Unusual Binding Mode of an HIV-1 Protease Inhibitor Explains its Potency against Multi-drug-resistant Virus Strains

Weber, Jan; Mesters, J.R.; Lepšı́k, Martin; Prejdová, Jana; Švec, Martin; Šponarová, Jana; Mlčochová, Petra; Skalická, Kristina; Střı́šovský, Kvido; Uhlı́ková, Táňa; Souček, Milan; Machala, Ladislav; M, Stanková; Vondrášek, Jiřı́; Klimkait, Thomas; Kraeusslich, H.-G.; Hilgenfeld, Rolf; Konvalinka, Jan

doi:10.1016/s0022-2836(02)01139-7

Cited by 48 publications

(54 citation statements)

References 53 publications

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“…The expression, refolding, and purification of HIV-1 PR, HIV-2 PR, and the HIV-1 PR variant (Q7K, L33I, L63I) bearing three mutations that minimize the autoproteolytic cleavage (30) were performed as described (31). PRs from mouse intracisternal A particles (MIA14 PR) (32), human cathepsin D, and pepsin were prepared as described (33,34).…”

Section: Methodsmentioning

confidence: 99%

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From nonpeptide toward noncarbon protease inhibitors: Metallacarboranes as specific and potent inhibitors of HIV protease

Cígler

Kožíšek

Řezáčová

et al. 2005

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

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273

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HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]di-ate, exhibited a Ki value of 2.2 nM and a submicromolar EC50 in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 Å resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3 subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition. rational drug design ͉ aspartic proteases ͉ carboranes ͉ x-ray structure analysis ͉ virostatics

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Section: Methodsmentioning

confidence: 99%

“…Inhibition of HIV PRs. The IC 50 and K i values were determined by spectrophotometric assay with the chromogenic substrate KARVNleNphEANle-NH 2 as described (31). The inhibition constants were estimated by using a competitive inhibition equation according to ref.…”

Section: Methodsmentioning

confidence: 99%

From nonpeptide toward noncarbon protease inhibitors: Metallacarboranes as specific and potent inhibitors of HIV protease

Cígler

Kožíšek

Řezáčová

et al. 2005

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

Self Cite

292

273

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“…The inhibition constants (K i ) were determined by spectrophotometric assay using the chromogenic peptide substrate LysAlaArgValNle*NphGluAlaNle-NH 2 as previously described. 11 Typically, 8-10 pmol of PR was added to 1 mL of 0.1 M sodium acetate buffer, pH 4.7, 0.3 M NaCl, and 4 mM EDTA, containing substrate at a concentration near the K m of the enzyme and various concentrations of inhibitor dissolved in DMSO. The final concentrations of DMSO were kept below 2.5% (v/v).…”

Section: Experimental Methodsmentioning

confidence: 99%

Inorganic Polyhedral Metallacarborane Inhibitors of HIV Protease: A New Approach to Overcoming Antiviral Resistance

et al. 2008

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HIV protease (PR) is a prime target for rational anti-HIV drug design. We have previously identified icosahedral metallacarboranes as a novel class of nonpeptidic protease inhibitors. Now we show that substituted metallacarboranes are potent and specific competitive inhibitors of drug-resistant HIV PRs prepared either by site-directed mutagenesis or cloned from HIV-positive patients. Molecular modeling explains the inhibition profile of metallacarboranes by their unconventional binding mode.

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“…The inhibition constants (K i values) were determined by spectrophotometric assay using the chromogenic peptide substrate KARVNle*NphEANle-NH 2 (where Nle is norleucine and Nph is p-nitrophenylalanine and the asterisk signifies the PR cleavage site) as previously described (39). Typically, 8 pmol of PR was added to 1 ml of 0.1 M sodium acetate buffer, pH 4.7, 0.3 M NaCl, and 4 mM EDTA, containing substrate at a concentration near the K m of the enzyme and various concentrations of inhibitor dissolved in dimethyl sulfoxide (DMSO).…”

Section: Generation Of Site-directed Mutants In the Recombinant Virusmentioning

confidence: 99%

Ninety-Nine Is Not Enough: Molecular Characterization of Inhibitor-Resistant Human Immunodeficiency Virus Type 1 Protease Mutants with Insertions in the Flap Region

Kožíšek

Šašková

Řezáčová

et al. 2008

J Virol

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Unusual Binding Mode of an HIV-1 Protease Inhibitor Explains its Potency against Multi-drug-resistant Virus Strains

Cited by 48 publications

References 53 publications

From nonpeptide toward noncarbon protease inhibitors: Metallacarboranes as specific and potent inhibitors of HIV protease

From nonpeptide toward noncarbon protease inhibitors: Metallacarboranes as specific and potent inhibitors of HIV protease

Inorganic Polyhedral Metallacarborane Inhibitors of HIV Protease: A New Approach to Overcoming Antiviral Resistance

Ninety-Nine Is Not Enough: Molecular Characterization of Inhibitor-Resistant Human Immunodeficiency Virus Type 1 Protease Mutants with Insertions in the Flap Region

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