Analysis and characterization of dimerization inhibition of a multi-drug-resistant human immunodeficiency virus type 1 protease using a novel size-exclusion chromatographic approach.
Active-site inhibitors of HIV-1 PR (protease) block viral replication by preventing viral maturation. However, HIV-1 often develops resistance to active-site inhibitors through multiple mutations in PR and therefore recent efforts have focused on inhibiting PR dimerization as an alternative approach. Dimerization inhibitors have been identified using kinetic analysis, but additional characterization of the effect of these inhibitors on PR by physical methods has been difficult. In the present study, we identified a PR(MDR) (multi-drug-resistant HIV-1 PR) that was highly resistant to autoproteolysis. Using this PR and a novel size-exclusion chromatographic approach that incorporated fluorescence and MS detection, we were able to demonstrate inhibition of dimerization using P27 (peptide 27), a peptide dimerization inhibitor of PR previously identified on the basis of kinetic analysis. Incubation of PR(MDR) with P27, or other dimerization inhibitors, led to a dose- and time-dependent formation of PR monomers based on the change in elution time by size exclusion and its similar elution time to engineered forms of monomeric PR, namely PR(T26A) and glutathionylated PR. In contrast, incubation of PR(MDR) with a potent active-site inhibitor did not change the elution time for the PR(MDR) dimer. The monomeric PR induced by P27 had fluorescent characteristics which were consistent with unfolded PR. Structure-activity studies identified the active regions of P27 and experiments were performed to examine the effect of other dimerization inhibitors on PR. The present study is the first characterization of dimerization inhibition of PR(MDR), a prime target for these inhibitors, using a novel size-exclusion chromatographic approach.
Davis
David A
DA
HIV and AIDS Malignancy Branch, Retrovirology Disease Section, Center for Cancer Research, National Cancer Institute, Building 10, Room 6N106, NIH (National Institutes of Health), Bethesda, MD 20892, USA. dadavis@helix.nih.gov
Tebbs
Irene R
IR
Daniels
Sarah I
SI
Stahl
Stephen J
SJ
Kaufman
Joshua D
JD
Wingfield
Paul
P
Bowman
Michael J
MJ
Chmielewski
Jean
J
Yarchoan
Robert
R
D002850:Chromatography, Gel / Q000379:methods*; D016333:HIV Protease / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D055503:Protein Multimerization / Q000187:drug effects*; D055550:Protein Stability / Q000187:drug effects; D013329:Structure-Activity Relationship
D010455:Peptides; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1