Inhibition of HIV replication in cell culture by the specific aspartic protease inhibitor pepstatin A

Helm, K. von der; Gürtler, Lutz; Eberle, Josef; Deinhardt, Friedrich

doi:10.1016/0014-5793(89)81368-7

Cited by 37 publications

(9 citation statements)

References 17 publications

(3 reference statements)

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“…The cells were incubated for 4 days at 37 °C without inhibitor; then culture supernatants of these cells were tested for virus pro-duction with an HIV gag p24 antigen capture assay (Abbott) [19]. From these data TCIDs0 was calculated.…”

Section: Hiv Infectivity Assaymentioning

confidence: 99%

“…The H I V proteinase is a member of the aspartic proteases and can be inhibited in vitro by 1 0 -S M Pepstatin A, a prototype of aspartic proteinase inhibitors [7,11,16,17]. This drug was applied at a concentration of 1 0 -4 M to HIV infected cell cultures and both the production and the infectivity of HIV particles were significantly decreased [19]. New aspartic protease inhibitors, specifically developed for the HIV proteinase, blocked the purified enzyme even at nanomolar concentrations and when applied to cell culture immediately after HIV infection exhibited antiviral activity and led to a reduction of HIV particle (gag protein p24) production [3,12,13,15].…”

Section: Introductionmentioning

confidence: 99%

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Analysis of non-infectious HIV particles produced in presence of HIV proteinase inhibitor

Schätzl

Gelderblom

Nitschko

et al. 1991

Archives of Virology

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Newly developed substrate analogue peptidomimetics are able to inhibit the human immunodeficiency virus, HIV-1 proteinase at nanomolar concentration. In HIV infected cell culture they exhibit antiviral activity. We have analyzed the non-infectious HIV particles produced in chronically HIV infected cell culture in presence of one of these inhibitors. The total production of virus particles was not substantially reduced in drug treated cultures, compared to non-inhibited control cultures, but the infectivity of these virus particles was reduced about 100 fold. The processing of gag and gag-pol protein precursor was inhibited; only borderline activity of reverse transcriptase (RT) could be detected in these particles and they contained nonprocessed gag precursor protein. Thin section electron microscopy of inhibitor-treated, HIV-infected cells revealed reduced viral cytopathogenicity and both inhibition of particle assembly and incomplete maturation of the particles formed. The HIV particles produced in the presence of the proteinase inhibitor were studded with envelope glycoprotein knobs and often comprised multiple budding regions, but were morphologically immature.

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Section: Hiv Infectivity Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Analysis of non-infectious HIV particles produced in presence of HIV proteinase inhibitor

Schätzl

Gelderblom

Nitschko

et al. 1991

Archives of Virology

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“…Here we present the first report of an aspartic protease specific inhibitor, pepstatin A, exhibiting slow-tight binding inhibition against xylanase from Thermomonospora sp. There are several reports on the inhibition of pepstatin against aspartic proteases (17,25,37,38,39), but this is the first study that explores the inhibition mechanism of this specific aspartic protease inhibitor against a xylanase. The inhibitor showed exceptionally high potency against Xyl I, the thermostable xylanase from a Thermomonospora sp., and its 1:1 molar ratio of interaction with the enzyme indicated its "tight binding" nature.…”

Section: Discussionmentioning

confidence: 99%

“…The aspartic protease inhibitor (pepstatin A) was well documented for its inhibitory activity toward pepsin, renin, cathepsin D, and human immunodeficiency virus 1 protease (37,38,39,25). The bifunctional nature of pepstatin A was established by its potency toward Xyl I, the xylanase purified from the Thermomonospora sp.…”

Section: Kinetic Analysis Of the Inhibition Of Xyl I-mentioning

confidence: 99%

“…The unusual potency of pepstatin toward aspartic proteases has been widely exploited as a research tool for unraveling the mechanism of this group of enzymes (18), its biological functions (19), and in affinity chromatography (20,21). It also has been tested as a therapeutic agent for experimental control of gastric ulcer (17,22), carrageenin edema (17), hypertension (23,24), and infectious diseases like human immunodeficiency virus (25). To our knowledge there are no reports of pepstatin inhibition on xylanolytic enzymes.…”

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confidence: 99%

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Inhibition of 1,4-β-d-Xylan Xylanohydrolase by the Specific Aspartic Protease Inhibitor Pepstatin

Vathipadiekal

Rao

2004

Journal of Biological Chemistry

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This is the first report that describes the inhibition mechanism of xylanase from Thermomonospora sp. by pepstatin A, a specific inhibitor toward aspartic proteases. The kinetic analysis revealed competitive inhibition of xylanase by pepstatin A with an IC 50 value 3.6 ؎ 0.5 M. The progress curves were time-depended, consistent with a two-step slow tight binding inhibition. The inhibition followed a rapid equilibrium step to form a reversible enzyme-inhibitor complex (EI), which isomerizes to the second enzyme-inhibitor complex (EI*), which dissociated at a very slow rate. The rate constants determined for the isomerization of EI to EI* and the dissociation of EI* were 15 ؎ 1 ؋ 10 ؊5 and 3.0 ؎ 1 ؋ 10 ؊8 s ؊1 , respectively. The K i value for the formation of EI complex was 1.5 ؎ 0.5 M, whereas the overall inhibition constant K i * was 28.0 ؎ 1 nM. The conformational changes induced in Xyl I by pepstatin A were monitored by fluorescence spectroscopy, and the rate constants derived were in agreement with the kinetic data. Thus, the conformational alterations were correlated to the isomerization of EI to EI*. Pepstatin A binds to the active site of the enzyme and disturbs the native interaction between the histidine and lysine, as demonstrated by the abolished isoindole fluorescence of o-phthalaldehyde-labeled xylanase. Our results revealed that the inactivation of xylanase is due to the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis, and a model depicting the probable interaction between pepstatin A with xylanase has been proposed.

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Human immunodeficiency virus protease: A target for aids therapy

Debouck

Metcalf

1990

Drug Development Research

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Human immunodeficiency virus, also called HIV, is the etiologic agent of acquired immune deficiency syndrome (AIDS). This retrovirus produces a small, dimeric aspartyl protease which specifically cleaves the precursor forms of the structural proteins and enzymes of the virus. This proteolytic activity is absolutely required for the production of mature, infectious viral particles and is therefore an attractive target for therapeutic intervention. Peptide analogues containing transition‐state mimics were synthesized and shown to inhibit the activity of the purified HIV protease in vitro to various extents. Most interestingly, the most potent inhibitors were shown to effectively block the protease in HIV‐infected cells and to impair the viral life cycle. Other approaches to interfere with the viral protease activity or production are also discussed.

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Inhibition of HIV replication in cell culture by the specific aspartic protease inhibitor pepstatin A

Cited by 37 publications

References 17 publications

Analysis of non-infectious HIV particles produced in presence of HIV proteinase inhibitor

Analysis of non-infectious HIV particles produced in presence of HIV proteinase inhibitor

Inhibition of 1,4-β-d-Xylan Xylanohydrolase by the Specific Aspartic Protease Inhibitor Pepstatin

Human immunodeficiency virus protease: A target for aids therapy

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