Assay for measuring the binding affinity of bioactive compounds to cardiolipin

Abstract

The present invention relates to a method for evaluating the binding affinity of a bioactive substance to cardiolipin based on the use of an acridinium salt as a fluorescent probe.

Description

Technical Field

[0001] The present invention relates to a method for evaluating the binding affinity of a bioactive substance to a negatively charged membrane. In particular, the present invention relates to the use of a fluorescent acridinium salt as a fluorescent probe for measuring the binding affinity of an organic or inorganic substance to cardiolipin. Background Art

[0002] Cardiolipin (CL) is a unique phospholipid localized in the inner mitochondrial membrane (IMM) in eukaryotic cells and in the cytoplasmic membrane in prokaryotic cells. CL provides mitochondrial membrane stability, dynamics, and is required for the optimal activity of several mitochondrial membrane proteins (e.g., electron transport chain (ETC) complexes I, III, IV, ATP synthase, cytochrome c). [1] Furthermore, CL stabilizes anaerobic respiratory complexes in bacteria. [2] Due to its unique structural properties and localization, CL is an attractive pharmacological target for mitochondrial-specific therapies in combination with antibiotics. [3-5] Furthermore, the mitochondrial toxicity of some drugs, such as anthracyclines and aminoglycosides, has been attributed to their ability to interact with CL [6-8], leading to life-threatening side effects such as heart failure and decreased renal function. [9,10]

[0003] Therefore, exploring binding to CL is crucial for screening novel, CL-targeted modulators of mitochondrial function and antibiotics, as well as for evaluating the potential of drugs to cause mitochondrial toxicity through interaction with CL.

[0004] Previously, through 1 H and 13 C NMR can be used to detect the binding of compounds to CL[11,12], but this method is time-consuming, semi-quantitative, and requires large amounts of the compound of interest and CL. 2+ It can be used as a probe to evaluate the binding of compounds to anionic lipids,

[13] but this method has obvious disadvantages: nonspecific Ca 2+ Binding to CL, lack of hydrophobic interaction with CL, and requirement for Ca 2+ electrode.

[0005] Other methods are based on intrinsic properties of the compound and include circular dichroism measurements

[14] or separation and quantification of unbound ligand.

[15]

[0006] Previously, 10-N-nonylacridine orange (NAO) has been used as a fluorescent probe for evaluating the binding of 3',6-dinonylacridine to anionic phospholipids,

[16] although NAO has significant disadvantages and, therefore, limited use as a probe for competition assays - the fluorescence intensity of NAO is relatively low and unstable due to its low solubility in aqueous media.

[0007] Therefore, a powerful method is highly desirable to allow for the rapid screening of compounds targeting CL and the quantitative characterization of binding affinity. Recently, we have claimed that acridinium salts with azetidine moieties as fluorescent dyes are superior to NAO in terms of photoluminescence quantum yield, stability and solubility.

[17]

[0008] We have surprisingly determined that certain 3,6-bis(azetidin-1-yl)-10-substituted acridin-10-ium salts are useful as fluorescent probes for assessing the binding affinity of organic and inorganic substances for cardiolipin.

[0009] Purpose of the Invention

[0010] It is an object of the present invention to provide an assay that can be used to characterize the binding affinity of organic and inorganic compounds to cardiolipin. Summary of the Invention

[0011] Aspects of the present invention relate to the development of an assay for quantifying the interaction of bioactive compounds with negatively charged phospholipids. In particular, 3,6-bis(azetidin-1-yl)-10-(3-(trimethylsilyl)propyl)acridin-10-ium iodide (I) is used as a fluorescent probe for determining the binding affinity of organic and inorganic compounds for CL.

