Chromogenic And Fluorogenic Peptide Substrates For The Detection Of Serine Protease Activity

Abstract

The present invention relates to chromogenic and fluorogenic substrates that can be used for the highly sensitive and selective detection of the activity of serine proteases. The present invention further relates to methods for the detection of the activity of serine proteases, said methods using the substrates of the present invention. Furthermore, the present invention relates to diagnostic kits and test strips using the above substrates, as well as uses of said substrates.

Claims

1. A compound having Formula (I) ##STR00002## wherein Peptide is a peptide or peptide derivative, or a salt of said peptide or peptide derivative.

2. The compound according to claim 1, wherein the C-terminal amino acid of said peptide or peptide derivative is Arg or Lys.

3. The compound according to claim 1 or claim 2, wherein the peptide or peptide derivative is selected from the group consisting of thrombin substrates, factor Xa substrates, trypsin substrates, chymotrypsin substrates, factor VIIa substrates, factor IXa substrates, factor XIa substrates, factor XIIa substrates, kallikrein substrates, plasmin substrates, tissue plasminogen activator substrates, activated protein C substrates, tryptase substrates, matriptase substrates, granzyme substrates, elastase substrates, and human complement protease C1r substrates.

4. The compound according to any one of claims 1 to 3, wherein Peptide is the peptide D-Phe-Pro-Arg (Compound 1) or a salt thereof, the peptide derivative Benzylsulfonyl-D-Arg-Gly-Arg (Compound 2) or a salt thereof, or the peptide derivative Benzyloxycarbonyl-D-Arg-Gly-Arg (Compound 2a) or a salt thereof.

5. A method for the detection of the activity of at least one serine protease in a sample, comprising the steps of contacting said sample with a compound according to any one of claims 1 to 4, and measuring the amount of resorufin released from said compound.

6. The method according to claim 5, wherein the at least one serine protease is a trypsin-like protease.

7. The method according to claim 5 or claim 6, wherein the at least one serine protease is selected from the group consisting of trypsins, chymotrypsins, elastases, thrombin, factor VIIa, factor IXa, factor Xa, factor XIa, factor XIIa, kallikreins, plasmin, tissue plasminogen activator, activated protein C, human complement protease C1r, tryptases, matriptases, and granzymes.

8. The method according to any one of claims 5 to 7, wherein the at least one serine protease is thrombin or factor Xa.

9. The method according to claim 8, wherein the compound is Compound 1 or a salt thereof, Compound 2 or a salt thereof, or Compound 2a or a salt thereof.

10. The method according to any one of claims 5 to 9, wherein the sample (i) is a sample having a high optical density and/or a high autofluorescence, and/or (ii) is in contact with a surface having a high autofluorescence, and/or (iii) contains at least one biological structure that is labeled with at least one chromogenic and/or fluorogenic substance.

11. The method according to any one of claims 5 to 10, wherein the sample is selected from the group consisting of whole blood, serum, plasma, urine, saliva, sputum, semen, lacrimal fluid, cerebrospinal fluid, defecation, cells and tissues.

12. A test strip having a surface on which a compound according to any one of claims 1 to 4 is immobilized.

13. A compound according to any one of claims 1 to 4 for use in the diagnosis of a conditions or disease in a subject that is characterized by abnormal levels of at least one serine protease.

14. Use of a compound according to any one of claims 1 to 4 for the detection of the activity of at least one serine protease in a sample.

15. A diagnostic kit, comprising at least one compound according to any one of claims 1 to 4.

Description

[0084] The figures show:

[0085] FIG. 1:

[0086] Activation of pro-fluorophore by proteolytic enzymes

[0087] After cleavage of the compounds of the present invention by a protease, an intermediate compound is formed which spontaneously converts to the chromogenic and fluorogenic compound resorufin.

[0088] FIG. 2:

[0089] Synthesis of Compound 1 of the present invention

[0090] Reagents and conditions: (a) TBTU, DIEA, DMF, rt, 12 h (93%); (b) cyanuric chloride, DMSO, rt, 1 h (30%); (c) K.sub.2CO.sub.3, DMF, rt, 12 h (95%); (d) TFA-DCM 1:1, rt, 3 h (88%); (e) TBTU, DIEA, DMF, rt, 12 h (73%); (f) THF-H.sub.2O, NaOH, 0 C., 3 h (79%); (g) TBTU, DIEA, DMF, rt, 12 h; (h) TFA-DCM 1:1, rt, 1 h (60% over 2 steps).

