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BioAssay: AID 504803

Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of the HTRA serine peptidase 1 (HTRA1)

Name: Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of the HTRA serine peptidase 1 (HTRA1). ..more
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 504803
Data Source: The Scripps Research Institute Molecular Screening Center (HTRA1_INH_FP_1536_1X%INH PRUN)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-06-08

Data Table ( Complete ):           Active    All
BioActive Compounds: 1710
Depositor Specified Assays
504817Summary of the probe development efforts to identify inhibitors of the HTRA serine peptidase 1 (HTRA1)summary
540248Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of the HTRA serine peptidase 1 (HTRA1)other
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Antonis S. Zervos, University of Central Florida
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: DK055734
Grant Proposal PI: Antonis S. Zervos, University of Central Florida
Grant Proposal PI: Antonis S. Zervos, University of Central Florida
External Assay ID: HTRA1_INH_FP_1536_1X%INH PRUN

Name: Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of the HTRA serine peptidase 1 (HTRA1).


High temperature-responsive antigen (HTRA) proteins comprise a family of four serine proteases that play critical roles in a diversity of biological processes, including protein quality control, especially under conditions of cellular stress (1, 2). One member, HTRA1, has been shown to play a role in osteoarthritis (3-6), Alzheimer's disease (7), and age-related macular degeneration (8-9). Overexpression of the HTRA1 gene or over-activity of the enzyme is always associated with these conditions. HTRA1 has also been shown to play a role in chemotherapy-induced cytotoxicity in ovarian (10) and gastric cancers (11) and in mesothelioma (12). HtrA1, an extracellular secreted protein, has a highly conserved chymotrypsin-like serine protease domain and a PDZ protein-protein interaction motif at its carboxyl terminus (1). The amino terminal region contains a predicted signal peptide, an IGF binding domain, as well as protease inhibitor domains (1). The normal function of HtrA1 is unclear, but is expected to involve cleavage of extracellular matrix proteins. A number of these target substrates have been identified and include C-polypeptides of fibril-forming types I, II and III procollagen (13), fibronectin (5) and proteoclycans. HTRA1 has also been shown to regulate TGF-beta signaling (14). As a result, a specific inhibitor for this serine protease would be invaluable in basic studies to elucidate the normal function of HTRA1 and its deregulation in the development and progression of human disease, and could potentially lead to the development of new and effective clinical interventions.


1. Clausen, T., Southan, C., and Ehrmann, M. (2002) The HtrA family of proteases: implications for protein composition and cell fate. Mol Cell 10, 443-455.
2. Ehrmann, M., and Clausen, T. (2004) Proteolysis as a regulatory mechanism. Annu Rev Genet 38, 709-724.
3. Hu, S. I., Carozza, M., Klein, M., Nantermet, P., Luk, D., and Crowl, R. M. (1998) Human HtrA, an evolutionarily conserved serine protease identified as a differentially expressed gene product in osteoarthritic cartilage. J. Biol. Chem. 273, 34406-34412.
4. Grau, S., Richards, P. J., Kerr, B., Hughes, C., Caterson, B., Williams, A. S., Junker, U., Jones, S. A., Clausen, T., and Ehrmann, M. (2006) The role of human HtrA1 in arthritic disease. J Biol Chem 281, 6124-6129.
5. Polur, I., Lee, P. L., Servais, J. M., Xu, L., and Li, Y. (2010) Role of HTRA1, a serine protease, in the progression of articular cartilage degeneration. Histol Histopathol 25, 599-608.
6. Tsuchiya, A., Yano, M., Tocharus, J., Kojima, H., Fukumoto, M., Kawaichi, M., and Oka, C. (2005) Expression of mouse HtrA1 serine protease in normal bone and cartilage and its upregulation in joint cartilage damaged by experimental arthritis. Bone 37, 323-336.
7. Grau, S., Baldi, A., Bussani, R., Tian, X., Stefanescu, R., Przybylski, M., Richards, P., Jones, S. A., Shridhar, V., Clausen, T., and Ehrmann, M. (2005) Implications of the serine protease HtrA1 in amyloid precursor protein processing. Proc Natl Acad Sci U S A 102, 6021-6026.
8. Marx, J. (2006) Gene offers insight into macular degeneration. Science 314, 405.
9. Yang, Z., Camp, N. J., Sun, H., Tong, Z., Gibbs, D., Cameron, D. J., Chen, H., et al. (2006) A variant of the HTRA1 gene increases susceptibility to age-related macular degeneration. Science 314, 992-993.
10. Chien, J., Staub, J., Hu, S. I., Erickson-Johnson, M. R., Couch, F. J., Smith, D. I., Crowl, R. M., Kaufmann, S. H., and Shridhar, V. (2004) A candidate tumor suppressor HtrA1 is downregulated in ovarian cancer. Oncogene 23, 1636-1644.
11. Bowden, M. A., Di Nezza-Cossens, L. A., Jobling, T., Salamonsen, L. A., and Nie, G. (2006) Serine proteases HTRA1 and HTRA3 are down-regulated with increasing grades of human endometrial cancer. Gynecol Oncol 103, 253-260.
12. Baldi, A., Mottolese, M., Vincenzi, B., Campioni, M., Mellone, P., Di Marino, M., di Crescenzo, V. G., et al. (2008) The serine protease HtrA1 is a novel prognostic factor for human mesothelioma. Pharmacogenomics 9, 1069-1077.
13. Murwantoko, Yano, M., Ueta, Y., Murasaki, A., Kanda, H., Oka, C., and Kawaichi, M. (2004) Binding of proteins to the PDZ domain regulates proteolytic activity of HtrA1 serine protease. Biochem J 381, 895-904.
14. Launay, S., Maubert, E., Lebeurrier, N., Tennstaedt, A., Campioni, M., Docagne, F., Gabriel, C., Dauphinot, L., Potier, M. C., Ehrmann, M., Baldi, A., and Vivien, D. (2008) HtrA1-dependent proteolysis of TGF-beta controls both neuronal maturation and developmental survival. Cell Death Differ 15, 1408-1416.


