Luminescent HTS for small molecule inhibitors of MT1-MMP transcription
The sustained presence of matrix metalloproteinases (MMPs) in a tumor environment is a characteristic of many cancer types. The expression of the MT1-MMP mRNA and the MT1-MMP protein closely correlates with increased tumor volume, tumor invasiveness, and the incidence of local and distant metastases. Tumorigenic MT1-MMP is effective in both its active and inactive states. MT1-MMP protects more ..
BioActive Compounds: 537
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Screening Centers Network (MLSCN)
Grant Number: XO1 MH78949-01
Assay Provider: Dr. Alex Strongin, Sanford-Burnham Medical Research Institute
The sustained presence of matrix metalloproteinases (MMPs) in a tumor environment is a characteristic of many cancer types. The expression of the MT1-MMP mRNA and the MT1-MMP protein closely correlates with increased tumor volume, tumor invasiveness, and the incidence of local and distant metastases. Tumorigenic MT1-MMP is effective in both its active and inactive states. MT1-MMP protects malignant cells against the host immune surveillance thus making tumor cells resistant to the anti-tumor immunity mechanisms. Proteolytically active MT1-MMP is trafficked to centrosomes. Through the proteolysis of centrosomal proteins MT1-MMP promotes mitotic spindle aberrations and causes aneuploidy.
A multiplexed cellular assay was developed to determine the effects of small molecule compounds on MT1-MMP transcription, as well as their effect on cell viability/proliferation on HT1080 cells that have been stably transfected with the luciferase gene under the control of the MT1-MMP promoter. The assay was performed in a 384 well format. By having two readouts, it is possible to eliminate from the hit list cytotoxic compounds that would also show a decrease in reporter gene level.
Cytotoxicity/Cell viability is measured using Alamar Blue (resazurin) that detects respiratory activity of live proliferating cells. The generation of the reduced product, resorufin, was detected in fluorescence mode. The data for this portion of the screen are located in AID 620, Fluorescent HTS Cytotoxicity/Cell viability assay (HT 1080 cells).
MT1-MMP gene transcription is measured using a luminescence readout. Induction of MT1-MMP transcription causes the elevation of luciferase transcription via a reporter gene construct. The amount of luciferase produced is quantified using SteadyLite HTS reagent. The data for this portion of the screen are located in this AID.
Compounds inhibiting luciferase activity would also appear as hits, i.e. false positives, of the reporter gene assay. To identify these compounds a luciferase functional assay was implemented and utilized in dose-response follow-up studies. The data for this portion of the screen are located in AID 773, Counter screen for luciferase-based assays positives.
This multiplexed screening was developed and performed at the Sanford-Burnham Center for Chemical Genomics (SBCCG) as part of the Molecular Library Screening Center Network (MLSCN).
1) HT1080 cells that had been stably transfected with the 7.2 kb 5'-flanking region of the MT1-MMP gene linked to the luciferase gene in the pGL3-Basic vector (Promega), were seeded into Greiner black, clear bottom plates (cat #781091). 7500 cells were seeded/well in 45ul of DMEM w/o phenol red supplemented with 10% FBS, 1mM Sodium Pyruvate, 2mM glutamine and 20ug/ml gentamicin.
2) Cells are left to adhere overnight at 37 Celsius degrees in a humid environment in the presence of 5% CO2.
3) After the overnight incubation, 5ul of diluted compound (final compound concentration is 10uM, final DMSO concentration is 1%) are added to appropriate wells.
4) 5ul of 10% DMSO are added to negative control wells.
5) 5ul of saturated sodium sulfate are added to positive control wells.
6) The cells are incubated with compound overnight at 37 Celsius degrees in a humid environment in the presence of 5% CO2.
7) After incubation 10ul of 16 fold diluted Alamar Blue (Biosource International DAL1100) are added to each well.
8) The plates are incubated for 4 hrs at 37 Celsius degrees in a humid environment in the presence of 5% CO2.
9) After incubation, the plates are read in a Perkin Elmer EnVision, multimode plate reader. The filters used were 531/25nm excitation, 595/60nm emission, and a 555nm dichroic mirror.
10) Data from the Alamar Blue portion of the assay can be found in AID 620, Cell viability/proliferation assay for HT1080 cells.
11) After the Alamar Blue portion of the assay is read, approximately 45ul of the each wells contents are removed using a Titertek MAP-C2 plate processor.
12) 10ul of SteadyLite HTS (Perkin Elmer #6016989) are added to each well.
13) Plates are then read using the ultra sensitive luminescence mode of the Perkin Elmer EnVision, multimode plate reader.
14) Data for the luminescence portion of the assay can be found in this AID.
Data calculation: Raw data are sent to our biological database (CBIS) whereby % activity of the compounds are calculated based on the intraplate controls of sodium sulfate (positive control) and 1% DMSO (negative control). Compounds that do not inhibit the transcription of MT1-MMP will have a low % activity, while inhibitors of transcription will have a high % activity.
For information regarding these cells or the reporter gene construct please contact:
Alex Strongin, Ph.D.
Cell Adhesion & Extracellular Matrix Biology
Sanford-Burnham Medical Research Institute
San Diego, CA 92037
Compounds with greater than 50% activity in reporter gene assay are defined as actives of the primary screening. For dose-response testing, compounds were selected based on the logical conjunction of the following criteria: a) >50% activity in the reporter gene portion of the assay, b) <50% activity in the cytotoxicity assay, c) inactive in AID 411 Luciferase Profiling Assay containing results of MLSCN compound collection partial screening performed by NCGC.
Compounds that show <50% inhibition at 50 uM concentration in the reporter gene portion assay were assigned inactive in the outcome column. Compounds with EC50<50 uM in the reporter gene portion assay remain active independent of the results of cytotoxicity or luciferase assays.
Activity scoring rules were devised to take into consideration specific efficacy of a compound and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Since the goal of the screening was to identify the compounds active in the reporter gene (RG) assay and inactive in the cytotoxicity (CT) or luciferase (Luc) testing, the specific outline of the scoring system utilized for the MT1-MMP transcription is as follows:
1) First tier (0-40 range) is reserved for primary screening data - the score is positively correlated with % repression of MT-MMP transcription and negatively correlated with % toxicity in the cytotoxicity arm of the multiplexed assay (AID 620) demonstrated by a compound at 10 uM concentration:
The score is calculated as (% Inhibition_RG - % Inhibition_CT)*0.4, where % Inhibition_RG and % Inhibition_CT is the efficacy values obtained in reporter gene and cytotoxicity arms of the multiplexed assay, respectively.
a. If the calculated score is less than 0, then the assigned score is 0
b. If the calculated score is more than 40, then the value equal 40 is assigned.
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. For compounds with IC50<50 uM, the score is linearly correlated with a compound's potency using the following equation
Score = 44 + 6*(pEC50_RG - 3)*2.6*(exp(-0.5*nH^2)-exp(-1.5*nH^2)) - 6*(pEC50_CT - 3) - 6*(pEC50_Luc - 3),
where pEC50_RG, pEC50_CT and pEC50_Luc are negative log(10) of the EC50 values for reporter gene, cytotoxicity (AID 620) and luciferase (AID 773) assays, respectively, expressed in mole/L concentration units; nH is Hill coefficient in reporter gene assay. If the calculated score is less than 41, then the value equal 41 is assigned.
This equation results in the Score values above 50 for compounds that are active in reporter gene assay (EC50>100 uM) and are not or much less potent in the cytotoxicity and luciferase assays.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)