[0091] FIG. 3:

[0092] Synthesis of Compound 2 of the present invention

[0093] Reagents and conditions: (a) NaOH, BzIs-CI, Et.sub.3N, acetone-water (27%); (b) NHS-ester: NHS, DCC, DME then NaHCO.sub.3 (19%); (c) TBTU, DIEA, DMF, rt, 12 h; (d) TFA-DCM 1:1, rt, 3 h (10% over 2 steps).

[0094] FIG. 4:

[0095] A. Absorption spectra of resorufin and compound 1 of the present invention; B. Enzymatic hydrolysis of 1 (5 M) in the presence of thrombin (100 pM) with Tris buffer pH 8.3 (.sub.ex=570 nm; .sub.em=583 nm) at 24 C.; inset: full emission spectra recorded after 60 min, with and without enzyme; C. Emission spectra demonstrating the stability of 1 in Tris buffer pH 8.3, at 24 C.

[0096] FIG. 5:

[0097] Kinetic parameters obtained for compound 1 (A) and compound 2 (B).

[0098] FIG. 6:

[0099] Fluorescence assay to evaluate the specificity of compound 1 toward thrombin

[0100] The enzymatic reactions were carried out in 50 mM Tris buffer pH 8.3 and 130 mM NaCl. The activity shows the increase of resorufin fluorescence over time.

[0101] FIG. 7:

[0102] Representative standard curve for determination of thrombin in water.

[0103] The mean change of resorufin fluorescence dF/min is plotted versus the corresponding thrombin concentration (in pM).

[0104] FIG. 8:

[0105] Calibration curve for determination of dabigatran concentration in human plasma

[0106] The measurements were carried out in triplicate. Plasma spiked with dabigatran was added to a thrombin solution and assayed at the 1:25 dilution. The thrombin solution had the following composition: human thrombin 100 pM; Tris buffer pH 8.3 50 mM; NaCl 130 mM; urea 500 mM; BSA 0.01%; polybrene 100 ng/mL; aprotinin 0.15 U/mL. Compound 1 of the present invention (5 M) was added after 5 min preincubation of plasma sample with thrombin solution. The inverted reaction rate of the enzyme with the substrate, determined from the increase of resorufin fluorescence in time, was plotted versus the dabigatran concentration. The picture shows the reaction wells with different dabigatran concentrations after 60 min (taken under UV-lamp).

[0107] FIG. 9:

[0108] Calibration curve for determination of dabigatran concentration in whole human blood

[0109] The fresh blood sample (20 L) was stabilized with 20 mM EDTA solution (2 L), which also contained dabigatran at desired concentration. The thrombin solution had the following composition: human thrombin 250 pM; Tris buffer pH 8.3 50 mM; NaCl 130 mM; urea 500 mM; BSA 0.01%; polybrene 100 ng/mL; aprotinin 40 mU/mL. The fluorogenic substrate 1 (10 M) was added after 5 min preincubation of blood sample with thrombin solution. The reaction rate of the enzyme with the substrate, determined from the increase of resorufin fluorescence in time, is plotted versus the dabigatran concentration.

[0110] FIG. 10:

[0111] Synthesis of the building block 17

[0112] Reagents and conditions: (a) TMS-CI, DIEA, 1,2-dichloroethane, Alloc-CI (85%); (b) PABA, TBTU, DIEA, DMF, rt, 12 h (73%); (c) LiCI, 2,6-lutidine, MsCI, DMF (40%); (d) Resorufin, K.sub.2CO.sub.3, DMF, rt, 12 h; (e) DBU, DCM, rt, 20 min (85%, 2 steps).

[0113] FIG. 11:

[0114] Alternative synthesis of Compound 1

[0115] Reagents and conditions: (a) TMS-CI, DIEA, 1,2-dichloroethane, Alloc-CI (80%); (b) L-Proline methyl ester, TBTU, DIEA, DMF, rt, 12 h; (c) THF-H2O, NaOH, 0 C., 4 h (54%); (d) 17, TBTU, DIEA, DMF, rt, 12 h (90%); (e) Pd[(Ph)3P]4, morpholin, THF/DMF 4:1, rt, 2 h (50%).