[0012]

[0013] One aspect relates to quantifying the interaction of endogenous compounds (e.g., cytochrome c, calcium and magnesium ions) with CL. Another aspect of the invention relates to exploring the ability of xenobiotics (e.g., anthracyclines, anthracenediones, aminoglycosides, ammonium salts, and phosphonium salts) to bind to CL, an essential negatively charged phospholipid in the inner mitochondrial membrane and the bacterial cytoplasmic membrane, based on the fluorescence properties of Compound I. DETAILED DESCRIPTION

[0014] While searching for fluorescent probes to determine the binding affinity of a range of organic and inorganic compounds to CL, we unexpectedly discovered that 3,6-bis(azetidin-1-yl)-10-(3-(trimethylsilyl)propyl)acridin-10-ium iodide (I) exhibited suitable properties. Our discovery was surprising, as no fluorescence-based assays on the market allow characterization of compound binding to CL.

[0015] We state that the described assay allows the determination of the binding affinity of compounds of interest for CL contained within the membrane of lipid vesicles. This method has significant advantages over previously used approaches: it allows quantitative characterization of binding to CL, it uses CL in a membrane model (lipid vesicle) similar to natural membranes, the reaction conditions (buffer, pH, temperature) can be flexibly modified, and the assay is relatively rapid and simple, making it suitable for screening large numbers of compounds.

[0016] This method is very useful for exploring compounds and antibiotics targeting mitochondria, especially CL, and for predicting the potential mitochondrial toxicity of drugs.

[0017] Description of the assay :

[0018] Lipid vesicles may consist of CL and an auxiliary phospholipid (preferably, phosphatidylcholine), or may have a more complex composition comprising several phospholipids, provided that the preparation is stable in the medium used. The CL content in liposomal preparations may range from 0.5 mol% to 50 mol% of the total phospholipids, thus covering the CL content present in natural membranes.

[18]

[0019] The assay can be performed in various buffer solutions, for example, HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, CAS No. 7365-45-9, >99%), TRIS (tris(hydroxymethyl)aminomethane), MES (2-(N-morpholino)ethanesulfonic acid, CAS No. 4432-31-9, >99%), ADA (2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid, CAS No. 26239-55-4, >99%), with a pH range of 6.0 to 8.0.

[0020] The final concentration of fluorescent probe I can range from 0.2 μM to 25 μM. The optimal incubation temperature is 37°C, as it represents physiological temperature and allows for shorter incubation times. Incubation of the compound with CL-containing liposomes can vary from 10 min to 1 h.

[0021] Materials needed:

[0022] A fluorometer capable of scanning emission intensity at the desired wavelength is required, as well as a plate shaker-thermostat. The assay is performed in a 96-well microplate for fluorescence-based assays. All buffers should be made from the highest quality reagents and ultrapure water (MilliQ, 18.2 MΩ·cm -1 , 25°C). In this assay, a 20 mM HEPES solution was used, and the pH was adjusted to 7.4 with a 0.1 M aqueous NaOH solution. In the assay, a stock solution of Compound I in ethanol or DMSO (dimethyl sulfoxide), a stock solution of lipid vesicles containing CL in 20 mM HEPES buffer, and a stock solution of the ligand in 20 mM HEPES buffer, ethanol, or DMSO were used.

[0023] Representative experimental protocol:

[0024] Before using the assay in different buffers and pH values, it is recommended to titrate CL-containing liposomes with I (purity >95%) in order to find the optimal CL:I molar ratio.

[0025] 1. Prepare stock solutions of I, lipid vesicles containing CL, and ligand in 20 mM HEPES buffer.

[0026] 2. Place a stock solution of CL-containing lipid vesicles (final CL concentration of 2.5 μM) in a 96-well microplate. Then titrate it with a stock solution of the ligand and gently mix by pipetting the solution up and down (approximately 5 times) with a micropipette. Also place triplicate CL-containing lipid vesicles without ligand (vehicle).

[0027] 3. Place the 96-well microplate in a plate shaker-incubator for 10 minutes (500 RPM, 37°C).

[0028] 4. Add an appropriate amount of I stock solution (final I concentration is 5 μM) to the wells (including the wells containing the vector) and gently mix by gently pipetting the solution up and down (about 5 times) with a micropipette;

[0029] 5. Incubate in a plate shaker-incubator for 15 minutes (500 RPM, 37°C);

[0030] 6. Measure fluorescence intensity at excitation and emission wavelengths of 497 and 529 nm, respectively;

[0031] 7. Normalize the obtained fluorescence intensity (0% - CL liposomes containing I without ligand (carrier), 100% - maximum effect of the ligand);

[0032] 8. Plot the normalized fluorescence intensity versus ligand concentration on a logarithmic scale;

[0033] 9. Fit the titration curve with an appropriate fitting model to calculate EC 50 value.

