibiting integrase to achieve the purpose of treating AIDS, but scientists do not know exactly the mechanism of these drugs and the mechanism of improving efficacy.
Because the structure of the integrase can only be determined by obtaining more high-quality crystals, in this study, researchers from Imperial College of Technology and Harvard University jointly borrowed the integrase from a retrovirus called prototype foam virus (PFV) and used it to make crystals.Although the scientific community does not know much about the prototype foam virus, based on the existing knowledge, researchers believe that this version of integrase is very similar to the integrase in HIV in structure and function.
The researchers conducted more than 40000 experiments in four years, and finally obtained seven kinds of crystals, and only one of them has enough mass, so that they can determine the three-dimensional structure of the integrase.Using the large synchrotron in the diamond light source center, the researchers collected the X-ray diffraction data of the crystal, and then determined the structure.Later, we observed for the first time how these antiretroviral drugs bind to integrase and block its activity.
This research shows that the integrase in HIV has a completely different structure from that previously expected, and its cracking means that scientists can fully understand the principle of action of related drugs from now on, in order to improve the efficacy and prevent HIV from developing drug resistance.
Design principle and action mechanism of inhibitors
According to the Daily Mail on February 2, 2010, scientists have successfully overcome a key problem that has plagued HIV research for more than 20 years. Their discovery is expected to develop a revolutionary new anti HIV drug to help patients get better treatment.
British and American researchers said they had cultivated a crystal that could clearly see the three-dimensional structure of integrase.This enzyme exists in retroviruses such as HIV, and is the target of new anti-cancer virus drugs.The research report was published in the authoritative journal Nature yesterday.
After more than 40000 experiments, researchers obtained high-quality crystals through which the three-dimensional structure of integrase can be clearly seen.Peter Cherepanov, a researcher at Imperial College London, said: "Although the initial progress was extremely slow and experienced many failures, we did not give up and finally achieved success."
Scientists from Imperial College London and Harvard University said that mastering the three-dimensional structure of integrase can help people fully understand how integrase inhibits drug action, how to improve drugs, and how to prevent HIV from producing antibodies to drugs.
At present, nearly 60 million people worldwide have been infected with HIV, and 25 million people have died of HIV since it began to spread.Although cocktail therapy can maintain the health of infected patients, it cannot be cured and prevented.[1]
