Methods for quantifying frizzled protein activity

Abstract

The present disclosure provides methods, compositions, and kits for measuring the activity of Frataxin (FXN) protein, e.g., FXN fusion protein. The present invention also provides methods for identifying compounds capable of modulating the activity of FXN protein, e.g., FXN fusion protein.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 940,214, filed November 25, 2019, the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list, which has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on November 24, 2020, named 130197-01020_SL.txt, and is 8,457 bytes in size.

[0005] introduction

[0006] Friedreich ataxia (FRDA) is a rare, inherited, progressive neurodegenerative disorder caused by mutations in the gene encoding ataxia protein (FXN). FXN is an essential and phylogenetically conserved protein present in cells throughout the body, with the highest levels found in the heart, spinal cord, liver, pancreas, and skeletal muscle. FXN is encoded in the cell nucleus, expressed in the cytoplasm, and imported into the mitochondria, where it is processed into its mature form. In humans, the full-length 210-amino acid hFXN (hFXN) is... 1-210 The protein (23.1 kDa) contains a typical mitochondrial targeting sequence (MTS) at its N-terminus. When introduced into the mitochondrial matrix, it is processed in a two-step cleavage by mitochondrial matrix processing peptidase (MPP). The resulting protein is a 130-amino acid, 14.2 kDa mature hFXN protein (hFXN... 81-210 ).

[0007] FXN fusion proteins are currently being investigated as an FXN replacement therapy to restore functional levels of FXN in the mitochondria of FRDA patients. FXN fusion proteins comprise an HIV-TAT peptide linked to the N-terminus of the full-length hFXN protein. The mechanism of action of FXN fusion proteins depends on the cell-penetrating ability of the HIV-TAT peptide to deliver the FXN fusion protein into cells, followed by processing into mature hFXN after translocation into the mitochondria. The FXN fusion protein is described in U.S. Provisional Patent Application No. 62 / 891,029 and U.S. Patent Application Serial No. 16 / 942,276, the entire contents of which are incorporated herein by reference.

[0008] To facilitate the development of FXN replacement therapies, there is a need for accurate and reliable methods to quantify the activity of FXN and FXN fusion proteins. For example, it is desirable to quantify the activity of various batches of FXN fusion proteins as a quality control metric for preparation methods used to produce FXN fusion proteins. It is also desirable to quantify the activity of various batches of FXN fusion proteins to identify appropriate storage conditions for preparing multiple batches of FXN fusion proteins. This invention addresses this unmet need.

[0009] Invention Summary

[0010] Therefore, in some aspects, this disclosure provides a method for measuring the activity of a conjugating protein (FXN), the method comprising: combining the FXN protein with a reducing agent and a reactive oxygen species (ROS) detection compound to produce an assay mixture, wherein the FXN protein promotes the oxidation of the reducing agent and the generation of ROS such that the amount of ROS generated is higher than the amount of ROS generated in an assay mixture containing the reducing agent and the ROS detection compound but not the FXN protein.

[0011] In some aspects, this disclosure provides a method for measuring the activity of a conjugating protein (FXN), the method comprising combining the FXN protein with a reducing agent and a reactive oxygen species (ROS) detection compound at a pH greater than about 7.4 to produce an assay mixture, wherein the FXN protein promotes the oxidation of the reducing agent and the generation of ROS such that the amount of ROS generated is higher than the amount of ROS generated in an assay mixture containing the reducing agent and the ROS detection compound but not the FXN protein.

[0012] In some embodiments, the method includes combining the FXN protein with a reducing agent and a reactive oxygen species (ROS) detection compound at a pH of about 7.9 or higher.

[0013] In some aspects, this disclosure provides a method for measuring the activity of a conjugating protein (FXN), the method comprising combining the FXN protein with a reducing agent and a reactive oxygen species (ROS) detection compound in the absence of metal ions to produce an assay mixture, wherein the FXN protein promotes the oxidation of the reducing agent and the generation of ROS such that the amount of ROS generated is higher than the amount of ROS generated in an assay mixture containing the reducing agent and the ROS detection compound but without the FXN protein.