[0034] Example:

[0035] Representative examples of dose-effect curves obtained using this assay are listed below. This method allows the study of small proteins (e.g., cytochrome c), inorganic cations (e.g., Ca 2+ Mg 2+ NH 4+ ), and the binding affinities of different xenobiotics (such as aminoglycosides, anthracyclines, anthracenediones, ammonium and phosphonium cations) for CL.

[0036] Example 1. Cytochrome c from horse heart, Sigma-Aldrich, CAS No.: 9007-43-6;

[0037] Example 2. Ammonium chloride for molecular biology, suitable for cell culture, ≥99.5%, Sigma-Aldrich, CAS No.: 12125-02-9;

[0038] Example 3. Anhydrous calcium chloride, BioReagent, suitable for insect cell culture, suitable for plant cell culture, ≥96.0%, Sigma-Aldrich, CAS No.: 10043-52-4;

[0039] Example 4. Anhydrous magnesium chloride, ≥98%, Sigma-Aldrich, CAS No.: 7786-30-3;

[0040] Example 5. Doxorubicin hydrochloride (Doxorubicin), (8S,10S)-10-(((2R,4S,5R,6S)-4-amino-5-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione hydrochloride, ≥95%, Fluorochem, CAS No.: 25316-40-9;

[0041] Example 6. Daunorubicin hydrochloride, (8S,10S)-8-acetyl-10-(((2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-6,8,11-trihydroxy-1-methoxy-7,8,9,10-tetrahydronaphthacene-5,12-dione hydrochloride, >98.0%, Tokyo Chemical Industry, CAS No.: 23541-50-6;

[0042] Example 7. Mitoxantrone dihydrochloride, l,4-dihydroxy-5,8-bis((2-((2-hydroxyethyl)amino)ethyl)amino)anthracene-9,10-dione dihydrochloride, >97.0%, Tokyo Chemical Industry, CAS No.: 70476-82-3;

[0043] Example 8. Gentamycin sulfate, (2R,3S,4R,5R)-2-(((1S,2R,3R,4S,6R)-4,6-diamino-3-(((2R,3R,6S)-3-amino-6-(1-(methylamino)ethyl)tetrahydro-2H-pyran-2-yl)oxy)-2-hydroxycyclohexyl)oxy)-5-methyl-4-(methylamino)tetrahydro-2H-pyran-3,5-diol sulfate, USP grade, Fluorochem, CAS No. 1405-41-0;

[0044] Example 9. Apramycin sulfate, (2R,3R,4S,5S,6S)-5-amino-2-(((2R,3S,4R,4aR,6S,7R,8aS)-7-amino-6-(((lR,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl)oxy)-4-hydroxy-3-(methylamino)octahydropyrano[3,2-b]pyran-2-yl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol sulfate, Sigma-Aldrich, CAS No. 65710-07-8;

[0045] Example 10. Streptomycin sulfate for protein research, 2-[(1R,2R,3S,4R,5R,6S)-3-(diaminomethylimino)-4-[(2R,3R,4R,5S)-3-[(2S,3S,4S,5R,6S)-4,5-dihydroxy-6-(hydroxymethyl)-3-(methylamino)oxolan-2-yl]oxy-4-formyl-4-hydroxy-5-methyloxolan-2-yl]oxy-2,5,6-trihydroxycyclohexyl]guanidine, >95.0%, Tokyo Chemical Industry, CAS No.: 3810-74-0;

[0046] Example 11. Kanamycin sulfate from Streptomyces kanamyceticus, powder, BioReagent, suitable for cell culture, suitable for plant cell culture, 2-(aminomethyl)-6-[4,6-diamino-3-[4-amino-3,5-dihydroxy-6-(hydroxymethyl)oxathin-2-yl]oxy-2-hydroxycyclohexyl]-oxathin-3,4,5-triol sulfate, Sigma-Aldrich, CAS No. 25389-94-0;

