Correlation between shiftide activity and HIV-1 integrase inhibition by a peptide selected from a combinatorial library |
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Authors: | Armon-Omer Ayelet Levin Aviad Hayouka Zvi Butz Karin Hoppe-Seyler Felix Loya Shoshana Hizi Amnon Friedler Assaf Loyter Abraham |
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Institution: | 1 Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel 2 Department of Organic Chemistry, Institute of Chemistry, The Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel 3 Molecular Therapy of Virus-Associated Cancers, German Cancer Research Center, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany 4 Department of Cell and Developmental Biology, The Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel |
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Abstract: | The human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein is an emerging target for the development of anti-HIV drugs. We recently described a new approach for inhibiting IN by “shiftides”—peptides that inhibit the protein by shifting its oligomerization equilibrium from the active dimer to the inactive tetramer. In this study, we used the yeast two-hybrid system with the HIV-1 IN as a bait and a combinatorial peptide aptamer library as a prey to select peptides of 20 amino acids that specifically bind IN. Five non-homologous peptides, designated as IN-1 to IN-5, were selected. ELISA studies confirmed that IN binds the free peptides. All the five peptides interact with IN with comparable affinity (Kd≈10 μM), as was revealed by fluorescence anisotropy studies. Only one peptide, IN-1, inhibited the enzymatic activity of IN in vitro and the HIV-1 replication in cultured cells. In correlation, fluorescence anisotropy binding experiments revealed that of the five peptides, only the inhibitory IN-1 inhibited the DNA binding of IN. Analytical gel filtration experiments revealed that only the IN-1 and not the four other peptides shifted the oligomerization equilibrium of IN towards the tetramer. Thus, the results show a distinct correlation between the ability of the selected peptides to inhibit IN activity and that to shift its oligomerization equilibrium. |
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Keywords: | Y2H yeast two-hybrid IN integrase HIV-1 human immunodeficiency virus type 1 LTR long terminal repeat LEDGF lens epithelium-derived growth factor IBD integrase binding domain Gal4-BD Gal4 binding domain trxA thioredoxin A NIH National Institutes of Health BSA bovine serum albumin MTT 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide MAGI multinuclear activation of a galactosidase indicator AZT 3&prime -azido-3&prime -deoxythymidine m o i multiplicity of infection PBS phosphate-buffered saline |
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