Single concentration confirmation of HCS identification of small molecules that inhibit hepatic lipid droplet formation
Medical researchers world-wide now use the term "epidemic" or "pandemic" to describe the alarming incidence of obesity in modern society, which is estimated to occur in 30% of the general US population. Perhaps, most alarming is the high incidence of obesity in children and adolescents, which indicates that medical problems associated with obesity (e.g., diabetes, metabolic syndrome, and heart more ..
BioActive Compounds: 569
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute(SBIMR, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH083261-01A1
Assay Provider: Dr. Patrick M. McDonough, Vala Sciences Inc.
Medical researchers world-wide now use the term "epidemic" or "pandemic" to describe the alarming incidence of obesity in modern society, which is estimated to occur in 30% of the general US population. Perhaps, most alarming is the high incidence of obesity in children and adolescents, which indicates that medical problems associated with obesity (e.g., diabetes, metabolic syndrome, and heart disease) will increase in the foreseeable future, as obesity often increases with age. The dominant cellular basis for obesity is increased accumulation of triglycerides in lipid droplets within the cell. The two major cell types in which lipid droplet formation leads to pathological problems are adipocytes and hepatocytes.
The overall goal of this project is to identify chemical probes for inhibiting lipid droplet formation in hepatocytes. Identification of such compounds will better our understanding of the biochemical/molecular pathways that lead to the development of fatty liver disease, and may identify lead compounds for consideration for therapeutic development.
The goal of this assay is to confirm hits from "High Throughput Imaging Assay for Hepatic Lipid Droplet Formation" (AID 1656).
Wu JC, Merlino G, Fausto N. Establishment and characterization of differentiated, nontransformed hepatocyte cell lines derived from mice transgenic for transforming growth factor alpha. Proc Natl Acad Sci U S A. Jan 18 1994;91(2):674-678.
de Alwis NM, Day CP. Non-alcoholic fatty liver disease: the mist gradually clears. J Hepatol. 2008;48 Suppl 1:S104-112.
Guo Y, Walther TC, Rao M, et al. Functional genomic screen reveals genes involved in lipid-droplet formation and utilization. Nature. May 29 2008;453(7195):657-661.
Beller M, Sztalryd C, Southall N, et al. COPI complex is a regulator of lipid homeostasis. PLoS Biol. Nov 25 2008;6(11):e292.
Szymanski KM, Binns D, Bartz R, et al. The lipodystrophy protein seipin is found at endoplasmic reticulum lipid droplet junctions and is important for droplet morphology. Proc Natl Acad Sci U S A. Dec 26 2007;104(52):20890-20895.
1) 384-well plates, black with clear bottom (Greiner# 781091).
2) AML12 cells (mouse hepatocytes) were obtained from the ATCC.
3) Culture Media: phenol-red free DMEM with L-glutamine, Pen-strep, and 10% Fetal Bovine Serum.
4) Oleic Acid Working Solution: water soluble Oleic Acid (Sigma, O1257, 5mM stock in PBS) diluted to 1mM in PBS.
5) Positive Control Working Solution: Triacsin-C (Sigma, T4540 1mg/ml stock in DMSO) diluted to 50uM in PBS. Additional DMSO is added to achieve a DMSO concentration of 2%.
6) Negative Control Working Solution: 2% DMSO in water.
7) Fixative Working Solution: 6% Paraformaldehyde (PFA) in PBS.
8) 1.5% DMSO: to balance DMSO concentration and assay well volume.
8) Permeabilization Buffer Working Solution: 0.1% BSA (Sigma, A7888) and 0.01% Saponin (Sigma, 84510) in PBS.
9) Lipid Stain Working Solution: BODIPY 493/503 (Invitrogen, D3922) diluted to 5 ug/mL in PBS.
10) Nuclear Stain Working Solution: DAPI (Invitrogen, D1306) diluted to 150 ng/ml in DAPI buffer (10mM TRIS, 10mM EDTA, 100mM NaCl, pH 7.4).
1) 50 ul of cell suspension (140,000 cells/ml in culture medium) was dispensed in each well of the assay plates using a Wellmate (Matrix) bulk dispenser.
2) Plates were incubated plates overnight or approx. 20 hours at 37 degrees C and 5% CO2.
