The amino acid Asn136 in HIV-1 reverse transcriptase (RT) maintains efficient association of both RT subunits and enables the rational design of novel RT inhibitors

Jan Balzarini, Joeri Auwerx, Fátima Rodríguez-Barrios, Allel Chedad, Viktor Farkas, Francesca Ceccherini-Silberstein, Carlos García-Aparicio, Sonsoles Velázquez, Erik De Clercq, Carlo Federico Perno, María José Camarasa, Federico Gago

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

The highly conserved Asn136 is in close proximity to the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-specific lipophilic pocket of human immunodeficiency virus type 1 (HIV-1) RT. Site-directed mutagenesis has revealed that the catalytic activity of HIV-1 RT mutated at position Asn136 is heavily compromised. Only 0.07 to 2.1% of wild-type activity is retained, depending on the nature of the amino acid change at position 136. The detrimental effect of the mutations at position 136 occurred when the mutated amino acid was present in the p51 subunit but not in the p66 subunit of the p51/p66 RT heterodimer. All mutant enzymes could be inhibited by secondgeneration NNRTIs such as efavirenz. They were also markedly more sensitive to the inactivating (denaturating) effect of urea than wild-type RT, and the degree of increased urea sensitivity was highly correlated with the degree of (lower) catalytic activity of the mutant enzymes. Replacing wild-type Asn136 in HIV-1 RT with other amino acids resulted in notably increased amounts of free p51 and p66 monomers. Our findings identify a structural/functional role for Asn136 in stabilization of the RT p66/p51 dimer and provide hints for the rational design of novel NNRTIs or drugs targeting either Asn136 in the β7-β8 loop of p51 or its anchoring point on p66 (the peptide backbone of His96) so as to interfere with the RT dimerization process and/or with the structural support that the p51 subunit provides to the p66 subunit and which is essential for the catalytic enzyme activity.

Original languageEnglish
Pages (from-to)49-60
Number of pages12
JournalMolecular Pharmacology
Volume68
Issue number1
DOIs
Publication statusPublished - Jul 2005

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Reverse Transcriptase Inhibitors
RNA-Directed DNA Polymerase
HIV-1
Amino Acids
efavirenz
Urea
Enzymes
Dimerization
Drug Delivery Systems
Site-Directed Mutagenesis
Peptides
Mutation
Human immunodeficiency virus 1 reverse transcriptase

ASJC Scopus subject areas

  • Pharmacology

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The amino acid Asn136 in HIV-1 reverse transcriptase (RT) maintains efficient association of both RT subunits and enables the rational design of novel RT inhibitors. / Balzarini, Jan; Auwerx, Joeri; Rodríguez-Barrios, Fátima; Chedad, Allel; Farkas, Viktor; Ceccherini-Silberstein, Francesca; García-Aparicio, Carlos; Velázquez, Sonsoles; De Clercq, Erik; Perno, Carlo Federico; Camarasa, María José; Gago, Federico.

In: Molecular Pharmacology, Vol. 68, No. 1, 07.2005, p. 49-60.

Research output: Contribution to journalArticle

Balzarini, J, Auwerx, J, Rodríguez-Barrios, F, Chedad, A, Farkas, V, Ceccherini-Silberstein, F, García-Aparicio, C, Velázquez, S, De Clercq, E, Perno, CF, Camarasa, MJ & Gago, F 2005, 'The amino acid Asn136 in HIV-1 reverse transcriptase (RT) maintains efficient association of both RT subunits and enables the rational design of novel RT inhibitors', Molecular Pharmacology, vol. 68, no. 1, pp. 49-60. https://doi.org/10.1124/mol.105.012435
Balzarini, Jan ; Auwerx, Joeri ; Rodríguez-Barrios, Fátima ; Chedad, Allel ; Farkas, Viktor ; Ceccherini-Silberstein, Francesca ; García-Aparicio, Carlos ; Velázquez, Sonsoles ; De Clercq, Erik ; Perno, Carlo Federico ; Camarasa, María José ; Gago, Federico. / The amino acid Asn136 in HIV-1 reverse transcriptase (RT) maintains efficient association of both RT subunits and enables the rational design of novel RT inhibitors. In: Molecular Pharmacology. 2005 ; Vol. 68, No. 1. pp. 49-60.
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abstract = "The highly conserved Asn136 is in close proximity to the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-specific lipophilic pocket of human immunodeficiency virus type 1 (HIV-1) RT. Site-directed mutagenesis has revealed that the catalytic activity of HIV-1 RT mutated at position Asn136 is heavily compromised. Only 0.07 to 2.1{\%} of wild-type activity is retained, depending on the nature of the amino acid change at position 136. The detrimental effect of the mutations at position 136 occurred when the mutated amino acid was present in the p51 subunit but not in the p66 subunit of the p51/p66 RT heterodimer. All mutant enzymes could be inhibited by secondgeneration NNRTIs such as efavirenz. They were also markedly more sensitive to the inactivating (denaturating) effect of urea than wild-type RT, and the degree of increased urea sensitivity was highly correlated with the degree of (lower) catalytic activity of the mutant enzymes. Replacing wild-type Asn136 in HIV-1 RT with other amino acids resulted in notably increased amounts of free p51 and p66 monomers. Our findings identify a structural/functional role for Asn136 in stabilization of the RT p66/p51 dimer and provide hints for the rational design of novel NNRTIs or drugs targeting either Asn136 in the β7-β8 loop of p51 or its anchoring point on p66 (the peptide backbone of His96) so as to interfere with the RT dimerization process and/or with the structural support that the p51 subunit provides to the p66 subunit and which is essential for the catalytic enzyme activity.",
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AU - Chedad, Allel

AU - Farkas, Viktor

AU - Ceccherini-Silberstein, Francesca

AU - García-Aparicio, Carlos

AU - Velázquez, Sonsoles

AU - De Clercq, Erik

AU - Perno, Carlo Federico

AU - Camarasa, María José

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N2 - The highly conserved Asn136 is in close proximity to the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-specific lipophilic pocket of human immunodeficiency virus type 1 (HIV-1) RT. Site-directed mutagenesis has revealed that the catalytic activity of HIV-1 RT mutated at position Asn136 is heavily compromised. Only 0.07 to 2.1% of wild-type activity is retained, depending on the nature of the amino acid change at position 136. The detrimental effect of the mutations at position 136 occurred when the mutated amino acid was present in the p51 subunit but not in the p66 subunit of the p51/p66 RT heterodimer. All mutant enzymes could be inhibited by secondgeneration NNRTIs such as efavirenz. They were also markedly more sensitive to the inactivating (denaturating) effect of urea than wild-type RT, and the degree of increased urea sensitivity was highly correlated with the degree of (lower) catalytic activity of the mutant enzymes. Replacing wild-type Asn136 in HIV-1 RT with other amino acids resulted in notably increased amounts of free p51 and p66 monomers. Our findings identify a structural/functional role for Asn136 in stabilization of the RT p66/p51 dimer and provide hints for the rational design of novel NNRTIs or drugs targeting either Asn136 in the β7-β8 loop of p51 or its anchoring point on p66 (the peptide backbone of His96) so as to interfere with the RT dimerization process and/or with the structural support that the p51 subunit provides to the p66 subunit and which is essential for the catalytic enzyme activity.

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