[0116] FIG. 12:

[0117] Synthesis of Compound 2a

[0118] Reagents and conditions: (a) TMS-CI, DIEA, 1,2-dichloroethane, Alloc-CI (53%); (b) NHS-ester: NHS, DCC; (c) DME, NaHCO3, glycine (57%); (d) 17, TBTU, DIEA, DMF, rt, 12 h; (e) Pd[(Ph)3P]4, morpholin, THF/DMF 4:1, rt, 2 h (50%).

[0119] FIG. 13:

[0120] Michaelis-Menten kinetic for factor Xa substrate Compound 2a.

[0121] FIG. 14:

[0122] Calibration curve for determination of rivaroxaban concentration in human plasma.

[0123] Plasma was spiked with different rivaroxaban concentrations and added to factor Xa solution (assayed at the 1:100 dilution). The factor Xa solution had the following composition: bovine factor Xa 5 nM; Tris buffer pH 8.3 50 mM; NaCl 130 mM; urea 500 mM; BSA 0.01%; polybrene 100 ng/mL; aprotinin 0.03 U/mL. The fluorogenic substrate 2a (5 M) was added after 5 min preincubation of plasma sample with factor Xa solution. The remaining factor Xa activity was plotted versus the rivaroxaban concentration. The experiment was done in triplicate.

[0124] FIG. 15:

[0125] Calibration curve for determination of rivaroxaban concentration in whole human blood.

[0126] Each sample of fresh blood (20 pM) was stabilized with 20 mM EDTA solution (2 L), which also contained rivaroxaban at desired concentration. The factor Xa solution had the following composition: bovine factor Xa 5 nM; Tris buffer pH 8.3 50 mM; NaCl 130 mM; urea 500 mM; BSA 0.01%; polybrene 100 ng/mL; aprotinin 30 mU/mL. The fluorogenic substrate 2a (5 pM) was added after 5 min preincubation of blood sample (20 L) with factor Xa solution (2 mL). Resorufin fluorescence increase rate (deltaF/min) was taken as factor Xa activity and plotted against rivaroxaban concentration.

[0127] The present invention will be further illustrated by the following examples without being limited thereto.

EXAMPLES

Example 1

Probe Design

[0128] Successful probes for biomolecular imaging applications need to fulfill several requirements: increase in emission intensity upon reaction with the enzyme, efficiency and stability. Herein, the model of a self-cleavable linker as spacer between peptide substrate and the fluorescent label was chosen. The prodrug linker p-aminobenzyl alcohol (PABA) allows coupling of peptides through its amino group and conjugation of alcohol and aniline-based fluorophores and drugs. The spacer is also beneficial to prevent steric hindrance around the cleavage site. As the fluorescent reporter molecule, resorufin (Res) was chosen which can be used to visualize enzyme activities. Very important features are good solubility in water, long emission wavelength and extremely efficient quenching via 7-hydroxy substitution.

[0129] To evaluate the efficiency of thrombin and factor Xa to activate the probes bearing the PABA spacer, D-Phe-Pro-Arg-PABA-Res (1) (thrombin substrate), BzIs-D-Arg-Gly-Arg-PABA-Res (2), and Cbz-D-Arg-Gly-Arg-PABA-Res (2a) (factor Xa substrates) were synthesized (FIG. 1).

Example 2

Synthesis

[0130] The synthesis of the building block 6, which was used for both enzyme substrates started from Boc-Arg(Boc)2-OH (3) (FIG. 2). It coupled to PABA under standard conditions affording the benzylic alcohol (4) in almost quantitative yield. The chlorination of the alcohol proved to be very tricky due to acid-labile Boc groups. Commonly used chlorination reagents (SOCl.sub.2/Et.sub.3N, CCl.sub.4/Ph.sub.3P) failed to deliver the desired compound. Even MsCl/Et.sub.3N, a mild chlorination reagent, afforded 5 in unsatisfactory yields (9-15%). The best results were obtained with a cyanuric chloride/DMSO mixture, which gave 5 in 30% yield. It is worthwhile mentioning that 0.5 eq of cyanuric chloride was optimal; more reagent led to dramatic decrease of isolated product. Alkylation of resorufin and Boc-deprotection proceeded smoothly yielding the conjugate 6 in very good yield.