[0014] In some aspects, this disclosure provides a method for measuring the activity of a conjugating protein (FXN), the method comprising combining the FXN protein with a reducing agent and a reactive oxygen species (ROS) detection compound in the absence of iron ions to produce an assay mixture, wherein the FXN protein promotes the oxidation of the reducing agent and the generation of ROS such that the amount of ROS generated is higher than the amount of ROS generated in an assay mixture containing the reducing agent and the ROS detection compound but without the FXN protein.

[0015] In some embodiments, the method further includes adjusting the pH of the mixture to a pH greater than about 7.4. In some embodiments, the method further includes adjusting the pH of the mixture to a pH of about 7.9 or higher.

[0016] In some embodiments, the method further includes adding metal ions to the assay mixture. In some embodiments, the metal ions are not Fe. 2+ In some implementations, the metal ion is selected from Mn. 2+ Fe 3+ Zn 2+ Cu 2+ and Mg 2+ In one implementation, the metal ion is Mn. 2+ In another embodiment, the metal is Fe. 3+ .

[0017] In some embodiments, the method further includes incubating the assay mixture. In some embodiments, the method further includes measuring the amount of ROS generated in the mixture. In some embodiments, the method further includes measuring the amount of ROS generated in the mixture over time.

[0018] In some embodiments, the method further includes measuring the amount of ROS generated at regular time intervals. In some embodiments, the method includes measuring the amount of ROS generated approximately every 0.1 seconds to approximately every 10 minutes, for example, approximately every 0.1 seconds to approximately every 2 seconds, approximately every 1 second to approximately every 10 seconds, approximately every 5 seconds to approximately every 60 seconds, approximately every 30 seconds to approximately every 5 minutes, or approximately every 2 minutes to approximately every 10 minutes, after incubation of the assay mixture has begun.

[0019] In some embodiments, the method further includes determining the maximum initial rate (Vi) of ROS production in the assay mixture. In some embodiments, the method further includes determining the time to reach the maximum initial rate (Ti). In some embodiments, the method further includes determining the unit activity of the FXN protein at a pH greater than about 7.4.

[0020] In some aspects, this disclosure provides a method for measuring the activity of a conjugating protein (FXN), the method comprising mixing the FXN protein with a reducing agent and a reactive oxygen species (ROS) detection compound to produce an assay mixture; and measuring the amount of ROS generated in the assay mixture over time, wherein the FXN protein promotes the oxidation of the reducing agent and the generation of ROS such that the amount of ROS generated is higher than the amount of ROS generated in an assay mixture containing the reducing agent and the ROS detection compound but not the FXN protein.

[0021] In some embodiments, the method further includes measuring the amount of ROS generated at regular time intervals. In some embodiments, the method includes measuring the amount of ROS generated about every 0.1 seconds to about 10 minutes, for example, about every 0.1 seconds to about 2 seconds, about 1 second to about 10 seconds, about 5 seconds to about 60 seconds, about 30 seconds to about 5 minutes, or about 2 minutes to about 10 minutes.

[0022] In some embodiments, the method further includes determining the maximum initial rate (Vi) of ROS production in the assay mixture. In some embodiments, the method further includes determining the time to reach the maximum initial rate (Ti). In some embodiments, the method further includes determining the unit activity of the FXN protein.

[0023] In some embodiments, the reducing agent is an organic compound. In some embodiments, the reducing agent is a reduced quinone compound. In some embodiments, the reduced quinone compound is selected from reduced benzoquinone compounds, reduced naphthoquinone compounds, and reduced anthraquinone compounds. In some embodiments, the reduced quinone compound is a reduced benzoquinone compound. In some embodiments, the reduced benzoquinone compound is hydroquinone.

