uHTS identification of inhibitors of MT1-MMP activation in a fluoresence assay
Twenty four individual MMPs are encoded by the human genome (1). MT1-MMP (aka MMP-14) is distinguished from soluble MMPs by a transmembrane domain and a cytoplasmic tail (2). MT1-MMP expression is associated with a variety of pathophysiological conditions and especially so with tumor growth, cancer cell invasion and metastasis (3-6). MT1-MMP accumulates predominantly at the leading and trailing more ..
BioActive Compounds: 222
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute(SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1R03DA033979-01 (Cycle 19)
Assay Provider: Vladislav Golubkov, Ph.D. Sanford Burnham Medical Research Institute
Twenty four individual MMPs are encoded by the human genome (1). MT1-MMP (aka MMP-14) is distinguished from soluble MMPs by a transmembrane domain and a cytoplasmic tail (2). MT1-MMP expression is associated with a variety of pathophysiological conditions and especially so with tumor growth, cancer cell invasion and metastasis (3-6). MT1-MMP accumulates predominantly at the leading and trailing edges in the migrating cells where the proteinase contributes most efficiently to the cleavage of extracellular matrix proteins, adhesion and signaling cell receptors, and the activation of soluble MMPs (3,4). Inhibition of MT1-MMP activity decreases malignant cell migration and invasion in vitro, and tumor growth and metastasis in vivo (7, 8). Because of its importance in cancer and other diseases, MT1-MMP is a promising drug target and its selective inhibitors are urgently needed (9).
1. Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer. 2002;2(3):161-74.
2. Holmbeck K, Bianco P, Yamada S, Birkedal-Hansen H. MT1-MMP: a tethered collagenase. J Cell Physiol. 2004;200(1):11-9.
3. Yoshida S, Takahashi H. Expression of extracellular matrix molecules in brain metastasis. J Surg Oncol. 2009;100(1):65-8.
4. Mimori K, Ueo H, Shirasaka C, Mori M. Clinical significance of MT1-MMP mRNA expression in breast cancer. Oncol Rep. 2001;8(2):401-3.
5. Rowe RG, Weiss SJ. Breaching the basement membrane: who, when and how? Trends Cell Biol. 2008;18(11):560-74.
6. Gilles C, Polette M, Seiki M, Birembaut P, Thompson EW. Implication of collagen type I-induced membrane-type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in the metastatic progression of breast carcinoma. Lab Invest. 1997;76(5):651-60.
7. Coussens LM, Fingleton B, Matrisian LM. Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science. 2002;295(5564):2387-92.
8. Devy L, Huang L, Naa L, Yanamandra N, Pieters H, Frans N, et al. Selective inhibition of matrix metalloproteinase-14 blocks tumor growth, invasion, and angiogenesis. Cancer Res. 2009;69(4):1517-26.
9. Zucker S, Cao J. Selective matrix metalloproteinase (MMP) inhibitors in cancer therapy: ready for prime time? Cancer Biol Ther. 2009;8(24):2371-3.
Assay Materials: HT1080 RFP-PRO-GFP cells (PI lab)
HT1080 RFP-L50D-GFP cells (PI Lab)
DMEM (Mediatech 10-013-CM)
DMEM (no phenol red) (Mediatech 17-205-CV)
Fetal Bovine Serum (Hyclone)
Penicillin Streptomycin solution (Invitrogen)
DPBS without calcium and magnesium (1X) (Hyclone)
Corning culture flasks
ATPLite (Perkin Elmer 6016739)
Corning CellBIND 1536-well black/clear (Corning 7320)
1-Prepare cell suspension as described in section F. Cell Culture.
2-Dispense 5 uL/well of assay media to column 1.
3-Dispense 5 uL/well of cells at 6X10^5 cells/mL of RFP-L50D-GFP to the column 2.
4-Dispense 5 uL/well of cells at 6X10^5 cells/mL of RFP-PRO-GFP to the columns 3-48.
5-Spin down plates on Eppendorf centrifuge 5810 at 500 rpm for 1 minute.
6-Put Kalypsys metal lids on plates, incubate plates at 37 degrees C with 5% CO2 for overnight.
7-Transfer test compounds to columns 5-48 and DMSO to columns 1-4 using the Labcyte ECHO 555. Transfer volume of test compound and DMSO is 50 nL, making 20 uM compound concentration at 1% DMSO final.
8-Spin down plates on Vspin at 1000 rpm for 1 minute.
9-Put Kalypsys metal lids on plates, incubate plates at 37 degrees C with 5% CO2 for overnight.
10-Read GFP and RFP fluorescence on Envison.
11-Add 3 ul of ATPLite on BioRAPTR FRD.
12-Centrifuge at 1000rpm for 2 minutes (no lid).
13-Cover and incubate for 10 minutes at room temp. Read luminescence on Viewlux.
Compounds with a ratio of GFP: Corrected/RFP: Corrected >= 30 % and ATPLite: Corrected >= -50 % and RFPex535: Corrected" between -50 and 50% are considered active in this assay
The experimental values were normalized by the difference between values from neutral and stimulator control wells in each plate. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing tip issues etc. Further information about data correction is available at http://www.genedata.com/products/screener.html.
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.
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 % activity in the assay:
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
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration 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)