[0131] As shown in the FIG. 2, the synthesis of the second building block started with the coupling of Boc-D-Phe-OH (7) with H-Pro-OMe (10) to provide the methyl ester 8 in good yield. The ester was hydrolyzed in THF/H.sub.2O mixture with NaOH at 0 C., providing the free dipeptide in 79% yield.

[0132] Coupling of the dipeptide 9 with the building block 6 was best achieved using TBTU as activator in the coupling reaction. COMU was also used, but some by-products which form are difficult to separate from the product. The conjugate was chromatographed on a column packed with C18 silica. The final Boc-deprotection with TFA/DCM mixture afforded 1 in good yield as TFA salt.

[0133] The Factor Xa substrate 2 was synthesized similarly (FIG. 3). H-D-Arg(Pbf)-OH was first protected with benzylsulfonyl chloride. The coupling with glycine via NHS-ester afforded dipeptide 13, which was further coupled to the building block 6 following the same procedure as used for the preparation of 1.

Example 3

Substrate Properties

[0134] The photophysical properties of compound 1 of the present invention, as well as its enzymatic conversion to fluorescent product resorufin were investigated. Compound 1 displays a blue shift in the absorption spectra (90 nm) relative to resorufin (FIG. 4A). It also has a negligible emission, if exited either at its maxima (480 nm) or at resorufin maxima (570 nm). Additionally, no spontaneous hydrolysis is observed during incubation with thrombin buffer (Tris pH 8.3), indicating high stability of the conjugate (FIG. 4C). Thrombin-induced substrate hydrolysis gives rise to a 300-fold increase in fluorescence, which demonstrates that quenching of resorufin upon alkylation is extremely efficient (FIG. 4B).

Example 4

Kinetic Measurements

[0135] Commercial substrate 1a, depicted in FIG. 2, was used as reference in the kinetic measurements. The kinetic values K.sub.M, k.sub.cat and k.sub.cat/K.sub.M were established for the fluorogenic compound 1 using Human Thrombin. In order to compare the results of 1 and 1a, the absorption of the released chromophores was measured during the enzymatic reaction (570 nm for resorufin and 380 nm for p-nitroaniline). Advantageously, thrombin turnover of 1 did not suffer at all, suggesting that the PABA linker plays a very important role in the recognition of the substrate by the enzyme, placing the fluorophore away from the active site.

[0136] The same parameters were also determined for 1 by measuring the emission of resorufin at 583 nm and similar values to the ones obtained from absorption were found (FIG. 5A).

[0137] The Factor Xa substrate 2 surprisingly showed a very low KM value, a relatively good turnover number k.sub.cat and excellent catalytic efficiency (FIG. 5B). A similar conjugate, with the same peptide sequence, but having p-nitroanilide as reporter, has a much higher K.sub.M (40 M) and the enzyme has poorer catalytic efficiency for this substrate (2.710.sup.6 M.sup.1 s.sup.1). Thus, compound 2 of the present invention performs better than the best factor Xa substrate so far reported.

Example 5

Selectivity of Fluoroqenic Compound 1 and LOD for Thrombin

[0138] the specificity of Compound 1 of the present invention toward thrombin was tested. Compound 1 (5 M) was incubated in the presence of thrombin (100 pM) and some possible interfering proteases and proteins, like trypsin, factor Xa, myoglobin, cytochrome C and BSA (100 pM). The increase of fluorescence in response to factor Xa, myoglobin, cytochrome C and BSA was negligible (FIG. 6). Only trypsin hydrolyzed Compound 1, but at a slower rate compared to thrombin (7.5-fold more selective for thrombin over trypsin). A commercial assay can detect 1 pM thrombin, by fishing it out of plasma samples using microwells coated with DNA-aptamer. The AMC-based substrate is converted by thrombin to fluorescent product after the enrichment step. The fluorescence assay according to the present invention allowed the detection of thrombin at the concentrations as low as 0.5 pM in water solution (FIG. 7), which was achieved without any enrichment step.

