Fluorescence Intensity-based biochemical primary high throughput screening assay to identify activators of kallikrein-7 (K7) zymogen
Name: Fluorescence intensity-based biochemical primary high throughput screening assay to identify activators of kallikrein-7 (K7) zymogen. ..more
BioActive Compounds: 3325
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: University of California, Santa Barbara
Assay Provider: Patrick Daugherty, University of California, Santa Barbara
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA034600-01
Grant Proposal PI: Patrick Daugherty, University of California, Santa Barbara
External Assay ID: K7ZYM_ACT_FLINT_1536_1X%INH
Name: Fluorescence intensity-based biochemical primary high throughput screening assay to identify activators of kallikrein-7 (K7) zymogen.
The objective of this project is to identify small molecule probes that selectively activate the chymotrypsin-like serine protease kallikrein-7 (K7) zymogen. Although zymogens are widely processed by proteolytic removal of a propeptide, this mechanism of activation is difficult to control in biological systems. Consequently, the biological functions of many proteases remain obscure. The chymotrypsin-like serine protease K7 is secreted as an inactive proenzyme/zymogen and highly expressed in the skin, esophagus, and kidney and at lower levels in many organs including the brain (1-2). Kallikreins are thought to be part of a proteolytic program that mediates cleavage of cell adhesion proteins in skin, including cadherins, to mediate skin desquamation (3). K7 has attracted attention since it has been shown to play critical roles in skin diseases and tumor progression. Elevated K7 activity has been associated with psoriasis, atopic dermatitis and a rare skin disease known as Netherton syndrome (4-5). In cancer, elevated K7 expression is strongly associated with increased tumor cell migration, invasion and metastasis (6-8). Elevated K7 has been observed in multiple cancer types (6-7, 9). Ovarian cancer patients with high K7 expression have a significantly reduced overall mean survival time (9). K7 stimulates migration and invasion of cultured prostate carcinoma (7) and glioblastoma cells (8). In addition to demonstrated roles in cancer and skin disease, deficient K7 activity may be associated with the progression of Alzheimer's disease (10-11). In a global search of gene expression changes as a function of late-onset Alzheimer's disease (AD) stage (Braak stage), kallikrein-7 was the only known peptidase transcript to decrease as stage progressed (10). Probe compounds that directly activate K7 without a requirement for proteolytic processing would serve as powerful tools to investigate the potential roles of K7 in amyloid beta and/or amyloid precursor protein processing. The chemical probes arising from this work could shed light on the functions of chymotrypsin-like serine proteases in health and disease, and provide insights that enable development of new therapeutics for cancer, skin diseases, and Alzheimer's disease.
1. Kishi, T., Soosaipillai, A., Grass, L., Little, S. P., Johnstone, E. M., and Diamandis, E. P. (2004) Development of an immunofluorometric assay and quantification of human kallikrein 7 in tissue extracts and biological fluids, Clin Chem 50, 709-716.
2. Shimizu-Okabe, C., Yousef, G. M., Diamandis, E. P., Yoshida, S., Shiosaka, S., and Fahnestock, M. (2001) Expression of the kallikrein gene family in normal and Alzheimer's disease brain, Neuroreport 12, 2747-2751.
3. Borgono, C. A., Michael, I. P., Komatsu, N., Jayakumar, A., Kapadia, R., Clayman, G. L., Sotiropoulou, G., and Diamandis, E. P. (2007) A potential role for multiple tissue kallikrein serine proteases in epidermal desquamation, J Biol Chem 282, 3640-3652.
4. Sales, K. U., Masedunskas, A., Bey, A. L., Rasmussen, A. L., Weigert, R., List, K., Szabo, R., Overbeek, P. A., and Bugge, T. H. (2010) Matriptase initiates activation of epidermal pro-kallikrein and disease onset in a mouse model of Netherton syndrome, Nat Genet 42, 676-683.
5. Stenshamn, K., Bongcam-Rudloff, E., Salmon Hillbertz, N., Nodtvedt, A., Bergvall, K., Hedhammar, A., and Andersson, G. (2006) Evaluation of kallikrein 7 as a disease-causing gene for canine atopic dermatitis using microsatellite-based association mapping, Anim Genet 37, 601-603.
6. Johnson, S. K., Ramani, V. C., Hennings, L., and Haun, R. S. (2007) Kallikrein 7 enhances pancreatic cancer cell invasion by shedding E-cadherin, Cancer 109, 1811-1820.
