AID	Name	Type	Comment
602344	Broad Institute Identification of HIV VIF Inhibitors Inhibitor Probe Project	summary

Keywords: VIF, multimerization, HIV, high content, imaging


Assay Overview: The goal of this project is to develop small molecules that antagonize Vif self-association and hence, viral infectivity. Basically, EGFP-V5-VIF and VIF-HA-REACh2 are coexpressed in HEK293T cells by transient transfection. Transfected cells are incubated with approximately 7.5 uM compounds for 24 hrs. Compounds that disrupt VIF self-association will lead to an increase in signal that will be detected by imaging on ImagExpress Micro (Molecular Devices). Image data is analyzed by MetaXpress software (Molecular Devices).


Expected Outcome: Inhibitors of VIF self-association will lead to an increase of signal.

Day 1:
Plate with CompacT SelecT (TAP Biosystem) HEK 293T cells at 8000 cells/well in 40 uL of growth medium [DMEM (VWR 45000-304) supplemented with 1x Anti-anti (Invitrogen 15240-062), 1x NEAA (Invitrogen 11140-050), and 10% FBS (HyClone SH30071.03)] in black clear bottom 384 plates (Aurora 1022-11330-s).
Incubate at 37oC for 24 hours.

Day 2:
Mix in a ratio of 1:2 (ug : uL) of testing DNA (mEGFP-V5-Vif : Vif-HA-REACh2 (1:4)) or control DNA (mEGFP-V5-Vif : pIRES-P (empty vector) (1:4)) prediluted in Optimem (Invitrogen 31985-070) with TurboFect (Fermentas, R0531). (e.g. 50 ng DNA with 0.1 uL TurboFect)
Mix and incubate for 15 minutes
Add 10 uL/well of the transfection mix to 384-well plates according to plate design with Combi (Thermo) at slow speed.
Shake plates for 30 seconds at 2000 rpm (Big Bear Automation HT-91000)

Incubate for 22 hrs at 37oC.


Day 3:
Pin compounds (3.75 mM) at 0.1 ul/well.

Incubate at 37oC for 24 hours.


Day 4:
Add 10 ul/well of fixation solution at final concentration of Hoeschet (1:2000, Invitrogen, H3570)/ Formaldehyde (2%, Sigma, 533998) with Combi at slow speed.

Seal the plates.

Plates are imaged with ImagExpress Micro (Molecular Devices) immediately or stored at 4oC before being imaged.

Images are analyzed with MetaExpress software (Molecular Devices)

PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in increasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Additive)' in Genedata (v7.0.3) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 50.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 50.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

tSamples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

Grant Number: 1 R21 NS067671-01