Multidrug Resistance to HIV-1 Protease Inhibition Requires Cooperative Coupling between Distal Mutations

Hiroyasu Ohtaka, Arne Schön, Ernesto I Freire

Research output: Contribution to journalArticle

Abstract

The appearance of viral strains that are resistant to protease inhibitors is one of the most serious problems in the chemotherapy of HIV-1/AIDS. The most pervasive drug-resistant mutants are those that affect all inhibitors in clinical use. In this paper, we have characterized a multiple-drug-resistant mutant of the HIV-1 protease that affects indinavir, nelfinavir, saquinavir, ritonavir, amprenavir, and lopinavir, This mutant (MDR-HM) contains six amino acid mutations (L10I/M46I/I54V/V82A/I84V/L90M) located within and outside the active site of the enzyme. Microcalorimetric and enzyme kinetic measurements indicate that this mutant lowers the affinity of all inhibitors by 2-3 orders of magnitude. By comparison, the multiiple-drug-resistant mutant only increased the Km of the substrate by a factor of 2, indicating that the substrate is able to adapt to the changes caused by the mutations and maintain its binding affinity. To understand the origin of resistance, three submutants containing mutations in specific regions were also studied, i.e., the active site (V82A/I84V), flap region (M46I/I54V), and dimerization region (L10I/L90M). None of these sets of mutations by themselves lowered the affinity of inhibitors by more than 1 order of magnitude, and additionally, the sum of the effects of each set of mutations did not add up to the overall effect, indicating the presence of cooperative effects. A mutant containing only the four active site mutations (V82A/I84V/M46I/I54V) only showed a small cooperative effect, suggesting that the mutations at the dimer interface (L10I/L90M) play a major role in eliciting a cooperative response. These studies demonstrate that cooperative interactions contribute an average of 1.2 ± 0.7 kcal/mol to the overall resistance, most of the cooperative effect (0.8 ± 0.7 kcal/mol) being mediated by the mutations at the dimerization interface. Not all inhibitors in clinical use are affected the same by long-range cooperative interactions between mutations. These interactions can amplify the effects of individual mutations by factors ranging between 2 and 40 depending on the inhibitor. Dissection of the energetics of drug resistance into enthalpic and entropic components provides a quantitative account of the inhibitor response and a set of thermodynamic guidelines for the design of inhibitors with a lower susceptibility to this type of mutations.

Original languageEnglish (US)
Pages (from-to)13659-13666
Number of pages8
JournalBiochemistry®
Volume42
Issue number46
DOIs
StatePublished - Nov 25 2003

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Multiple Drug Resistance
Mutation
Dimerization
Pharmaceutical Preparations
Nelfinavir
Saquinavir
Lopinavir
Indinavir
Ritonavir
Dissection
Enzyme kinetics
Chemotherapy
Substrates
Protease Inhibitors
Dimers
Catalytic Domain
Thermodynamics
Amino Acids
Human immunodeficiency virus 1 p16 protease
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Multidrug Resistance to HIV-1 Protease Inhibition Requires Cooperative Coupling between Distal Mutations. / Ohtaka, Hiroyasu; Schön, Arne; Freire, Ernesto I.

In: Biochemistry®, Vol. 42, No. 46, 25.11.2003, p. 13659-13666.

Research output: Contribution to journalArticle

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