AID	Name	Type	Comment
651609	Broad Institute MLPCN HIV Entry Inhibitor Probe Project	summary	Summary

Keywords: cell fusion, Env protein, HIV, luciferase

Assay Overview:

Although some inhibitors of HIV-1 entry exist, these are not suitable for use in a prophylactic setting because of limitations in potency, breadth and/or adaptability to a microbicide formulation. HIV-1 Envs expressed on the surface of cells can mediate the fusion of cells with target cells expressing the CD4 and CCR5/CXCR4 receptors, such as CEM lymphoblasts. We have established an assay that monitors the cell-cell fusion activity of HIV-1 Envs with a firefly luciferase readout. In our primary high-throughput screen (HTS), a modified HeLa cell line that inducibly expresses the Envs of a primary HIV-1 strain (HIV-1JRFL) will be co-cultivated with receptor-bearing target cells (CEM21) in the presence of the compounds to be screened. CEM21 expresses a Tet-activator responsive Luciferase reporter. This is a Tetracycline regulated system and when the Tet-activator from the Env-expressing cell line is transferred into the target cell upon successful fusion, the Luciferase reporter will be activated. Detection of Luciferase will occur using the SteadyGlo reagent (Promega, Madison, WI). Maraviroc, a known CCR5 antagonist, that blocks Env-mediated cell fusion will be used as a positive control.

Expected Outcome: Compounds that inhibit cell fusion will cause a decrease in Luciferase signal.

HIV Fusion Assay Protocol (Streamlined version for HTS)

1.Grow JRFL effector cells in DMEM passage medium with Doxycycline. Split 1:3 or 1:4 for passing. For HTS: 4 days prior to plating, thaw 3 vials (1.0 x 10;7 cells/ vial) into 4 triple flasks with 120 mL DMEM passage medium/flask. After 3 days, transfer to 3 Hyperflasks (HF) with ~550 mL RPMI induction media (~33 million cells per HF)

2.Day 0: Pass JRFL cells to DoxFree. Wash cells 2x with PBS & add 15 ml Accutase per triple to detach cells. Stop Accutase by adding RPMI induction media. Spin down cells & seed 9-10 x10;6 cells in 120 ml of RPMI induction medium in triple flask.

3.Day 1: Plating of JRFL cells. Wash cells with PBS & add 15 ml Accutase for triple or 50 ml for HF to detach cells (@ 37 degrees C). Stop by adding RPMI induction medium. Spin down the cells at 1200 rpm for 5 min & re-suspend in phenol red-free RPMI induction medium (with Puromycin).

4.Count cells, adjust to 1.25 x10;5 cells/mL & seed 2,500 cells per well in 20 microl of RPMI (no Phenol Red) induction medium in Aurora white, opaque 384 well plates.

5.Day 2: Pinning & adding CEM21 cells Spin down & count CEM21 cells. Adjust to 5.0x10;5 cells/ml in RPMI fusion medium. Dispense 5,000 cells at 10 microl/well in RPMI induction media. Pin transfer compounds to the plate. Positive control is Maraviroc to a final concentration of ~300 nM. Incubate the plate 37 degrees C O/N.

6.Day 3: SteadyGlo & Read. Add 10 microl of room temperature Steady-Glo substrate. Wait 10 minutes & read luminescence with ultrasensitive settings on Perkin-Elmer EnVision with 0.5 second read time per well.


Media Recipes:

DMEM passage medium (1 L):
DMEM 882 mL
Heat Inactivated-FBS 100 mL
Pen-Strep-L-Glu 10 mL
*Doxycycline (1 mg/mL) 2 mL
Puromycin (10 mg/mL) 100 microL
Geniticin (50 mg/mL)4 mL


RPMI induction medium (DoxFree) (1 L):
RPMI (-Phenol red)890 mL
10 % Tet approved HI-FBS100 mL
Pen-Strep-L-Glu 10 mL
Puromycin (10 mg/mL)100 microL


Notes
.Doxycycline is light sensitive so minimize light exposure
.All cells have to be in exponential growth phase. JRFL & Hela-F-Luc cells are usually split 1:3 every 2-3 days, CEM 21 are split 1:10 every 3 days. Cells should not be allowed to overgrow in the flasks.
.CEM21 cells take 1-2 weeks after thawing to grow at an optimal rate
.Cells must be spun down after dissociation to remove StemPro Accutase from the medium.
.Cells can be allowed to fuse 16 to 24 hours.
-Effector (JRFL) cells should be propagated for no more than 30 passages or 3 months whichever is sooner.

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

EXPECTED OUTCOME: Active compounds result in decreasing 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.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.


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 100.

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

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

Grant Number: 1 R03 DA034601-01