Example 6

[0139] Dabigatran is a commonly used anticoagulant in the clinic. While routine monitoring of dabigatran is not recommended, the determination of its blood level in specific situations (such as bleeding complications, emergency, self-compliance) and/or patient populations (such as the elderly, renal impairment) may increase drug safety. Specific assays for dabigatran have not been established along with drug development and further clinical trials are required to determine the relation of assay results to bleeding or thrombotic complications. In many laboratories only qualitative coagulation-based tests are available, such as prothrombin time (PT) assay or the activated partial thromboplastin time (APTT) assay. Unfortunately these tests often give false-negative results. Other coagulation-based test, such as thrombin clotting time (TCT) detects only minimal dabigatran plasma levels.

[0140] Ecarin chromogenic assay (ECA) uses a p-nitroanilide substrate and determines accurately therapeutic and supratherapeutic dabigatran levels in plasma.

[0141] Described herein is an assay that uses Compound 1 of the present invention for quantification of dabigatran in plasma, and, most importantly, in whole blood, as a key step for the development of a point-of-care test.

[0142] First it was tested how our Compound 1 works in the presence of human plasma. Human plasma was spiked with dabigatran (25-500 ng/mL) and added to a thrombin solution. After incubation for 5 min, the substrate 1 is added and the fluorescence increase at 585 nm is measured over time. A calibration curve could be constructed, which can be used to determine the dabigatran concentration in an unknown sample (FIG. 8, lower part). The results of a measurement can even be visualized by naked eye under an UV-lamp (FIG. 8, upper part).

[0143] The next step was to construct a similar calibration curve, but using whole blood instead of plasma.

[0144] The experimental procedure is similar to the one with plasma. Fresh blood portions (20 L) were spiked with dabigatran solution (2 L) and added to thrombin (2 mL) in a single-use fluorescence plastic cuvette. After a short preincubation at room temperature, the fluorogenic substrate was added and the fluorescence change was monitored using a portable fluorescence device (Aquafluor from Turner Designs). Advantageously, it was found that the rate of the enzymatic reaction decreases linearly with the increasing dabigatran concentration (FIG. 9).

Example 7

Alternative Synthesis of Compound 1; Synthesis of Compound 2a

[0145] An analog strategy to synthesize the thrombin and factor Xa substrates of the present invention is described. The following pathway allows to obtain the desired compounds in gram scale. In this case the factor Xa substrate has a Z protecting group, instead of BzIs, which does not significantly affect its performance.

[0146] The commercial Fmoc-Arg-OH was protected with alloc following a literature described procedure, with minor modifications (FIG. 10; building block 17). Compound 14 is coupled to PABA linker as already described (cf. Example 2). A key step of this synthesis represents the chlorination step of the benzylic alcohol. The MsCl/Lutidine method is very mild and gives reproducible yields (40%) in this case, also due to the fact that the substrate does not have acid labile protecting groups. This method did not work when Boc protecting groups were used. Fmoc and alloc protecting groups are fully compatible with the resorufin alkylation reaction conditions (although some Fmoc deprotection is observed when the reaction is left overnight). Resorufin conjugates are also stable under strong DBU basic conditions, during Fmoc deprotection. Building block 17 is then obtained in very good yield (85% over 2 steps), and is used for the synthesis of both Compound 1 and Compound 2a.

[0147] The alloc protection of D-Phe-OH is carried out similarly to the protection of arginine. The coupling to L-proline methyl ester and the hydrolysis of the dipeptide is described in the previous synthesis (cf. Example 2). The key step represents the final alloc deprotection in the presence of Pd-catalyst and morpholin. 20% DMF were used as a co-solvent, due to the formation of intermediates during the reaction, which are not soluble in THF. Fortunately, resorufin is not released during deprotection, which is otherwise very difficult to separate from the product. Thrombin substrate 1 is obtained in good yield (50%) and with high purity (FIG. 11).

[0148] In the current approach, for the synthesis of factor Xa substrate, it is started from Z-D-Arg-OH, which is commercially available. The previously used analog BzIs-D-Arg-OH is obtained synthetically in 27% yield. Alloc protection is performed as described above. The intermediate 18 couples to glycine via NHS ester in good yield (57%). In the case of BzIs protecting group, the average yield in the same step was less than 20%. Most importantly, the final deprotection step proceeds smoothly, yielding substrate 2a in very high purity (FIG. 12). Particularly in the case of factor Xa substrate 2, the final step was critical, where prolonged reaction time under acidic conditions was necessary to remove the Pbf protecting group. This led to resorufin release and low purity of the final product.

