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Molecular Pharmacology

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Research ArticleArticle

Mutational Analysis of Trp-229 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) Identifies This Amino Acid Residue as a Prime Target for the Rational Design of New Non-Nucleoside RT Inhibitors

Heidi Pelemans, Robert Esnouf, Erik De Clercq and Jan Balzarini
Molecular Pharmacology May 2000, 57 (5) 954-960;
Heidi Pelemans
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Robert Esnouf
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Erik De Clercq
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Jan Balzarini
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Abstract

Trp-229 is part of the non-nucleoside reverse transcriptase inhibitor (NNRTI)-binding pocket of HIV type 1 (HIV-1) reverse transcriptase (RT), and is also part of the “primer grip” of HIV-1 RT. Using site-directed mutagenesis, seven RT mutants were constructed bearing the mutations 229Phe, 229Tyr, 229Ile, 229His, 229Lys, 229Cys, and 229Gln. We found that all of the mutants showed severely compromised RNA- and DNA-dependent DNA polymerase activities (<2% of wild-type activity). The recombinant 229Phe and 229Tyr RT enzymes were among the mutant enzymes with the highest activity (0.7 and 1.1% of wild-type activity, respectively) and we evaluated these for resistance against several NNRTIs. No resistance was found for the 229Phe RT, but the 229Tyr RT showed a ∼20-fold resistance against UC-781 and lower resistance against emivirine and nevirapine. Attempts to make recombinant virus strains bearing the single 229Phe or 229Tyr RT mutation failed. Experiments in which we varied the pentenyl ether substituent of the thiocarboxanilide UC-781 revealed that Trp-229 can be specifically targeted by NNRTIs and that an alkenyloxy group length of five atoms assures an optimal interaction of the thiocarboxanilides with Trp-229. Our findings indicate that Trp-229, when combined with other crucial immutable amino acids (i.e., Tyr-318), is an appropriate candidate for the targeted design of new NNRTIs.

Footnotes

  • Send reprint requests to: Jan Balzarini, Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail: Jan.Balzarini{at}reg.kuleuven.ac.be

  • This research was supported by Funds of the Flemish Geconcerteerde Onderzoeksacties (GOA 95/5), the Flemish Fonds voor Wetenschappelijk Onderzoek (G.0104.98), the Biomedical Health Program of the European Commission, and European Union Contract IC18-CT98-0380.

  • Abbreviations:
    RT
    reverse transcriptase
    NNRTI
    non-nucleoside reverse transcriptase inhibitor
    UC-781
    N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]-2-methyl-3-furan-carbothioamide
    RDDP
    RNA-dependent DNA polymerase
    DDDP
    DNA-dependent DNA polymerase
    Ni-NTA
    nickel-nitrilotriacetic acid
    • Received October 1, 1999.
    • Accepted January 21, 2000.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 57 (5)
Molecular Pharmacology
Vol. 57, Issue 5
1 May 2000
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Research ArticleArticle

Mutational Analysis of Trp-229 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) Identifies This Amino Acid Residue as a Prime Target for the Rational Design of New Non-Nucleoside RT Inhibitors

Heidi Pelemans, Robert Esnouf, Erik De Clercq and Jan Balzarini
Molecular Pharmacology May 1, 2000, 57 (5) 954-960;

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Research ArticleArticle

Mutational Analysis of Trp-229 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) Identifies This Amino Acid Residue as a Prime Target for the Rational Design of New Non-Nucleoside RT Inhibitors

Heidi Pelemans, Robert Esnouf, Erik De Clercq and Jan Balzarini
Molecular Pharmacology May 1, 2000, 57 (5) 954-960;
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