SAR analysis of compounds that inhibit Human Immunodeficiency Virus Fusion -Set 2
The fusion-active conformation of the envelope protein gp41 of HIV-1 consists of an N-terminal trimeric a-helical coiled coil domain, and three anti-parallel C-terminal helices which 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. This structure forms as a more ..
BioActive Compounds: 39
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1R21NS059403-01
Assay Provider: Dr. Miriam Gochin, Touro University-California, Vallejo, CA
The fusion-active conformation of the envelope protein gp41 of HIV-1 consists of an N-terminal trimeric a-helical coiled coil domain, and three anti-parallel C-terminal helices which 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. This structure forms as a result of a conformational change in gp41, triggered by gp120 and co-receptor binding to host cell receptors. Prevention of six-helix bundle formation has been recognized as an important mechanism for viral fusion inhibition
A metallopeptide-based fluorescence assay has been developed to detect HIV-1 fusion inhibitors, by the screening of small molecules that bind to the hydrophobic pocket of gp41. The assay involves two peptides representing the inner N-terminal-heptad-repeat (HR1) coiled coil and the outer C-terminal-heptad-repeat (HR2) helical domains of the gp41 six-helix bundle which forms prior to fusion. The HR1 peptide is modified with a metal-ligated dye complex, which maintains structural integrity and permits association with a fluorescein-labeled HR2 peptide concomitant with fluorescence quenching. Compounds able to disrupt six-helix bundle formation can act as fusion inhibitors, and can be detected in the assay from an increase in the fluorescence that is correlated with compound's efficacy.
This confirmatory, concentration-response assay has been developed and performed to confirm the hits originally identified in "uHTS fluorescence assay for the identification of Human Immunodeficiency Virus Fusion Inhibitors" (AID1986) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.
1) Env2.0 peptide
2) C18-Aib(FL) peptide
3) Assay Buffer: 25 mM Tris-acetate pH 7.0, 0.01% Tween-20
4) The Fe(II) complex [Fe(II)(env2.0)3]2+ is prepared by addition of a one-third stoichiometry of freshly prepared ferrous ammonium sulfate to peptide in 25 mM Tris-acetate pH 7.0
Mix equal parts of the Env2.0/Fe complex and C18-Aib(FL) (60 uM) just prior to the assay
1) Using a Labcyte Echo, DMSO and test compounds are transferred to wells of a black, Corning 1536 well assay plate. DMSO only is transferred to columns 1-3 and 46-48(Control wells), while varying volumes of test compounds are transferred to columns 4-45 to achieve the desired test concentrations. Compounds are transferred from a 2 mM stock to give the stated final concentration. Test compound wells in the assay plate are back-filled with DMSO to equalize final assay concentrations.
2) Immediately following Echo Transfer, 2 ul of assay buffer is added to columns 1 and 2.
3) Add 2 ul of 60 uM C18-Aib(FL) into columns 3-48of a black, Corning (#2725) 1536 well assay plate.
4) Add 2 ul of 4 uM Env2.0/Fe complex into all columns (1-48)
5) Incubate lidded plate for 30 minutes at room temp.
6) Read plate on a BMG PHERAstar at 485/520nm in Fluorescence Intensity mode
i. Positioning delay = 0.0
ii. Flashes/well = 10
Compounds with a demonstrated IC50 <= 100 uM are defined as actives in this assay.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data and is not applicable in this assay.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in this assay demonstrates a Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account the items discussed above is
Score = 82 + 3*(pIC50 - 3)*QC,
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 85 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)