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BioAssay: AID 1986

uHTS fluorescence assay for the identification of Human Immunodeficiency Virus Fusion Inhibitors.

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 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 ..
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 Tested Compounds
 Tested Compounds
All(292329)
 
 
Active(890)
 
 
Inactive(291441)
 
 
 Tested Substances
 Tested Substances
All(292483)
 
 
Active(891)
 
 
Inactive(291592)
 
 
AID: 1986
Data Source: Burnham Center for Chemical Genomics (BCCG-A227-GP41-Inhibitors)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-10-14
Modify Date: 2010-12-30

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 890
Depositor Specified Assays
AIDNameTypeComment
435029SAR analysis of compounds that inhibit Human Immunodeficiency Virus Fusion, cell-cell fusion assayconfirmatory
1991Summary assay for the identification of Human Immunodeficiency Virus Fusion Inhibitors.summary
493095SAR analysis of compounds that inhibit Human Immunodeficiency Virus Fusion -Set 2confirmatory
434967SAR analysis of compounds that inhibit Human Immunodeficiency Virus Fusion.confirmatory
435031SAR analysis of compounds that inhibit Human Immunodeficiency Virus Fusion, cytoxicity assayconfirmatory
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries 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 alpha-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.
Protocol
Primary, single concentration screen:
Assay materials:
1) Env2.0 peptide
Seq: Ac-MTWBEWDREIBNYTSLIC(FAM)-NH2
2) C18-Aib(FL) peptide
Seq: bpy-GQAVEAQQHLLQLTVWGIKQLQARILAVEKK-NH2
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

Procedure
Mix equal parts of the Env2.0/Fe complex and C18-Aib(FL) (60 uM) just prior to the assay
1) Dispense 4 ul of 60 uM C18-Aib(FL) into columns 1 and 2 of a black, Corning (#2725) 1536 well assay plate.
2) Add 4 ul of 60 uM C18-Aib(FL)/4 uM Env2.0/Fe complex into columns 3-48
3) Using a HighRes biosolutions pintool dispense 40 nl of 2 mM compounds in DMSO to columns 5-48
4) Using a HighRes biosolutions pintool dispense 40 nl of DMSO to columns 1-4.
5) Incubate lidded plate for 30 minutes at room temp.
6) Read plate on a BMG PHERAstar at 485/520nm in Fluorescence Intensity mode
Positioning delay = 0.0
Flashes/well = 10

Dose Response:
Assay materials:
5) Env2.0 peptide
Seq: Ac-MTWBEWDREIBNYTSLIC(FAM)-NH2
6) C18-Aib(FL) peptide
Seq: bpy-GQAVEAQQHLLQLTVWGIKQLQARILAVEKK-NH2
7) Assay Buffer: 25 mM Tris-acetate pH 7.0, 0.01% Tween-20
8) 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

Procedure
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 3-48 of 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
Comment
Compounds that demonstrated % activity of >= 50 % and <=125 % at 20 uM concentration are defined as actives in this assay. Compounds that demonstrate >125% efficacy are fluorescent compounds interfering with the assay thus are considered inactive. Compounds that were retested in duplicate at 18.6 uM were marked as active if the %Inhibition >= 50%.

In the dose response assay compounds with an IC50 < 99 uM are considered to be active.

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
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. The score is correlated with % Inhibition in the assay demonstrated by a compound at 20 uM concentration or in the confirmation assay at 18.6 uM:
a. If primary % inhibition is less than 0 %, then the assigned score is 0
b. If primary % inhibition is greater than 125 %, then the assigned score is 40
c. If primary % inhibition is between 0 % and 125 %, then the calculated score is (% Inhibition)*0.32
2) Second tier (41-80 range) is reserved for dose-response confirmation data
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 the assay demonstrates the 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 = 44 + 6*(pEC50 - 3)*QC,
Where pEC50 is a negative Log(10) of the EC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 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.

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_QualifierThis qualifier is to be used with the next TID, IC50. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
2IC50*IC50 value determined using sigmoidal dose response equationFloatμM
3Std.Err(IC50)Standard Error of IC50 valueFloatμM
4nHHill coefficient determined using sigmoidal dose response equationFloat
5%inhibition at 20uM (20μM**)% inhibition in primary screeningFloat
6Ave %inhibition of Repeats at 18.6uM (18.6μM**)Average % Inhibition of the repeatsFloat%
7Std.Err(Repeats) at 18.6uM (18.6μM**)Average standard error of the repeatsFloat
8Mean High (20μM**)Mean Absorbance of negative controls in the corresponding plateFloatRFU
9STD Deviation High (20μM**)Standard deviation (n=64) of negative controls in the corresponding plateFloatRFU
10Mean Low (20μM**)Mean Absorbance of positive controls in the corresponding plateFloatRFU
11STD Deviation Low (20μM**)Standard deviation (n=64) of positive controls in the corresponding plateFloatRFU

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R21NS059403-01

Data Table (Concise)
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