Fluorogenic peptide substrates for in solution and solid phase factor Xa measurements

Abstract

The measurement of Factor Xa (FXa) enzymatic activity using novel fluorogenic peptide substrates that have a C-terminus cleavable fluorophore and optionally the ability to attach to a solid support. Fluorogenic measurements increase sensitivity and flexibility of measurements of enzymatic reactions over traditional absorbance-based approaches. The measurement of FXa generation is applicable to a range of biological reactions.

Claims

1. A fluorogenic peptide substrate having the formula
peptide-fluorophore-(linker)n-(X)m wherein X is an attachment group; the peptide comprises a C-terminus; and the fluorophore is cleavable at the C-terminus.

2. The fluorogenic peptide substrate of claim 1 wherein the fluorophore is selected from the group consisting of ACC, AMC, and AFC.

3. The fluorogenic peptide substrate of claim 1 wherein the fluorophore is excitable by at least one of a UV-light source and a violet light source.

4. The fluorogenic peptide substrate of claim 1 wherein the fluorophore comprises an amine group capable of being functionalized and conjugated with the at least one linker and the at least one attachment group, X.

5. The fluorogenic peptide substrate of claim 1 wherein: n is an integer from 0 to 4; and m is an integer from 0 to 4.

6. The fluorogenic peptide substrate of claim 1 wherein the C-terminus is Arg.

7. The fluorogenic peptide substrate of claim 1 wherein the peptide comprises the sequence DArg-Gly-Arg.

8. The fluorogenic peptide substrate of claim 1 wherein the peptide comprises the sequence Ile-Glu(gamma-pip)-Gly-Arg.

9. The fluorogenic peptide substrate of claim 1 wherein: the peptide comprises the sequence Ile-Glu(gamma-OR); and R is selected from the group consisting of H and CH.sub.3.

10. The fluorogenic peptide substrate of claim 1 wherein the linker is selected from the group comprising PEG and CC.

11. The fluorogenic peptide substrate of claim 1 wherein the linker is spherical PEG synthesized generating microfluidic droplets.

12. The fluorogenic peptide substrate of claim 1 wherein the linker is rectangular PEG synthesized by stop-flow lithography.

13. The fluorogenic peptide substrate of claim 1 wherein the fluorophore is configured to be cleaved at the C-terminus by FXa.

14. The fluorogenic peptide substrate of claim 1 wherein, upon cleavage at the C-terminus, the fluorophore is configured to remain bound to the linker.

15. The fluorogenic peptide substrate of claim 1 wherein the attachment group X comprises at least one of NH.sub.2, -biotin, COOH, SH, CM, -acrylate, -click, maleimide, -alkyne, -ITC, NHS, -SMCC, -ALD, -EPOX, -ester, -hydrazide, OH, -SIL, and -VA.

16. The fluorogenic peptide substrate of claim 1 wherein the peptide comprises an N-terminus.

17. The fluorogenic peptide substrate of claim 16 wherein the N-terminus comprises at least one of Z, Suc, Lys, Bz, and Cbz group.

18. A method of measuring enzymatic activity comprising using at least one of the peptide of the fluorogenic peptide substrate of claim 1 and the fluorogenic peptide substrate of claim 1 to detect at least one protein in a clotting cascade comprising FXa.

19. The method of claim 18 wherein the at least one protein comprises FXa, FVIII, and FIX.

20. The method of claim 18 wherein the method is used to detect the at least one protein in a patient with hemophilia.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0040] FIG. 1 shows the conceptual diagram of some embodiments. There is the peptide sequence, linked at the C-terminus to the cleavable fluorophore, optionally attached to a linker and/or an attachment group that allows for attachment to a solid support.

[0041] FIG. 2 shows an example of some types of solid supports that can be utilized with some embodiments. It includes spherical and rectangular PEG microparticles and also microspheres.

[0042] FIG. 3 shows cleavage of the peptides in solution and linked to a solid support. Cleavage on the solid support leaves the fluorophore on the support, thus allowing for fluorescence measurements on the solid support.

[0043] FIG. 4 shows some of the peptide sequences described in some embodiments. The peptides show an N-terminal group for increasing the stability of the peptide, the cleavage sequence, the fluorophore (selected from -AMC, -ACC, and -AFC), linkers, and also the attachment chemistry. The peptides in this figure are examples and other combinations are possible based on the information provided.

[0044] FIG. 5 shows a partial list of attachment chemistries and also linkers.

[0045] FIG. 6 shows the structures of the three fluorophores utilized in some embodiments.