Example 8

Kinetic Measurements of Compound 2a

[0149] The kinetic parameters were determined as described for the other substrates (cf. Example 4). The results of Michaelis-Menten kinetic is depicted in FIG. 13. They clearly indicate that the substrate performance is not significantly affected by changing from BzIs to Cbz protecting group (parameters for 2a K.sub.M=5.5 M; k.sub.cat=4.9 s.sup.1; k.sub.cat/K.sub.M=0.8910.sup.6 M.sup.1 s.sup.1).

Example 9

[0150] The anticoagulant rivaroxaban can be determined in human plasma and whole blood, similarly to dabigatran. Either plasma or fresh blood was spiked with rivaroxaban and the resulting mixture was added to a factor Xa solution. In this case, substrate 2a was used to determine the residual enzyme activity. Factor Xa has been used at a higher concentration, if compared to thrombin, due to the fact that substrate 2a has a k.sub.cat lower than substrate 1. The results for plasma fit to the following exponential decay: y=2.92*exp(x/64.75)+0.1 (FIG. 14). The calibration curve for blood shows a linear dependence (FIG. 15).

CONCLUSION

[0151] Herein described is the synthesis and the application of a new thrombin substrate 1 based on 3 modules: resorufin fluorophore, self-cleavable PABA linker and recognition tripeptide. Similarly, new factor Xa substrates 2 and 2a were synthesized.

[0152] The new substrates are chemically stable toward spontaneous hydrolysis. Fluorogenic Compounds 1, 2 and 2a do not lose their specificity for thrombin and factor Xa correspondingly, if compared to the commercial substrates, due to PABA linker incorporation. Furthermore, 1, 2 and 2a are chromogenic and fluorogenic substrates: upon reaction with the enzyme it results in more than 300-fold increase in fluorescence; simultaneously a color change from yellow to purple is observed. It could also be shown that compound 1 is 7.5 times more specific for thrombin if compared to trypsin and 400 times more specific for thrombin if compared to factor Xa. Surprisingly, as low as 0.5 pM thrombin in water could be detected using the substrate 1. This sensitivity is way lower than the aptamer-based methods reported in the literature. Taking advantage of its high selectivity and sensitivity, fluorogenic substrate 1 was applied for quantification of a commonly used anticoagulant dabigatran in the therapeutic range (27-411 ng/mL) in plasma and whole blood. Compounds 2 and 2a could be used similarly for detection of another important anticoagulant rivaroxaban. The whole blood assay was also adapted for use at the point of care. To our knowledge, this is the first fluorogenic assay which can measure directly the dabigatran and rivaroxaban concentration without separating the red blood cells.

ABBREVIATIONS

[0153] Alloc: Allyloxycarbonyl [0154] AMC: 7-Amino-4-methylcoumarin [0155] Boc: tert.-Butyloxycarbonyl [0156] BzIs: benzylsulfonyl [0157] COMU: 1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholinocarbenium hexafluorophosphate [0158] DBU: 1,8-Diazabicyclo[5.4.0]undec-7-en [0159] DCC: N,N-Dicyclohexylcarbodiimide [0160] DCM: Dichloromethane [0161] DIEA: N,N-Diisopropylethylamine [0162] DME: Dimethoxyethane [0163] DMF: Dimethylformamide [0164] DMSO: Dimethyl sulfoxide [0165] EDTA: Ethylenediaminetetraacetic acid [0166] em: emission [0167] ex: excitation [0168] Et: ethyl [0169] FRET: Frster resonance energy transfer [0170] Ms: Methanesulfonyl [0171] NHS: N-Hydroxysuccinimide [0172] NP: nanoparticle [0173] PABA: p-aminobenzyl alcohol [0174] Pbf: 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl [0175] Ph: phenyl [0176] Res: resorufin [0177] rt: room temperature [0178] TBTU: N,N,N,N-Tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate [0179] TFA: Trifluoroacetic acid [0180] THF: Tetrahydrofuran [0181] TMS: Trimethylsilyl