[0047] Example 12. Tobramycin, (2S,3R,4S,5S,6R)-4-amino-2-{[(1S,2S,3R,4S,6R)-4,6-diamino-3-{[(2R,3R,5S,6R)-3-amino-6-(aminomethyl)-5-hydroxyoxan-2-yl]oxy}-2-hydroxycyclohexyl]oxy}-6-(hydroxymethyl)oxan-3,5-diol, Sigma-Aldrich, CAS No. 32986-56-4;

[0048] Example 13. Nonyltriphenylphosphonium bromide, >97.0%, CAS No. 60902-45-6. Prepared according to

[19] by treating triphenylphosphine with 1-bromononane in toluene.

[0049] Example 14. Dodecyltrimethylammonium chloride purum pure, ≥98.0% anhydrous basis, Sigma-Aldrich, CAS number: 112-00-5.

[0050] Figure 1 shows the concentration-effect curves obtained in a competition binding assay for the endogenous CL ligand, cytochrome c (A), and monovalent and divalent cations (B), using CL-DOPC liposomes (25:75 mol %) and I as a CL-specific fluorescent probe.

[0051] Figure 1A depicts the binding of cytochrome c to CL in 20 mM HEPES buffer at pH 7.4, using 2.5 μM CL and 5 μM I as the fluorescent probe. Specifically, the normalized fluorescence intensity of I was plotted against various cytochrome c concentrations on a logarithmic scale and fitted with a four-parameter logistic (4PL) curve to calculate the EC 50 value.

[0052] Figure 1B depicts the Ca2+ / Ca ... 2+ Mg 2+ and NH4 + The binding of CL was investigated using 2.5 μM CL and 5 μM I as fluorescent probes. In particular, the normalized fluorescence intensity of I was calculated for various Ca2+ Mg 2+ and NH4 + Concentrations were plotted on a logarithmic scale and fitted with a four-parameter logistic (4PL) or five-parameter logistic (5PL) curve to calculate the EC 50 Under the same experimental conditions, the EC 50 can be compared and related to the affinity of the compound for CL. 2+ With a ratio of Mg 2+ ≈30% lower EC 50 , and therefore has a higher affinity for CL. In turn, monovalent NH4 + The affinity of ions for Cl is greater than that for Ca 2+ ions 23 times lower.

[0053] Figure 2 shows the concentration-effect curves obtained in the competition binding assay of xenobiotics - mitochondria-targeted nonyltriphenylphosphonium bromide and dodecyltrimethylammonium chloride (A), anthracyclines and anthracenediones (B), and aminoglycosides (C) - to CL using CL-DOPC liposomes (25:75 mol%) and I as a CL-specific fluorescent probe.

[0054] Figure 2A depicts the binding of nonyltriphenylphosphonium bromide and dodecyltrimethylammonium chloride to CL in 20 mM HEPES buffer at pH 7.4, using 2.5 μM CL and 5 μM I as fluorescent probes. Specifically, the normalized fluorescence intensity of I was plotted against various concentrations of nonyltriphenylphosphonium bromide and dodecyltrimethylammonium chloride on a logarithmic scale and fitted with a four-parameter logistic (4PL) curve to calculate the EC 50 Under these conditions, the affinity of nonyltriphenylphosphonium bromide for CL is ≈30% higher than that of dodecyltrimethylammonium chloride.

[0055] Figure 2B shows the binding of daunorubicin, doxorubicin, and mitoxantrone to CL in 20 mM HEPES buffer at pH 7.4, using 2.5 μM CL and 5 μM I as fluorescent probes. In particular, the normalized fluorescence intensity of I was plotted against various daunorubicin, doxorubicin, and mitoxantrone concentrations on a logarithmic scale and fitted with a four-parameter logistic (4PL) curve to calculate the EC 50 Among this group of compounds, mitoxantrone has the highest affinity for CL, with an EC 50 The value was two times lower than that of daunorubicin, and the EC 50 The value is three times lower.

