A fluorescence assay for rapid detection of ligand binding affinity to HIV-1 gp41.
The fusion-active conformation of the envelope protein gp41 of HIV-1 consists of an N-terminal trimeric alpha-helical coiled-coil domain and three anti-parallel C-terminal helices that fold down the grooves of the coiled-coil to form a six-helix bundle. Disruption of the six-helix bundle is considered to be a key component of an effective non-peptide fusion inhibitor. In the current study, a fluorescence resonance energy transfer (FRET) experiment for the detection of inhibitor binding to the gp41 N-peptide coiled-coil of HIV-1 was performed, utilizing peptide inhibitors derived from the gp41 C-terminal helical region. The FRET acceptor is a 31-residue N-peptide containing a known deep hydrophobic pocket, stabilized into a trimer by ferrous ion ligation. The FRET donor is a 16-18-residue fluorophore-labeled C-peptide, designed to test the specificity of the N-C interaction. Low microM dissociation constants were observed, correlated to the correct sequence and helical propensity of the C-peptides. Competitive inhibition was demonstrated using the assay, allowing for rank ordering of peptide inhibitors according to their affinity in the 1-20 microM range. The assay was conducted by measuring fluorescence intensity in 384-well plates. The rapid detection of inhibitor binding may permit identification of novel drug classes from a library.
Gochin
Miriam
M
Department of Basic Sciences, Touro University, Vallejo, CA 94592, USA. mgochin@touro.edu
Savage
Ryan
R
Hinckley
Spencer
S
Cai
Lifeng
L
D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D015700:HIV Envelope Protein gp41 / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D020539:Sequence Analysis, Protein
D019380:Anti-HIV Agents; D015700:HIV Envelope Protein gp41; D008024:Ligands; D010455:Peptides