An assay for measuring binding affinity for cardiolipin of biologically active compounds

Abstract

The present invention relates to a method for the evaluation of binding affinity of biologically active substances for cardiolipin based on acridinium salt utilization as a fluorescent probe.

Claims

1. An assay method, comprising: incubating a solution comprising a compound of interest, a lipid vesicle containing cardiolipin, and 3,6-di(azetidin-1-yl)-10-(3-(trimethylsilyl)propyl)acridin-10-ium iodide (I) ##STR00002## and measuring fluorescence intensity at an excitation wavelength of 497 nm and at an emission wavelength of 529 nm, respectively.

2. The method of claim 1, wherein the compound of interest is an endogenous compound.

3. The method of claim 1, wherein the compound of interest comprises a metal cation.

4. The method of claim 1, wherein the compound of interest is an xenobiotic.

5. The method of claim 1, wherein the compound of interest comprises an ammonium or phosphonium cation.

6. The method of claim 1, wherein the compound of interest is an anthracycline or anthracenedione.

7. The method of claim 1, wherein the compound of interest is an aminoglycoside.

8. A kit comprising: 3,6-di(azetidin-1-yl)-10-(3-(trimethylsilyl)propyl)acridin-10-ium iodide; a buffer; and a material containing cardiolipin.

9. (canceled)

10. The method of claim 1, wherein the solution further comprises a buffer.

11. The method of claim 1, wherein 3,6-di(azetidin-1-yl)-10-(3-(trimethylsilyl)propyl)acridin-10-ium iodide is in a concentration of from 0.2 μM to 25 μM in the solution.

12. The method of claim 1, wherein the solution is incubated at 37° C.

13. The method of claim 1, further comprising determining an EC.sub.50 value of the compound of interest after the measuring step.

14. The method of claim 1, wherein, before the incubating step, the method further comprises: mixing the compound of interest and a lipid vesicle containing cardiolipin to form an intermediate solution; incubating the intermediate solution; and adding 3,6-di(azetidin-1-yl)-10-(3-(trimethylsilyl)propyl)acridin-10-ium iodide into the intermediate solution.

Description

[0044] FIG. 1 represents concentration-effect curves obtained in the competition binding assay of endogenous CL ligands—cytochrome c (A) and monovalent and bivalent cations (B) using CL-DOPC liposomes (25:75 mol %) and I as the CL-specific fluorescent probe.

[0045] FIG. 1A depicts cytochrome c binding with CL in 20 mM HEPES buffer at pH 7.4, using 2.5 μM CL and 5 μM I as a fluorescent probe. Particularly, normalized fluorescence intensity of I is plotted against various cytochrome c concentrations on a logarithmic scale and fit with four parameter logistic (4PL) curve allowing calculating EC.sub.50 value.

[0046] FIG. 1B depicts Ca.sup.2+, Mg.sup.2+ and NH.sub.4.sup.+ binding with CL in 20 mM HEPES buffer at pH 7.4, using 2.5 μM CL and 5 μM I as a fluorescent probe Particularly, normalized fluorescence intensity of I is plotted against various Ca.sup.2+, Mg.sup.2+ and NH.sub.4.sup.+ concentrations on a logarithmic scale and fit with four parameter logistic (4PL) or five parameter logistic (5PL) curve allowing calculating EC.sub.50 values. Under the same experimental conditions obtained EC.sub.50 can be compared and related to the compounds' affinity for CL. Herein, Ca.sup.2+, has ≈30% lower EC.sub.50 than Mg.sup.2+ and, therefore, higher affinity for CL. In turn, monovalent NH.sub.4.sup.+ ions have 23-fold lower affinity for CL than Ca.sup.2+ ions.

[0047] FIG. 2 represents concentration-effect curve obtained in the competition binding assay of xenobiotics—mitochondria-targeted nonyltriphenylphosphonium bromide and dodecyltrimethylammonium chloride (A), anthracyclines and anthracenedione (B), and aminoglycosides (C) for CL using CL-DOPC liposomes (25:75 mol %) and I as the CL-specific fluorescent probe.

[0048] FIG. 2A depicts nonyltriphenylphosphonium bromide and dodecyltrimethylammonium chloride binding with CL in 20 mM HEPES buffer at pH 7.4, using 2.5 μM CL and 5 μM I as a fluorescent probe. Particularly, normalized fluorescence intensity of I is plotted against various nonyltriphenylphosphonium bromide and dodecyltrimethylammonium chloride concentrations on a logarithmic scale and fit with four parameter logistic (4PL) curve allowing calculating EC.sub.50 values. Under these conditions nonyltriphenylphosphonium bromide's affinity for CL is ≈30% higher than that of dodecyltrimethylammonium chloride.

