Development of a microtiter plate fluorescent assay for inhibition studies on the HTLV-1 and HIV-1 proteinases

Bagossi, Péter; Kádas, János; Miklóssy, Gabriella; Boross, Péter; Weber, Irene T.; Tőzsér, József

doi:10.1016/j.jviromet.2004.03.001

Cited by 35 publications

(25 citation statements)

References 21 publications

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“…To assay the HIV-1 and HTLV-1 PR inhibitors, a microtiter plate reader assay using fluorescent Dabcyl/Edans-tagged analog of the capsid2nucleocapsid substrate was used (RE(Edans)TKVL2VVQPK-(Dabcyl)R, where the arrow represents the cleavable bond) (35). Briefly, enzyme, substrate, and inhibitor were incubated in 250 mM phosphate buffer, pH 5.6, containing 5% glycerol, 1 mM EDTA, 5 mM DTT, 500 mM NaCl, 1% Me 2 SO in 96-well microtiter plates.…”

Section: Methodsmentioning

confidence: 99%

“…Our previous studies on HIV-1 and HIV-2 PRs indicated that reduced peptide bond-containing substrate analogs act as potent inhibitors of the retroviral proteinases in the presence of high salt (30). IB268 and IB269 were potent inhibitors of the wild type HTLV-1 PR in the HPLC assay, with K i values below 50 nM (35). They were also the best inhibitors of the wild type HTLV-1 PR in the fluorometric assay, which was performed in a substantially lower ionic strength (Fig.…”

Section: Mutations Affecting the S3/s1-binding Sites Of Htlv-1 Protease-mentioning

confidence: 98%

“…We have tested these inhibitors and two reduced peptide-containing HTLV-1 inhibitors on the wild type and the mutant HTLV-1 PRs. The assays were performed in a high-throughput microtiter plate fluorescent assay system (35). Except for indinavir, the HIV-1 PR inhibitors were not effective against the wild type HTLV-1 PR (Fig.…”

Section: Mutations Affecting the S3/s1-binding Sites Of Htlv-1 Protease-mentioning

confidence: 99%

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Narrow Substrate Specificity and Sensitivity toward Ligand-binding Site Mutations of Human T-cell Leukemia Virus Type 1 Protease

Kádas

Weber

Bagossi

et al. 2004

Journal of Biological Chemistry

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Human T-cell leukemia virus type 1 (HTLV-1) is associated with a number of human diseases; therefore, its protease is a potential target for chemotherapy. To compare the specificity of HTLV-1 protease with that of human immunodeficiency virus type 1 (HIV-1) protease, oligopeptides representing naturally occurring cleavage sites in various retroviruses were tested. The number of hydrolyzed peptides as well as the specificity constants suggested a substantially broader specificity of the HIV protease. Amino acid residues of HTLV-1 protease substrate-binding sites were replaced by equivalent ones of HIV-1 protease. Most of the single and multiple mutants had altered specificity and a dramatically reduced folding and catalytic capability, suggesting that mutations are not well tolerated in HTLV-1 protease. The catalytically most efficient mutant was that with the flap residues of HIV-1 protease. The inhibition profile of the mutants was also determined for five inhibitors used in clinical practice and inhibitor analogs of HTLV-1 cleavage sites. Except for indinavir, the HIV-1 protease inhibitors did not inhibit wild type and most of the mutant HTLV-1 proteases. The wild type HTLV-1 protease was inhibited by the reduced peptide bond-containing substrate analogs, whereas the mutants showed various degrees of weakened binding capability. Most interesting, the enzyme with HIV-1-like residues in the flap region was the most sensitive to the HIV-1 protease inhibitors and least sensitive to the HTLV-1 protease inhibitors, indicating that the flap plays an important role in defining the specificity differences of retroviral proteases.

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Section: Methodsmentioning

confidence: 99%

Section: Mutations Affecting the S3/s1-binding Sites Of Htlv-1 Protease-mentioning

confidence: 98%

Section: Mutations Affecting the S3/s1-binding Sites Of Htlv-1 Protease-mentioning

confidence: 99%

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Narrow Substrate Specificity and Sensitivity toward Ligand-binding Site Mutations of Human T-cell Leukemia Virus Type 1 Protease

