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US-12625142-B2 - Assay for measuring binding affinity for cardiolipin of biologically active compounds

US12625142B2US 12625142 B2US12625142 B2US 12625142B2US-12625142-B2

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.

Inventors

  • Pavels Arsenjans
  • Pavels Dimitrijevs

Assignees

  • LATVIAN INSTITUTE OF ORGANIC SYNTHESIS

Dates

Publication Date
20260512
Application Date
20210524
Priority Date
20200820

Claims (13)

  1. 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) measuring fluorescence intensity at an excitation wavelength of 497 nm and at an emission wavelength of 529 nm, respectively.
  2. 2 . The method of claim 1 , wherein the compound of interest is an endogenous compound.
  3. 3 . The method of claim 1 , wherein the compound of interest comprises a metal cation.
  4. 4 . The method of claim 1 , wherein the compound of interest is an xenobiotic.
  5. 5 . The method of claim 1 , wherein the compound of interest comprises an ammonium or phosphonium cation.
  6. 6 . The method of claim 1 , wherein the compound of interest is an anthracycline or anthracenedione.
  7. 7 . The method of claim 1 , wherein the compound of interest is an aminoglycoside.
  8. 8 . The method of claim 1 , wherein the solution further comprises a buffer.
  9. 9 . 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.
  10. 10 . The method of claim 1 , wherein the solution is incubated at 37° C.
  11. 11 . The method of claim 1 , further comprising determining an EC 50 value of the compound of interest after the measuring step.
  12. 12 . 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.
  13. 13 . A kit comprising: 3,6-di(azetidin-1-yl)-10-(3-(trimethylsilyl)propyl)acridin-10-ium iodide; a buffer; and a material containing cardiolipin.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a U.S. national phase filing under 35 U.S.C. § 371 of International Application No. PCT/IB2021/054479, filed May 24, 2021, which claims priority to Latvian Application No. LVP2020000056, filed Aug. 20, 2020. The International Application was published in English on Feb. 24, 2022 as WO 2022/038424 under PCT Article 21 (2). The contents of the prior applications are incorporated herein by reference in their entirety. FIELD OF THE INVENTION The invention relates to a method for the evaluation of binding affinity of biologically active substances with negatively charged membrane. In particular, the invention relates to the fluorescent acridinium salt utilization as a fluorescent probe for measuring binding affinity of organic or inorganic substances for cardiolipin. BACKGROUND OF THE INVENTION Cardiolipin (CL) is a unique phospholipid which is localized in the inner mitochondrial membrane (IMM) in eukaryotes and in the cytoplasmic membrane of prokaryotes. CL provides mitochondrial membrane stability, dynamics and is required for optimal activity of several mitochondrial membrane proteins (e.g. electron transport chain (ETC) complexes I, III, IV, ATP synthase, cytochrome c). [1] Also, CL stabilizes anaerobic respiratory complexes in bacteria. [2] Because of its distinctive structural properties and localization CL is an attractive pharmacological target for mitochondria specific therapies along with antibiotic treatment. [3-5] Moreover, mitochondrial toxicity of some drugs, e.g. anthracyclines and aminoglycosides, is attributed to their ability to interact with CL [6-8] leading to life-threatening side effects such as heart failure and decline of renal function. [9, 10] Consequently, exploring binding with CL is crucial for screening new, CL-targeted modulators of mitochondrial functions and antibiotics, as well as for evaluating drugs' potential to cause mitochondrial toxicity by interacting with CL. Previously, compounds binding with CL was detected by 1H and 13C NMR [11,12], but this method is time-consuming, semi quantitative and requires large amount of both compound of interest and CL. Also, Ca2+ can be used as a probe for evaluating compounds binding with anionic lipids, [13] but this method has significant disadvantages: non-specific Ca2+ binding to CL, lacking hydrophobic interaction with CL and requirement of Ca2+ electrode. Other methods are based on a compound's intrinsic properties and includes circular dichroism measurements [14] or separation and quantification of unbound ligand. [15] Earlier, 10-N-nonyl acridine orange (NAO) has been used as a fluorescent probe for the evaluation of 3′,6-dinonyl neamine binding to anionic phospholipids, [16] although NAO has significant drawbacks and, therefore, a limited use as a probe for competition assays—fluorescence intensity of NAO is relatively low and unstable due to low solubility in aqueous medium. Therefore, there is a great demand for a robust method that would allow rapid compound screening for CL targeting as well as binding affinity quantitative characterization. Recently we claimed acridinium salts bearing azetidine fragments as fluorescent dyes superior to NAO in photoluminescence quantum yield, stability and solubility. [17] THE PRESENT INVENTION We have surprisingly determined that certain 3,6-di(azetidin-1-yl)-10-substituted-acridin-10-ium salts can be used as fluorescent probe for evaluating binding affinity of organic and inorganic substances for cardiolipin. OBJECTS OF THE INVENTION It is an object of the present invention to provide an assay, useful for characterization of binding affinity of organic and inorganic compounds for cardiolipin. SUMMARY OF THE INVENTION Aspects of the invention relate to the development of an assay for the quantification of interaction of biologically active compounds with negatively charged phospholipids. In particular, utilization of 3,6-di(azetidin-1-yl)-10-(3-(trimethylsilyl)propyl)acridin-10-ium iodide (I) as a fluorescent probe for the determination of binding affinity of organic and inorganic compounds for CL. One aspect relates to the quantification of interaction of endogenous compounds (e.g. cytochrome c, calcium and magnesium ions) with CL. Another aspect of the invention relates to the exploring xenobiotics' ability to bind with CL (e.g. anthracyclins, anthracenediones, aminoglycosides, ammonium and phosphonium salts), which is an essential negatively charged phospholipid in the mitochondrial inner membrane and bacterial cytoplasmic membrane, based on fluorescent properties of compound I. DETAILED DESCRIPTION OF THE INVENTION Searching for the fluorescent probe for the determination of binding affinity of a series of organic and inorganic compounds with CL we unexpectedly discovered that 3,6-di(azetidin-1-yl)-10-(3-(trimethylsilyl)propyl)-acridin-10-ium iodide (I) exhibits appropriate properties. Our finding is astonishing, because there i