[0049] FIG. 2B shows daunorubicin, doxorubicin and mitoxantrone binding with CL in 20 mM HEPES buffer at pH 7.4, using 2.5 μM CL and 5 μM I as a fluorescent probe. Particularly, normalized fluorescence intensity of I is plotted against various daunorubicin, doxorubicin and mitoxantrone concentrations on a logarithmic scale and fit with four parameter logistic (4PL) curve allowing calculating EC.sub.50 values. Mitoxantrone has the highest affinity for CL in this set of compounds, with twice as low EC.sub.50 value compared to daunorubicin and thrice as low EC.sub.50 value compared to doxorubicin.

[0050] FIG. 2C shows apramycin, gentamycin, streptomycin and tobramycin binding with CL in 20 mM HEPES buffer at pH 7.4, using 2.5 μM CL and 5 μM I as a fluorescent probe. Particularly, normalized fluorescence intensity of I is plotted against various daunorubicin, doxorubicin and mitoxantrone concentrations on a logarithmic scale and fit with four parameter logistic (4PL) or five parameter logistic (5PL) curve allowing calculating EC.sub.50 Gentamicin has the highest affinity for CL in this series of aminoglycosides and the highest affinity among this set of xenobiotics, although gentamycin exhibits ≈3 times lower affinity toward CL than it's natural ligand cytochrome c. In turn, streptomycin has the lowest affinity for CL among studied aminoglycosides.

[0051] Table 1. lists EC.sub.50 values obtained by the competition binding assay in 20 mM HEPES buffer at pH 7.4 (physiological pH) and 6.8 (pH of the intermembrane space in mitochondria [20]) using 2.5 μM CL and 5 μM I as a fluorescent probe. Among studied compounds cytochrome c, a natural CL ligand, exhibited the lowest EC.sub.50 value, thus the highest binding affinity for CL. Inorganic cations showed much higher EC.sub.50 values (in mM range). Mitoxantrone has EC.sub.50 values in micromolar range and higher affinity for CL than doxorubicin and daunorubicin.

[0052] All studied aminoglycosides bind with CL but with different affinities—gentamicin exhibiting the highest and streptomycin exhibiting the lowest affinity toward CL. Mitochondria targeted nonyltriphenylphosphonium bromide and dodecyltrimethylammonium chloride have EC.sub.50 values in micromolar range although lower affinities than studied aminoglycosides or anthracyclines. Nonyltriphenylphosphonium bromide and dodecyltrimethylammonium chloride were the only compounds studied whose interaction with CL was affected by changes of pH.

TABLE-US-00001 TABLE 1 EC.sub.50 values obtained from competitive binding assay EC.sub.50 ± SD, μM Example Compound pH = 7.4 pH = 6.8 1 Cytochrome C 0.32 ± 0.06 0.28 ± 0.02 2 NH.sub.4Cl 31853.33 ± 2.30   n.t.* 3 CaCl.sub.2 1334.33 ± 10.10  1285.67 ± 129.5  4 MgCl.sub.2 1932.33 ± 20.51  n.t. 5 Doxorubicin 6.94 ± 0.90 5.86 ± 0.25 6 Daunorubicin 4.35 ± 1.25 n.t. 7 Mitoxantrone 2.04 ± 0.30 n.t. 8 Gentamycin sulfate 0.98 ± 0.14 1.01 ± 0.12 9 Apramycin sulfate 3.34 ± 0.95 n.t. 10 Streptomycin sulfate 9.82 ± 1.76 n.t. 11 Kanamycin sulfate 4.32 ± 1.85 n.t. 12 Tobramycin 4.37 ± 0.32 n.t. 13 Nonyltriphenylphosphonium bromide 19.76 ± 0.75  36.65 ± 2.93  14 Dodecyltrimethylammonium chloride 30.71 ± 2.75  74.41 ± 8.47  Values are shown as the means ± S.D. from 3 independent experiments. *n.t. - not tested.

[0053] Those values and binding curves were particularly encouraging, bearing in mind that developed assay allows studying and comparing organic and inorganic compound affinity for cardiolipin.

[0054] While the invention has been described with respect to certain embodiments, the description is intended to be exemplary, rather than limiting. Modifications and changes may be made within the scope of the invention, which is defined in the appended claims.

[0055] The invented procedure can be used for determination of substances' binding affinity for cardiolipin using a fluorescent probe e.g. compound I in an assay kit.

[0056] Wherein an assay kit comprising: fluorescent probe, appropriate buffer, cardiolipin containing material for the determination of organic and inorganic compounds binding to cardiolipin.

REFERENCES

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