Kádas

Weber

Bagossi

et al. 2004

Journal of Biological Chemistry

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“…Although the design and synthesis of inhibitors specific for HTLV-1 PR have been carried out, most of the compounds are active only in micromolar concentration (9,10). The best statine-containing inhibitor has a K i of 50 nM under high-salt conditions (7) but of only 2.3 M in a low-salt buffer (11). In comparison, a number of subpicomolar inhibitors of HIV-1 PR have been developed by using the principles of rational drug design (12).…”

mentioning

confidence: 99%

Crystal structure of human T cell leukemia virus protease, a novel target for anticancer drug design

Laco

Jaskólski

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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The successful development of a number of HIV-1 protease (PR) inhibitors for the treatment of AIDS has validated the utilization of retroviral PRs as drug targets and necessitated their detailed structural study. Here we report the structure of a complex of human T cell leukemia virus type 1 (HTLV-1) PR with a substratebased inhibitor bound in subsites P5 through P5 . Although HTLV-1 PR exhibits an overall fold similar to other retroviral PRs, significant structural differences are present in several loop areas, which include the functionally important flaps, previously considered to be structurally highly conserved. Potential key residues responsible for the resistance of HTLV-1 PR to anti-HIV drugs are identified. We expect that the knowledge accumulated during the development of anti-HIV drugs, particularly in overcoming drug resistance, will help in designing a novel class of antileukemia drugs targeting HTLV-1 PR and in predicting their drug-resistance profile. The structure presented here can be used as a starting point for the development of such anticancer therapies.inhibitor ͉ leukemia ͉ retroviral protease H uman T cell leukemia virus type 1 (HTLV-1) is a retrovirus that is epidemiologically associated with mature CD3 ϩ CD4 ϩ T cell-type leukemia͞lymphoma (ATL), as well as with tropical spastic paraparesis͞myelopathy (1, 2). It is estimated that up to 30 million people worldwide are infected with HTLV, with ATL being particularly prevalent in Japan (3). Only an estimated 3-5% of people infected with the virus develop ATL in their lifetime, but for those that do, the prognosis is poor (4). Although a number of treatments for ATL, such as combination chemotherapy, monoclonal antibodies directed against the ␣ chain of the interleukin 2 receptor, and antiviral therapy involving IFN-␣ and zidovudine, are used clinically, they show only very limited efficacy (3). Novel approaches under investigation use proteasome inhibitors (5) and Tax-targeted immunotherapy (4), but they have not yet been tested in practice. It is clear that new anti-ATL targets need to be found.In common with other retroviruses, HTLV-1 encodes a protease (PR) necessary for its maturation. Because inhibition of the enzyme has been shown to prevent viral proliferation, development of inhibitors targeting HTLV-1 PR is an attractive new path for chemotherapy (6). HTLV-1 PR is a homodimer, with each chain containing 125 residues. The enzymatic properties of HTLV-1 PR, including its substrate specificity, have already been studied in considerable detail (7,8). Although the design and synthesis of inhibitors specific for HTLV-1 PR have been carried out, most of the compounds are active only in micromolar concentration (9, 10). The best statine-containing inhibitor has a K i of 50 nM under high-salt conditions (7) but of only 2.3 M in a low-salt buffer (11). In comparison, a number of subpicomolar inhibitors of HIV-1 PR have been developed by using the principles of rational drug design (12).Structural investigations of HTLV-1 PR have not been su...

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“…However, significant differences are seen in some of the pockets that accommodate the inhibitor side chains. These differences explain why various anti-HIV drugs in current clinical use, such as amprenavir, saquinavir, indinavir, ritonavir, and nelfinavir, fail to inhibit HTLV-1 PR, 7 and suggest the chemical and steric prerequisites of an optimal inhibitor. We found that two residues, Trp98 and Leu57 of HTLV-1 PR, are particularly responsible for the specific requirements of HTLV-1 PR (Fig.…”

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confidence: 99%

Lessons Learned Fighting HIV Can Be Applied to Anti-Cancer Drug Design

Gustchina

Jaskólski²,

Wlodawer³

2006

Cell Cycle

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Development of a microtiter plate fluorescent assay for inhibition studies on the HTLV-1 and HIV-1 proteinases

Cited by 35 publications

References 21 publications

Narrow Substrate Specificity and Sensitivity toward Ligand-binding Site Mutations of Human T-cell Leukemia Virus Type 1 Protease

Narrow Substrate Specificity and Sensitivity toward Ligand-binding Site Mutations of Human T-cell Leukemia Virus Type 1 Protease

Crystal structure of human T cell leukemia virus protease, a novel target for anticancer drug design

Lessons Learned Fighting HIV Can Be Applied to Anti-Cancer Drug Design

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