[0024] In some embodiments, the ROS detection compound is a fluorescent probe or a chemiluminescent probe. In some embodiments, the ROS detection compound is a superoxide anion detection reagent or a hydrogen peroxide detection reagent. In some embodiments, the ROS detection compound is selected from coelenterazine; dihydroethidium; 2',7'-dichlorodihydrofluorescein (H2DCF), luciferin, luminol, cypridina fluorescein analog (CLA), cypridina fluorescein methoxy analog (MCAL), methylthiazolium diphenyl-tetrazole bromide (MTT), p-nitrotetrazole blue (NBT), RedoxSensor Red CC-1, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazole-5-carboxyaniline (XTT), and their structural variants and analogs. In some embodiments, the ROS detection compound is 2',7'-dichlorodihydrofluorescein diacetate (H2DCF).

[0025] In some embodiments, the reducing agent and the ROS detection compound are the same compound. In other embodiments, the reducing agent and the ROS detection compound are different compounds. In one embodiment, the reducing agent is dihydroquinone, and the ROS detection compound is 2',7'-dichlorodihydrofluorescein (H2DCF).

[0026] In some embodiments, the FXN protein is a conjugate protein (FXN). In some embodiments, the FXN protein is an FXN fusion protein comprising a full-length hFXN and a cell-penetrating peptide (CPP). In some embodiments, the CPP comprises a peptide selected from CPPs listed in the Cell-Penetrating Peptides CPPsite2.0 database. In some embodiments, the CPP comprises a peptide selected from HIV-TAT, glycopeptide, mestoparan, transport peptide, penetratin, polyarginine, VP22, and variants or derivatives thereof.

[0027] In some embodiments, CPP comprises HIV-TAT or its variants or derivatives. In one embodiment, the FXN fusion protein comprises SEQ ID NO: 12.

[0028] In some aspects, this disclosure provides a method for quality control of samples containing conjugation protein (FXN), the method comprising measuring the activity of the FXN protein according to the method of this disclosure.

[0029] In some aspects, this disclosure provides compositions for measuring the activity of FXN proteins, said compositions comprising FXN proteins, a reducing agent, and a reactive oxygen species (ROS) detection compound, wherein said composition has a pH greater than about 7.4. In some embodiments, the pH of the composition is about 7.9 or higher.

[0030] In some embodiments, the composition further comprises metal ions. In some embodiments, the metal ions are not Fe. 2+ In some implementations, the metal ion is selected from Mn. 2+ Fe 3+ Zn 2+ Cu 2+ and Mg 2+ In one embodiment, the metal ion is Fe. 3+ In another embodiment, the metal ion is Mn. 2+ .

[0031] In some aspects, this disclosure also provides compositions for measuring the activity of FXN proteins, said compositions comprising FXN proteins, a reducing agent, and a reactive oxygen species (ROS) detection compound, said compositions being substantially free of metal ions.

[0032] In some aspects, this disclosure also provides compositions for measuring the activity of FXN proteins, said compositions comprising FXN proteins, a reducing agent, and a reactive oxygen species (ROS) detection compound, said compositions being substantially free of iron ions.

[0033] In some embodiments, the pH of the composition is greater than about 7.4. In some embodiments, the pH of the composition is about 7.9 or higher.

[0034] In some embodiments, the reducing agent is an organic compound. In some embodiments, the reducing agent is a reduced quinone compound. In some embodiments, the reduced quinone compound is selected from reduced benzoquinone compounds, reduced naphthoquinone compounds, and reduced anthraquinone compounds. In some embodiments, the reduced quinone compound is a reduced benzoquinone compound. In some embodiments, the reduced benzoquinone compound is hydroquinone.

