Fluorescence polarization-based biochemical primary high throughput screening assay to identify inhibitors that disrupt the binding of a cyclic peptide (Tn6) to the fibrin proteolytic product D-Dimer and fragment E complex [DD(E )]
Name: Fluorescence polarization-based biochemical primary high throughput screening assay to identify inhibitors that disrupt the binding of a cyclic peptide (Tn6) to the fibrin proteolytic product D-Dimer and fragment E complex [DD(E )]. ..more
BioActive Compounds: 760
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: Harvard Medical School/Massachusetts General Hospital
Assay Provider: Peter Caravan, Harvard Medical School/Massachusetts General Hospital
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: R21 NS075627
Grant Proposal PI: Peter Caravan, Harvard Medical School/Massachusetts General Hospital
External Assay ID: FIBRIN-TN6_INH_FP_1536_1X%INH PRUN
Name: Fluorescence polarization-based biochemical primary high throughput screening assay to identify inhibitors that disrupt the binding of a cyclic peptide (Tn6) to the fibrin proteolytic product D-Dimer and fragment E complex [DD(E )].
Fibrin is an insoluble biopolymer that is formed by the proteolytic action of thrombin on the clotting factor fibrinogen. It constitutes the major protein component of blood clots and is generally associated with pathologies such as ischemic stroke, myocardial infarction, pulmonary embolism, and deep vein thrombosis [for review see (1-4)]. Evidence of fibrin has also been found in most solid tumors (5-8). Recent studies indicate a link between the presence of fibrin(ogen) and metastatic potential (9-10). Because this protein represents such an important biomarker of disease, there exists a great need within the medical community to rapidly and reliably image fibrin by non-invasive means. One approach to solving this problem would be to identify small molecule motifs that exhibit fibrin-specific binding properties. When conjugated to an optical, magnetic resonance (MR), or radioactive tracer, such a species would permit the direct detection of fibrin in vivo by observing its accumulation at clot sites (11-12). The development of new fibrin-binding probes could also provide important insights into yet unknown biological roles that fibrin plays in various diseases. The goal of this project is to develop a solution-based assay using binding probe Tn6 that will identify novel fibrin-binding molecules that can be modified for application in the clinical imaging of thrombus (blood clot) in humans. Additionally, this project aims to develop a series of secondary biochemical assays in order to 1) validate those compounds that are indicated as potential hits by the primary screen and 2) eliminate compounds that exhibit non-specific binding behavior, acute toxicity, or other unfavorable properties.
1. Abad Rico, J. I., Llau Pitarch, J. V., and Rocha, E. (2010) Overview of venous thromboembolism, Drugs 70 Suppl 2, 3-10.
2. Labropoulos, N., Spentzouris, G., Gasparis, A. P., and Meissner, M. (2010) Impact and clinical significance of recurrent venous thromboembolism, Br J Surg 97, 989-999.
3. Murray, V., Norrving, B., Sandercock, P. A., Terent, A., Wardlaw, J. M., and Wester, P. (2010) The molecular basis of thrombolysis and its clinical application in stroke, J Intern Med 267, 191-208.
4. Undas, A., and Ariens, R. A. (2011) Fibrin clot structure and function: a role in the pathophysiology of arterial and venous thromboembolic diseases, Arterioscler Thromb Vasc Biol 31, e88-99.
5. Brown, L. F., Dvorak, A. M., and Dvorak, H. F. (1989) Leaky vessels, fibrin deposition, and fibrosis: a sequence of events common to solid tumors and to many other types of disease, Am Rev Respir Dis 140, 1104-1107.
6. Dvorak, H. F. (1986) Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing, N Engl J Med 315, 1650-1659.
7. Dvorak, H. F. (1987) Thrombosis and cancer, Hum Pathol 18, 275-284.
8. Nagy, J. A., Brown, L. F., Senger, D. R., Lanir, N., Van de Water, L., Dvorak, A. M., and Dvorak, H. F. (1989) Pathogenesis of tumor stroma generation: a critical role for leaky blood vessels and fibrin deposition, Biochim Biophys Acta 948, 305-326.
9. Palumbo, J. S. (2008) Mechanisms linking tumor cell-associated procoagulant function to tumor dissemination, Semin Thromb Hemost 34, 154-160.
10. Palumbo, J. S., Talmage, K. E., Massari, J. V., La Jeunesse, C. M., Flick, M. J., Kombrinck, K. W., Jirouskova, M., and Degen, J. L. (2005) Platelets and fibrin(ogen) increase metastatic potential by impeding natural killer cell-mediated elimination of tumor cells, Blood 105, 178-185.
11. Spuentrup, E., Botnar, R. M., Wiethoff, A. J., Ibrahim, T., Kelle, S., Katoh, M., Ozgun, M., Nagel, E., Vymazal, J., Graham, P. B., Gunther, R. W., and Maintz, D. (2008) MR imaging of thrombi using EP-2104R, a fibrin-specific contrast agent: initial results in patients, Eur Radiol 18, 1995-2005.
12. Vymazal, J., Spuentrup, E., Cardenas-Molina, G., Wiethoff, A. J., Hartmann, M. G., Caravan, P., and Parsons, E. C., Jr. (2009) Thrombus imaging with fibrin-specific gadolinium-based MR contrast agent EP-2104R: results of a phase II clinical study of feasibility, Invest Radiol 44, 697-704.
PRUN, primary,Fibrin, DD(E), fibrin proteolytic fragment, fluorescence polarization, FP, FITC, TRITC, Primary, high throughput screen, HTS, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this biochemical assay is to identify compounds that bind to Fibrin proteolytic fragment-DD(E), a protein substrate prepared from human-derived polymerized fibrin clots. In this assay, Fibrin-DD(E) protein is incubated with test compounds for a defined period, followed by addition of the fluorescent probe tetramethylrhodamine Tn6 (TRITC-Tn6). The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value. As designed, test compounds that bind to Fibrin-DD(E) will prevent Fibrin-DD(E)-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds will be tested in singlicate at a final nominal concentration of 8.3 uM.
Displacement assay starts by the addition of Fibrin - DD(E) (final concentration 2uM) in assay buffer (50mM Tris base, 100 mM NaCl, 2 mM CaCl2, and 0.01% Triton X-100, pH 7.8) to all wells, followed by the addition of equal volume of inhibitor peptide EP-2104R (final concentration 10uM) to high control wells and equal volume of assay buffer to sample wells and low control wells. Compounds in DMSO are transferred to plate and incubated for a period of 10 min, at which time TRITC-TN6 probe is added to all wells (final concentration of 0.1uM). After 3 hrs incubation at room temperature fluorescence polarization is measured using Perkin Elmer EnVision with TRITC filter set.
%_Inhibition = ( FP_Test_Compound - MedianFP_Sample_Field ) / ( MedianFP_High_Control - MedianFP_Sample_Field ) * 100
Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing EP-2104R peptide.
Low_Control is defined as wells containing DMSO.
Sample_Field is defined as wells containing Test Compounds
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity 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:
Fibrin-DD(E ) (Supplied by Assay Provider)
TRITC-Tn6 peptide (Supplied by Assay Provider)
EP-2104R peptide (Supplied by Assay Provider)
TRIZMA(R) Base BioXtra >99% (Sigma-Aldrich, part T6791)
Tritontrade mark X-100 BioXtra (Sigma-Aldrich, part T9284)
Sodium Chloride BioXtra >99.5% (Sigma-Adrich, part S7653)
Calcium Chloride Dihydrate BioXtra >99% (Sigma-Aldrich, part C5080)
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)