HtrA1, HTRA1, High temperature-responsive antigen, serine protease, serine peptidase, protease, peptidase, hydrolase, extracellular, ARMD7, L56, ORF480, PRSS11, inhibitor, inh, inhibit, osteoarthritis, Alzheimer's, age-related macular degeneration, MD, eye, cancer, osteoarthritis, PDZ, FP-TAM, FP-Rh, FP, fluorophosphonate, fluorescence fluor, fluorescent, polarization, activity-based probe, activity-based protein probe, ABPP, biochemical, primary, singlicate, HTS, high throughput screen, 1536, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Assay Overview:

The purpose of this biochemical assay is to identify compounds that act as inhibitors of the serine protease HTRA1. In this assay, recombinant HTRA1 protein is incubated with test compounds and an Rh-conjugated activity-based probe. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value. The assay is performed by incubating test compounds with HTRA1 for a defined period, followed by addition of the FP-rhodamine probe and measurement of fluorescence polarization at a specific time point. As designed, test compounds that act as HTRA1 inhibitors will prevent HTRA1-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds are tested in singlicate at a final nominal concentration of 5.96 uM.

Protocol Summary:

Prior to the start of the assay, 4 uL of Assay Buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.01% Pluronic F-127, and 1 mM DTT) containing 1.06 uM of HTRA1 protein was dispensed into 1536 microtiter plates. Next, 30 nL of test compound in DMSO or DMSO alone (0.59% final concentration) were added to the appropriate wells and incubated for 30 minutes at 25 C.

The assay was started by dispensing 1.0 uL of 250 nM FP-TAM probe in Assay Buffer to all wells. Plates were centrifuged and after 30 minutes of incubation at 25 C, fluorescence polarization was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525 nm, emission = 598 nm). Fluorescence polarization was read for 15 seconds for each polarization plane (parallel and perpendicular).

Prior to further calculations, the following formula was used to calculate fluorescence polarization (FP):

FP = ( Raw1 - Raw2 ) / ( Raw1 + Raw2 )


Raw1 is defined as the S channel.
Raw2 is defined as the P channel.

The percent inhibition for each compound was calculated as follows:

%_Inhibition = 100 * ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control )


Low_Control is defined as wells containing HTRA1 and DMSO.
Test_Compound is defined as wells containing HTRA1 in the presence of test compound.
High_Control is defined as wells containing no HTRA1 protein.

A mathematical algorithm was used to determine nominally active compounds. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. 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.

PubChem Activity Outcome and Score:

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-10, and for inactive compounds 10-0.

List of Reagents:

Recombinant HTRA1 protein (supplied by Assay Provider)
FP-TAM probe (supplied by Assay Provider)
Tris HCl (Sigma, part T3038)
NaCl (Sigma, part S6546)
Pluronic acid (Invitrogen, part P6866)
DTT (Invitrogen, part 15508-013)
1536-well plates (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. 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, and 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. The compound SR-01000800927-1 (SID 49725089) was not screened because it produces a known fluorescence artifact.
Result Definitions
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition at 5.96 uM (5.96μM**)Normalized percent inhibition of the primary screen at a compound concentration of 5.96 micromolar.Float%

** Test Concentration.
Additional Information
Grant Number: DK055734

Data Table (Concise)