[0035] In some embodiments, the ROS detection compound is a fluorescent probe or a chemiluminescent probe. In some embodiments, the ROS detection compound is a superoxide anion detection reagent or a hydrogen peroxide detection reagent. In some embodiments, the ROS detection compound is selected from coelenterate; ethidium dihydrofluorescein; 2',7'-dichlorodihydrofluorescein (H2DCF), luciferin, luminol, luciferin analogue (CLA), luciferin methoxy analogue (MCAL), methylthiazolium diphenyl-tetrazole bromide (MTT), p-nitrotetrazole blue (NBT), RedoxSensor Red CC-1, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazole-5-carboxyaniline (XTT), and their structural variants and analogues. In some embodiments, the ROS detection compound is 2',7'-dichlorodihydrofluorescein diacetate (H2DCF).

[0036] In some embodiments, the ROS detection compound is the same compound. In some embodiments, the reducing agent and the ROS detection compound are different compounds. In one embodiment, the reducing agent is dihydroquinone and the ROS detection compound is 2',7'-dichlorodihydrofluorescein (H2DCF).

[0037] In some embodiments, the FXN protein is a conjugate protein (FXN). In some embodiments, the FXN protein is an FXN fusion protein comprising a full-length hFXN and a cell-penetrating peptide (CPP). In some embodiments, the CPP comprises a peptide selected from CPPs listed in the Cell-Penetrating Peptides CPPsite2.0 database. In some embodiments, the CPP comprises a peptide selected from HIV-TAT, glycopeptide, meliostepin, transport peptides, penetrantin, polyarginine, VP22, and variants or derivatives thereof.

[0038] In some embodiments, the CPP comprises HIV-TAT or its variants or derivatives. In some embodiments, the FXN fusion protein comprises SEQ ID NO: 12.

[0039] In some aspects, this disclosure also provides compositions comprising FXN protein, wherein the FXN protein exhibits a specific activity for hydroquinone reduction in the range of about 1000 mU / mg FXN protein to about 6500 mU / mg FXN protein. In some embodiments, the specific activity for hydroquinone reduction is in the range of about 1200 mU / mg to about 6000 mU / mg. In some embodiments, the specific activity is in the range of about 1400 mU / mg to about 5900 mU / mg.

[0040] In some aspects, this disclosure also provides a kit for determining the activity of FXN proteins, the kit comprising a reducing agent, a ROS detection compound, and instructions for combining the reducing agent, the ROS detection compound, and the FXN protein to produce an assay mixture.

[0041] In some aspects, this disclosure also provides a kit for determining the activity of FXN protein, the kit comprising a reducing agent, a ROS detection compound, and a combination of the reducing agent, the ROS detection compound, and FXN protein to produce an assay mixture; and instructions for determining the Vi of FXN protein in the assay mixture.

[0042] In some embodiments, the kit further includes a metal ion compound. In some embodiments, the metal ion compound comprises a compound selected from Mn. 2+ Fe 3+ Zn 2+ Cu 2+ and Mg 2+ The ions. In one embodiment, the metal ion compound contains Fe. 3+ In another embodiment, the metal ion compound comprises Mn. 2+ .

[0043] In some embodiments, the reducing agent is an organic compound. In some embodiments, the reducing agent is a reduced quinone compound. In some embodiments, the reduced quinone compound is selected from reduced benzoquinone compounds, reduced naphthoquinone compounds, and reduced anthraquinone compounds. In some embodiments, the reduced quinone compound is a reduced benzoquinone compound. In some embodiments, the reduced benzoquinone compound is hydroquinone.

[0044] In some embodiments, the ROS detection compound is a fluorescent probe or a chemiluminescent probe. In some embodiments, the ROS detection compound is a superoxide anion detection reagent or a hydrogen peroxide detection reagent. In some embodiments, the ROS detection compound is selected from coelenterate; ethidium dihydrofluorescein; 2',7'-dichlorodihydrofluorescein (H2DCF), luciferin, luminol, luciferin analogue (CLA), luciferin methoxy analogue (MCAL), methylthiazolium diphenyl-tetrazole bromide (MTT), p-nitrotetrazole blue (NBT), RedoxSensor Red CC-1, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazole-5-carboxyaniline (XTT), and their structural variants and analogues. In some embodiments, the ROS detection compound is 2',7'-dichlorodihydrofluorescein diacetate (H2DCF).