3) 32.5nl of 10mM compound was added to columns 3 through 24 of the assay plates using the Echo550 Acoustic Dispenser for a final concentration of 5.2uM. This adds 0.05% DMSO to the wells.
4) 6.25ul of negative control (2% DMSO) working solution was manually added to column 2 for a final assay concentration of 0.2% DMSO.
5) 6.25 ul of the positive control (50 uM Triacsin-C) working solution was added to column 1 of each plate manually for a final assay concentration of 5 uM Triacsin-C and 0.2% DMSO.
6) 6.25 ul of oleic acid working solution was added to all wells using the Biomek FX (Beckman Coulter) with 384-head dispenser for a final assay oleic acid concentration of 100 uM.
7) 6.25 ul of 1.5% DMSO was added to columns 3-24 using the Biomek FX with 384-head dispenser for a final assay DMSO concentration of 0.2%.
7) Plates were incubated overnight at 37 degree C and 5% CO2.
8) Media was aspirated leaving 20 ul liquid in each well using a Titertek plate washer.
9) 40 ul of fixative working solution was added to each well using a Wellmate bulk dispenser for a final concentration of 4% PFA and plates were allowed to fix for 40 minutes at room temperature.
10) Fixative was aspirated and plates were washed twice with 50ul PBS leaving 20 ul liquid in each well using a Titertek plate washer.
11) 40 ul of permeabilization buffer working solution was added to each well using the Wellmate bulk dispenser and plates were permeabilized for 15 minutes at room temperature.
12) Permeabilization buffer was aspirated and plates were washed twice with 50 ul PBS leaving 20 ul liquid in each well using a Titertek plate washer.
13) 40 ul of lipid stain working solution was added to each well using the Wellmate bulk dispenser for a final concentration of 3.3 ug/ml and plates were incubated for 1 hour at room temperature.
14) Stain solution was aspirated and plates were washed twice with 50ul PBS leaving 20 ul liquid in each well using a Titertek plate washer.
15) 40 ul of DAPI working solution was added using a Wellmate bulk dispenser for a final DAPI concentration of 100 ng/ml and plates were sealed.
HCS System Settings and Image Analysis Protocol:
1) Image acquisition was performed on an Opera QEHS (Perkin Elmer) with 45 plate capacity loader/stacker and the following settings:
- 20x 0.45 NA air objective
- Acquisition camera set to 2-by-2 binning for an image size of 688 by 512 pixels
- 2 channels acquired sequentially: Exp1Cam1 = Bodipy 493/503 (lipid droplets) using 488 nm laser excitation and 540/70 emission filters, Exp2Cam4 = DAPI (nuclei) using 365 nm Xenon lamp excitation and 450/50 emission filters
- 4 fields per well
2) Image analysis was performed using Acapella (Perkin Elmer). The following Acapella software settings were used:
- Nuclear Detection Algorithm G
- Threshold Adjustment: 1.5
- Nuclear Splitting Adjustment: 5
- Minimum Nuclear Area: 70
- Minimum Nuclear Contrast: 0.1
- Cytoplasm Threshold Adjustment: 0.05
- Cytoplasm Individual Threshold Adjustment: 0.1
LIPID DROPLET DETECTION
- Minimum Lipid Area in Pixels 8
- Intensity Threshold Value 400
3) Metrics calculated from
NUCLEI IMAGES: Cell Count ("Count"), Nuclei Area ("Area_Nm"), Integrated Intensity of the Nuclei ("TiiNINm"), Average Intensity of the Nuclei ("ApiNiNm")
BODIPY IMAGES: Lipid Area ("Area_Lm"), Integrated Intensity of Lipid ("TiiLiLm")
4) Actives were determined using CBIS software (ChemInnovations) by calculating the %Inhibition of the "TiiLiLm" metric.
Compounds with a %Inhibition >= 50% at 5.2 uM concentration and greater than 300 cells in all four replicates are defined as actives in this assay. Wells with cell counts <300 in the 4 acquired images were flagged cytotoxic/low cell count. All flagged wells were excluded from hit selection and were assigned an outcome of "inconclusive".
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 and single-concentration confirmation screening data.
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
d. If outcome of the confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30.
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
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 and is not applicable in this assay
** Test Concentration.
Data Table (Concise)