[0046] FIG. 7 shows the chemical structures for Suc-Ile-Glu(gamma-pip)-Gly-Arg-Acc-Peg2-NH.sub.2 and Suc-Ile-Glu(gamma-pip)-Gly-Arg-Acc-Peg4-NH.sub.2.

[0047] FIG. 8 shows the chemical structure for Z-D-Arg-Gly-Arg-ACC-PEG4-NH.sub.2.

[0048] FIG. 9 shows the chemical structure for Z-D-Arg-Gly-Arg-ACC-PEG.sub.2-NH.sub.2.

[0049] FIG. 10 shows the chemical structure for Z-D-Arg-Gly-Arg-ACC-PEG(n)-biotin.

[0050] FIG. 11 shows the excitation and emission spectra for 7-amino-4-trifluoromethylcoumarin and 7-amino-4-methylcoumarin.

[0051] FIG. 12 shows the excitation and emission spectra for ACC and AFC.

[0052] FIG. 13 shows the chromogenic pNA and ACC peptides' sensitivity to varying FXa concentrations 3900 ng/mL, 390 ng/mL, 39 ng/mL, 3.9 ng/mL, 0.39 ng/mL, and 0, respectively. A base 10 log scale is used on the x-axis. Graph (a) shows the limit of detection of pNA peptide is between 0.39 and 3.9 ng/mL, (P<0.0210). Graph (b) shows the limit of detection of Z-ACC is between 0 and 0.39 ng/mL (P<0.0004).

[0053] FIG. 14 shows the Z-ACC-linked microparticles response to varying FXa concentrations detected using miniature flow cytometer. The Z-ACC peptide microparticles were reacted with varying FXa concentrations (3900 ng/mL, 390 ng/mL, 39 ng/mL, 3.9 ng/mL, 0.39 ng/mL and 0). An increase of signal intensity was observed with increasing FXa concentration. All points are statistically different. 0 and 0.39 ng/mL are significant with P<0.0001.

DETAILED DESCRIPTION

[0054] The desired peptides and peptides linked to microparticles have specific cleavage sequences for FXa. In particular, they all contain a cleavable fluorophore at the C-terminus selected from AMC, ACC, or AFC. Cleavage of the peptide bond between the terminal amino acid and the fluorophore releases the fluorophore, leading to a right shifting of the spectra and increase in detectable fluorescence.

[0055] The ACC fluorophore is bifunctional and may be utilized when a solid support is utilized in some embodiments. Cleavage of the substrate allows the ACC fluorophore to remain on the solid support, allowing for detection along with the solid support. This is particularly important with a microparticle-based flow assay, which allows for measurement of concentrated ACC fluorophore on the microparticles.

[0056] The ACC fluorophore may be utilized when high sensitivity and assay dynamic range are desired. In particular, the ACC fluorophore has minimal spectral overlap between the uncleaved and cleaved states at the emission maxima (460 nm) of the fluorophore. This lack of a spectral tail for the uncleaved fluorophore make it such that, in some embodiments, there may be a large difference between the uncleaved and cleaved substrates. This is particularly important when a broad FXa assay range with high sensitivity is desired.

[0057] With the use of the ACC fluorophore on solid support, in some embodiments, a linker is also present. In some embodiments, the linker may be made of PEG that is spaced of two to four PEG molecules long. This will allow it to maintain functionality in enzymatic cleavage processes by offering a set distance between the peptide and the solid support. In some embodiments, the ACC fluorophore is further conjugated to another PEG molecule that increases the linker length on the solid support even further. CC linkers can also be utilized as well or other PEG spacer lengths. The synthesized peptide may be attached to a PEG molecule with a molecular weight (MW) that may be greater than 1 kilodaltons (kD) in some embodiments. The solid support may further be made of PEG to minimize non-specific binding interactions and to decrease autofluorescence in some embodiments.

[0058] The attachment chemistry on the peptide may be a molecule that is easily conjugated. It is, in some embodiments, selected from the following list: NH.sub.2, COOH, SH, -SCM, -acrylate, -click, maleimide, -alkyne, -ITC, NHS, -SMCC, -ALD, -EPOX, -ester, hydrazide, OH, -SIL, -VA. Easy to synthesize chemistry is best in some embodiments. For instance, the amine NH.sub.2 functionality is readily added during peptide synthesis, in some embodiments. Amine groups may be linked to succinimidyl chemistries readily.

[0059] One of the sequences is DArg-Gly-Arg preceding the fluorophore in some embodiments. Another sequence is Ile-Glu(gamma-pip)-Gly-Arg preceding the fluorophore in some embodiments. Other embodiments utilize the sequence Ile-Glu(gamma-OR) where RH or RCH.sub.3 or where R is a 50:50 mixture of H or CH.sub.3. Some of the sequences, such as DArg-Gly-Arg, Ile-Glu(gamma-pip)-Gly-Arg, and Ile-Glu(gamma-OR) allow for maximal sensitivity and specificity to FXa.