7. Mo, L., Zhang, J., Shi, J., Xuan, Q., Yang, X., Qin, M., Lee, C., Klocker, H., Li, Q. Q., and Mo, Z. (2010) Human kallikrein 7 induces epithelial-mesenchymal transition-like changes in prostate carcinoma cells: a role in prostate cancer invasion and progression, Anticancer Res 30, 3413-3420.
8. Prezas, P., Scorilas, A., Yfanti, C., Viktorov, P., Agnanti, N., Diamandis, E., and Talieri, M. (2006) The role of human tissue kallikreins 7 and 8 in intracranial malignancies, Biol Chem 387, 1607-1612.
9. Psyrri, A., Kountourakis, P., Scorilas, A., Markakis, S., Camp, R., Kowalski, D., Diamandis, E. P., and Dimopoulos, M. A. (2008) Human tissue kallikrein 7, a novel biomarker for advanced ovarian carcinoma using a novel in situ quantitative method of protein expression, Ann Oncol 19, 1271-1277.
10. Bossers, K., Wirz, K. T., Meerhoff, G. F., Essing, A. H., van Dongen, J. W., Houba, P., Kruse, C. G., Verhaagen, J., and Swaab, D. F. (2010) Concerted changes in transcripts in the prefrontal cortex precede neuropathology in Alzheimer's disease, Brain 133, 3699-3723.
11. Diamandis, E. P., Scorilas, A., Kishi, T., Blennow, K., Luo, L. Y., Soosaipillai, A., Rademaker, A. W., and Sjogren, M. (2004) Altered kallikrein 7 and 10 concentrations in cerebrospinal fluid of patients with Alzheimer's disease and frontotemporal dementia, Clin Biochem 37, 230-237.
PRUN, primary, K7, K7 zymogen, Kallikrein-7 KLK7, Serine protease-6, chymotrypsin-like, serine protease, cancer, skin diseases, Alzheimer's disease, inhibitor, inhibition, primary, primary screen, HTS, high throughput screen, 1536, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to identify compounds that act as activators of K7 zymogen. In this assay, compounds are incubated with K7 zymogen and an EDANS-DABCYL substrate probe that exhibits an increase in emission signal of the EDANS fluorophore upon complete proteolysis. As designed, test compounds that act as activators of K7 zymogen will hydrolyze the EDANS-DABCYL substrate, thereby decreasing the proportion of unhydrolyzed substrate probe in the well, and increasing the fluorescence emission. Compounds will be tested in singlicate at a final nominal concentration of 7 uM.
Prior the start of the assay 3 uL of Master Mix containing 5 nM K7ZYM (proKLK7) and 1 uM EDANS-DABCYL peptide substrate in Assay Buffer (50 mMTris,10 mM NaCl at pH 8.5) were added to all wells on columns 3-48. Three uL of the control mix containing 5 nM Active K7 (K7ZYM was activated following Vendor's protocol) and 1uM EDANS-DABCYL peptide substrate in Assay Buffer (previously incubated at 37 C for 2hrs) were added to all wells in columns 1-3. The assay started by the addition of 21 nl of compounds . Plates were centrifuged and fluorescence intensity was recorded (T0). Plates were incubated at room temperature for 4 hrs at which time fluorescence intensity (T4). Fluorescence intensity at T0 and T4 was read on a PerkinElmer EnVision using excitation wavelength 355 nm +/- 40nm, emission wavelength 520 nm +/- 8 nm and mirror wavelength cutoff 400 nm.
The % inhibition for each well at T0 and T4 was then calculated as follows:
%_Inhibition = ( RFU_Test_Compound - MedianRFU_Low_Control ) / ( MedianRFU_High_Control - MedianRFU_Low_Control ) * 100
Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing active K7.
Low_Control is defined as wells containing DMSO.
The final % inhibition was calculated as follows:
Final_%_Inhibition = %_Inhibition_Test_Compound_T4 - %_Inhibition_Test_Compound_T0
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the Primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested , and (2) three times their standard deviation (taking into account only compounds which % inhibition was greater than the average minus three times the standard deviation of the low control % inhibition and smaller than the sum of average and three times the standard deviations of the high control % inhibition). The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The activity score range for active compounds is 100-1, for inactive 1-0.
List of Reagents:
proKLK7 (R&D Systems, part # 2426-SE)
EDANS-DABCYL peptide substrate (Supplied by Assay Provider)
Tris (Fisher, part # BP125-5)
CaCl2 (Fisher, part # BP510-500)
NaCl (Fisher, part # S641-500)
Brij-35 (Sigma-Aldrich, part # P1254)
Thermolysin (R&D Systems, part # 3097-ZN)
EDTA (Fisher, part # S25687)
1536 well plate (Corning, part # 7261)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
** Test Concentration.
Data Table (Concise)