Thrombin 1536 HTS Dose Response Confirmation
Prothrombin, a 72 kDa blood zymogen (plasma concentration = 2 uM, (1)) is converted to thrombin by factor Xa (FXa) in the prothrombinase complex on platelets by cleavage of R271 and R320. Thrombin then further processes itself by cleavage at R155 and R284 in order to remove prothrombin fragment 1 and fragment 2, which contain the kringle 1 and 2 domains, leaving fully active alpha-thrombin (2). more ..
BioActive Compounds: 223
Molecular Library Screening Center Network (MLSCN)
Penn Center for Molecular Discovery (PCMD)
Assay Provider: Scott L. Diamond, University of Pennsylvania
MLSCN Grant: X01-MH076406-01
Prothrombin, a 72 kDa blood zymogen (plasma concentration = 2 uM, (1)) is converted to thrombin by factor Xa (FXa) in the prothrombinase complex on platelets by cleavage of R271 and R320. Thrombin then further processes itself by cleavage at R155 and R284 in order to remove prothrombin fragment 1 and fragment 2, which contain the kringle 1 and 2 domains, leaving fully active alpha-thrombin (2). alpha-thrombin goes to activate other coagulation factors that are involved in its own production, including the cofactors factor V and factor VIII, as well as activating platelets through the protease activated receptor-1 (PAR-1) (3).
With respect to formation of the secondary blood clot, alpha-thrombin catalyzes activation of fibrinogen to fibrin, cleaving peptides 14-16 amino acids in length, called fibrinopeptides, from the Aalpha and Bbeta subunits of fibrinogen (4). Fibrinogen (plasma concentration = 10.6 uM (5)) is a dimeric protein consisting of three pairs of disulfide-bonded polypeptide chains, referred to as Aalpha, Bbeta, and gamma, which form a complex with a total MW of approximately 340 kDa (6).
Specifically, the sites uncovered by the release of the fibrinopeptides, which are located in central regions of their respective chains, allow for noncovalent multimer formation of fibrin polymers by interactions with complementary central and outer regions of other fibrin monomers, forming an extensive fibrin meshwork. In this way alpha-thrombin produces a fibrin clot that is stabilized by the crosslinking activity of FXIIIa, which is also activated by alpha-thrombin from its inactive zymogen, factor XIII (7). The fibrin monomers produced by alpha-thrombin form a noncovalent meshwork with other fibrin molecules to produce a fibrin clot that stabilizes the primary platelet plug already in place at the site of injury, and reduces the volume of the plug in order to arrest blood loss (8).
HTS was performed using 217,350 compounds of the MLSCN library individually plated into 10ul 1536 compound plates at a concentration of 2.5 mM each, which were diluted 500-fold into 5 ul 1536 well assay plates (final concentration 5 uM each compound). This assay was performed as a counterscreen to compare with a separate HTS campaign to isolate inhibitors of the strong protein-protein interactions among fibrin monomers responsible for fibrin gel formation. Since any inhibitors of the proteolytic activity of alpha-thrombin would inhibit an assay to detect fibrin formation, this counterscreen will identify direct alpha-thrombin inhibitors. The assay used to test for percent inhibition was a fluorescence assay utilizing hydrolysis of Boc-Val-Pro-Arg-AMC, as first described by Morita, et al. (9).