[0060] For stability, in some embodiments, the N-terminus may have the equivalent of a capping group, which may include the Z, Suc, Lys, Bz, or Cbz groups. In this manner, the peptide may be protected from degradation and may have the optimal stability in biological reactions. Other N-terminal protecting or capping groups may be utilized as well and, in some embodiments the approach is consistent with existing peptide synthesis methods.

[0061] In some embodiments, the solid support may be made from polymerized. PEG microparticles. In some embodiments, PEG microparticles have low autofluorescence, are porous, have low non-specific binding, and can have different functionalities. In particular, PEG with acrylate groups may be utilized to form hydrogel particles using ultraviolet (UV) exposure. PEG microparticles thus have desirable attributes for biological applications.

[0062] Peptides can be readily incorporated into these hydrogels with the use of bifunctional linkers such as ACRYL-PEG-SCM functionality. The amine functionalized the peptide can be reacted with the -SCM group and then incorporated into the polymerization mixture. This requires reacting at pH>8, typically with the use of sodium bicarbonate at 0.1M, freeing up the electron pair on the amine group for the reaction.

[0063] PEG microparticles can be spherical or rectangular. Spherical microparticles may be generated through the generation of microfluidic droplets. This approach utilizes a microfluidic device that is fabricated with polydimethylsiloxane (PDMS). The PDMS device is fabricated utilizing an SU-8 master mold. The microfluidic device may be fabricated using replica molding. A mixture of PDMS prepolymer and curing agent (10:1, Sylgard 184, Dow Corning Co) is mixed, degassed and poured onto the SU-8 master and cured at 65 C. Droplets are formed using a droplet junction and then polymerized using UV light in the channel with a photoinitiator added to the mixture.

[0064] Rectangular microparticles may be synthesized by stop-flow lithography. In this approach, a PDMS channel is utilized and synchronized valves and shutters control passage of the PEG prepolymer mixture into the chip. UV light exposure polymerizes the microparticles through a photomask placed in the field stop position of the illuminating path. Slide-based polymerization may also be utilized to polymerize rectangular microparticles or particles of different shapes. This is done by allowing UV light to go through a photomask to pattern a PEG prepolymer mixture. The polymerized microparticles may be washed and utilized for assays.

[0065] Performance testing of the peptides may be done using purified FXa and plotting Michaelis-Menten curves and comparing the turnover rate (kcat). This reaction may be done at 37 C. and monitored over time for cleavage rate. A spectrophotometer, such as the SpectraMax M2 in kinetic mode may be utilized with excitation at 405 nm and emission at 450 nm.

[0066] The peptide substrates and peptides immobilized on a solid support may also be utilized for testing in FVIII or FIX assays. This includes taking a plasma sample, diluting it in a buffer such as 10 mM Tris with 01% BSA and then mixing it with FIXa, PL, and Ca2+ to form the tenase complex. The tenase complex then cleaves FX to make FXa, which then cleaves the substrate to give rise to fluorescence that is measured and is directly proportional to the plasma's FVIII level in some embodiments. PEG-based microparticles may be read out using a flow cytometer or micro-flow cytometer. In solution measurements may be done via a spectrofluorometer such as the SpectraMax M2.

[0067] Some embodiments may be applied to the measurement of FXa, FVIII, FIX, and other proteins in the clotting cascade that involves FXa. The fluorogenic nature of the reaction may allow for high sensitivity and dynamic range, which is particularly applicable for measurement of hemophilia patient in various settings and low levels of blood factors in some embodiments. The compatibility with solid phase reactions, may allow it to be utilized in a broad range of assay formats for measurements of various blood factors. Often times in diagnostic assays, hemolysis is an issue. Traditional chromogenic substrates, such as pNA, absorb at the same wavelength as hemoglobin. The fluorogenic substrates described here are less susceptible to hemolysis.

[0068] FIGS. 6-10 highlight the cleavable fluorophore, linkers, and attachment groups in accordance with some embodiments.

[0069] FIG. 12 shows the excitation and emission spectra for ACC and AFC. The ACC absorption spectra at a concentration of 3900 ng/mL 1210 is compared to the control 1220. The ACC emission spectra at a concentration of 3900 ng/mL 1230 is compared to the control 1240. The AFC absorption spectra at a concentration of 3900 ng/mL 1250 is compared to the control 1260. The AFC emission spectra at a concentration of 3900 ng/mL 1270 is compared to the control 1280.

[0070] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation and/or engineering, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the claims that follow the reference list.