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This Article Full Text Full Text (PDF) Supplemental Research Data All Versions of this Article: ps.036079.108v1 17/9/1555    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Sasková, K. G. Articles by Konvalinka, J. PubMed PubMed Citation Articles by Sasková, K. G. Articles by Konvalinka, J. Social Bookmarking                   What's this?

Enzymatic and structural analysis of the I47A mutation contributing to the reduced susceptibility to HIV protease inhibitor lopinavir

Klára Grantz aková^1,2, Milan Koíek^1,2, Martin Lepík¹, Jií Brynda^1,3, Pavlína ezáová^1,3, Jana Václavíková¹, Ron M. Kagan⁴, Ladislav Machala⁵, and Jan Konvalinka^1,2,3

¹ Gilead Sciences and IOCB Research Center, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague 6, Czech Republic
² Department of Biochemistry, Faculty of Science, Charles University, Prague 2, Czech Republic
³ Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague 6, Czech Republic
⁴ Department of Infectious Diseases, Quest Diagnostics Inc., San Juan Capistrano, California 92675, USA
⁵ AIDS Center at the Clinic of Infectious Diseases, University Clinic Bulovka, Prague 8, Czech Republic

(RECEIVED April 29, 2008; FINAL REVISION June 11, 2008; ACCEPTED June 11, 2008)

Lopinavir (LPV) is a second-generation HIV protease inhibitor (PI) designed to overcome resistance development in patients undergoing long-term antiviral therapy. The mutation of isoleucine at position 47 of the HIV protease (PR) to alanine is associated with a high level of resistance to LPV. In this study, we show that recombinant PR containing a single I47A substitution has the inhibition constant (K_i ) value for lopinavir by two orders of magnitude higher than for the wild-type PR. The addition of the I47A substitution to the background of a multiply mutated PR species from an AIDS patient showed a three-order-of-magnitude increase in K_i in vitro relative to the patient PR without the I47A mutation. The crystal structure of I47A PR in complex with LPV showed the loss of van der Waals interactions in the S2/S2' subsites. This is caused by the loss of three side-chain methyl groups due to the I47A substitution and by structural changes in the A47 main chain that lead to structural changes in the flap antiparallel β-strand. Furthermore, we analyzed possible interaction of the I47A mutation with secondary mutations V32I and I54V. We show that both mutations in combination with I47A synergistically increase the relative resistance to LPV in vitro. The crystal structure of the I47A/I54V PR double mutant in complex with LPV shows that the I54V mutation leads to a compaction of the flap, and molecular modeling suggests that the introduction of the I54V mutation indirectly affects the strain of the bound inhibitor in the PR binding cleft.

Keywords: HIV protease inhibitors; antiviral resistance development; X-ray structure; molecular recognition; enzyme kinetics

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