1. F. C. McDuffie et al., Thromb Res 16, 759 (1979).
2. T. Morita, C. M. Jackson, J Biol Chem 261, 4015 (Mar 25, 1986).
3. E. Di Cera, Q. D. Dang, Y. M. Ayala, Cell Mol Life Sci 53, 701 (Sep, 1997).
4. K. Bailey, F. R. Bettelheim, L. Lorand, W. R. Middlebrook, Nature 167, 233 (Feb 10, 1951).
5. Y. Takeda, J Clin Invest 45, 103 (Jan, 1966).
6. H. A. Scheraga, Biophys Chem 112, 117 (Dec 20, 2004).
7. J. E. Folk, S. I. Chung, Adv Enzymol Relat Areas Mol Biol 38, 109 (1973).
8. L. L. Shen, J. Hermans, J. McDonagh, R. P. McDonagh, M. Carr, Thromb Res 6, 255 (Mar, 1975).
9. T. Morita, H. Kato, S. Iwanaga, K. Takada, T. Kimura, J Biochem 82, 1495 (Nov, 1977).
Human alpha-thrombin was purchased from Haematologic Technologies Inc. (Cat no. HCT-0020). Substrate Boc-Val-Pro-Arg-AMC was from Bachem (Cat no. I-1120.0050). Assay buffer consisted of 50 mM Tris, pH 7.5, 150mM NaCl, 0.05% Tween 20. 1536-well black plates were from Corning (Item no. 3728).
alpha-thrombin (5.5 ng/mL) was incubated with Boc-Val-Pro-Arg-AMC substrate (15 uM) in 5 uL of assay buffer (see above) for 30 min at room temperature. Hits from 1536 HTS were confirmed on single compounds by IC50 determination as described below.
1.Serial dilute single compounds at 50x concentration in DMSO (16 two-fold dilutions from 2.5 mM to 75 nM)
2.Fill low-volume plate with 4 uL water using Multidrop-micro
3.Add 5 uL assay buffer to columns 1 and 23 using Multidrop-384
4.Add 200 nL of compound (in DMSO from step 1) using 2 transfers of 100 nL with the Evolution 1536 pintool (washed with isopropanol after each transfer)
5.Add 1 uL of Boc-Val-Pro-Arg-AMC substrate (150 uM in 5x assay buffer) using Multidrop-micro
6.Add 5 uL enzyme (11 ng/mL in assay buffer) using Multidrop-384
7.Incubate for 2 hr at room temperature
8.Read fluorescence (excitation 355, emission 460) on Envision reader
IC50 plates contained compounds in columns 3-22, controls (enzyme, no compound) in columns 2 and 24, and blanks (no enzyme) in columns 1 and 23. Each column 3-22 contained 16 two-fold dilutions of a single compound, ranging in concentration from 50 uM to 1.5 nM. Percent activity was calculated for each dilution of each compound from the signal in fluorescence units (FU) and the mean of the plate controls and the mean of the plate blanks using the following equation:
% Activity = 100*((signal-blank mean)/(control mean-blank mean))
Dose response curves of percent activity were fit using XLfit equation 205 (four parameter logistic fit with maximum percent activity and minimum percent activity fixed at 100 and 0, respectively).
The activity score reported here is based on follow-up IC50 testing on compounds that showed >40% inhibition in the primary HTS:
IC50 scores were calculated as follows:
(1) Score = 5.75 x (pIC50-3), where pIC50 = -log(10) of IC50 in mol/L
(2) For IC50 >50 uM, score was calculated from percent activity at maximum concentration tested in assay (50 uM):
Score = [5.75 x (0-3)] + [(100-percent activity at max concentration)/1.75]
Compounds that gave percent inhibition >40 in the primary HTS were judged to be hits and these compounds were selected for follow-up IC50 testing. IC50 values were determined as described in protocol above. The percent activity at the maximum concentration is reported and can be used to estimate the potency of compounds for which the IC50 values were >50 uM.
Activity outcome is reported as follows:
(1) IC50 <50 uM in all three IC50 determinations = active
(2) IC50 >50 uM, percent inhibition 30-50% at 50 uM = inconclusive
(3) IC50 >50 uM, percent inhibition <30% at 50 uM = inactive
This assay was submitted to the PCMD by Scott Diamond, assay development and HTS were conducted by Paul Riley and Abhishek Pratarphekar, and data were submitted by Andrew Napper and Paul Riley, all of the University of Pennsylvania.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)