[0056] Figure 2C shows the binding of apramycin, gentamicin, streptomycin, and tobramycin to CL in 20 mM HEPES buffer at pH 7.4, using 2.5 μM CL and 5 μM I as fluorescent probes. In particular, the normalized fluorescence intensity of I was plotted on a logarithmic scale against various daunorubicin, doxorubicin, and mitoxantrone concentrations and fitted with a four-parameter logistic (4PL) or five-parameter logistic (5PL) curve to calculate the EC 50 Gentamicin has the highest affinity for CL among this group of aminoglycosides and the highest affinity among this group of xenobiotics, although its affinity for CL is ≈3-fold lower than that for its natural ligand, cytochrome c. In turn, streptomycin has the lowest affinity for CL among the aminoglycosides studied.

[0057] Table 1 lists the EC obtained by competition binding assays using 2.5 μM CL and 5 μM I as fluorescent probes in 20 mM HEPES buffer at pH 7.4 (physiological pH) and 6.8 (pH of the mitochondrial intermembrane space

[20] ). 50 Among the compounds studied, the natural CL ligand cytochrome c exhibited the lowest EC 50 value, and therefore has the highest binding affinity for CL. Inorganic cations exhibit much higher EC 50 values ​​(in the mM range). Mitoxantrone has an EC in the micromolar range 50 values, and its affinity to CL is higher than that of doxorubicin and daunorubicin.

[0058] All aminoglycosides studied bind to CL, but with varying affinities—gentamicin exhibits the highest affinity for CL, while streptomycin exhibits the lowest. Mitochondrial-targeting nonyltriphenylphosphonium bromide and dodecyltrimethylammonium chloride have EC values ​​in the micromolar range. 50 The binding affinity of CL was similar to that of the aminoglycosides or anthracyclines studied, although the affinity was lower than that of the aminoglycosides or anthracyclines studied. Nonyltriphenylphosphonium bromide and dodecyltrimethylammonium chloride were the only compounds studied whose interaction with CL was affected by changes in pH.

[0059] Table 1. EC obtained from competition binding assays 50 value

[0060]

[0061] Values ​​are shown as mean ± SD from 3 independent experiments.

[0062] *nt - not tested.

[0063] These values ​​and binding curves are particularly encouraging considering that the developed assay allows the study and comparison of the affinity of organic and inorganic compounds for cardiolipin.

[0064] While the invention has been described in terms of certain embodiments, the description is intended to be illustrative rather than restrictive. Modifications and variations are possible within the scope of the invention as defined in the following claims.

[0065] The procedures of the present invention can be used to determine the binding affinity of a substance for cardiolipin using a fluorescent probe (eg, Compound I in the assay kit).

[0066] Among them, a determination kit includes: a fluorescent probe, a suitable buffer, and a material containing cardiolipin, which is used to determine organic and inorganic compounds that bind to cardiolipin.

[0067] References:

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Claims

1. An assay for determining the binding of a bioactive substance to cardiolipin using 3,6-bis(azetidin-1-yl)-10-(3-(trimethylsilyl)propyl)acridin-10-ium iodide (I) as a fluorescent probe.

2. Use of compound I as claimed in claim 1 for quantifying the interaction between endogenous compounds and cardiolipin.

3. Use of the compound I as claimed in claim 1 for quantifying the interaction between metal cations and cardiolipin.

4. Use of the compound 1 as claimed in claim 1 for quantifying the interaction between xenobiotics and cardiolipin.

5. Use of compound I as claimed in claim 1 or 4 for the quantitative determination of the interaction of ammonium and phosphonium cations with cardiolipin.

6. Use of compound I as claimed in claim 1 or 4 for quantifying the interaction of anthracyclines and anthracenediones with cardiolipin.

7. Use of the compound I as claimed in claim 1 or 4 for quantifying the interaction between aminoglycosides and cardiolipin.

8. A kit comprising: Compound I for use in determining the binding of a biologically active substance to cardiolipin according to claim 1.

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