[0045] In some embodiments, the reducing agent and the ROS detection compound are the same compound. In some embodiments, the reducing agent and the ROS detection compound are different compounds. In some embodiments, the reducing agent is dihydroquinone and the ROS detection compound is 2',7'-dichlorodihydrofluorescein (H2DCF).

[0046] In some aspects, this disclosure also provides a method for identifying compounds capable of modulating the activity of a concoction protein (FXN), the method comprising the steps of: (i) combining the FXN protein with a test compound, a reducing agent, and a reactive oxygen species (ROS) detection compound to produce an assay mixture, wherein the FXN protein promotes the oxidation of the reducing agent and the generation of ROS; (ii) measuring the amount of ROS generated in the presence of the test compound; (iii) comparing the amount of ROS generated in the presence of the test compound with the amount of ROS generated in the absence of the test compound; and (iv) identifying the test compound as a compound capable of modulating the activity of the FXN protein when the amount of ROS generated in the presence of the test compound differs from the amount of ROS generated in the absence of the test compound.

[0047] In some embodiments, the method further includes incubating the assay mixture. In some embodiments, step (ii) includes measuring the amount of ROS generated in the assay mixture over time. In some embodiments, step (ii) includes measuring the amount of ROS generated at regular time intervals. In some embodiments, step (ii) includes measuring the amount of ROS generated every 0.1 seconds to every 10 minutes, for example, about every 0.1 seconds to about every 2 seconds, about every 1 second to about every 10 seconds, about every 5 seconds to about every 60 seconds, about every 30 seconds to about every 5 minutes, or about every 2 minutes to about every 10 minutes.

[0048] In some embodiments, the method further includes determining the maximum initial rate (Vi) of ROS production in the assay mixture. In some embodiments, the method further includes determining the time to reach the maximum initial rate (Ti). In some embodiments, the method further includes determining the unit activity of the FXN protein.

[0049] In some embodiments, the method further includes the steps of: comparing the Vi of the FXN protein in the presence of a test compound with the Vi of the FXN protein in the absence of a test compound; and identifying the test compound as a compound capable of regulating the activity of the FXN protein when the Vi of the FXN protein in the presence of the test compound is different from the Vi of the FXN protein in the absence of the test compound.

[0050] In some aspects, this disclosure includes identifying compounds capable of modulating the activity of a concordia protein (FXN), the method comprising the steps of: (i) combining the FXN protein with a test compound, a reducing agent, and a reactive oxygen species (ROS) detection compound to produce an assay mixture, wherein the FXN protein promotes the oxidation of the reducing agent and the generation of ROS; (ii) measuring the amount of ROS generated in the presence of the test compound and determining the Vi of the FXN protein; (iii) comparing the Vi of the FXN protein in the presence of the test compound with the Vi of the FXN protein in the absence of the test compound; and (iv) identifying the test compound as a compound capable of modulating the activity of the FXN protein when comparing the Vi of the FXN protein in the presence of the test compound with the Vi of the FXN protein in the absence of the test compound.

[0051] In some embodiments, the method further includes adjusting the pH of the assay mixture to a pH greater than about 7.4. In some embodiments, the method further includes adjusting the pH of the assay mixture to a pH of about 7.9 or higher.

[0052] In some embodiments, the method further includes adding metal ions to the assay mixture. In some embodiments, the metal ions are selected from Mn. 2+ Fe 3+ Zn 2+ Cu 2+ and Mg 2+ In one implementation, the metal ion is Mn. 2+ In another embodiment, the metal ion is Fe. 3+ .

[0053] In some embodiments, the reducing agent is an organic compound. In some embodiments, the reducing agent is a reduced quinone compound. In some embodiments, the reduced quinone compound is selected from reduced benzoquinone compounds, reduced naphthoquinone compounds, and reduced anthraquinone compounds. In some embodiments, the reduced quinone compound is a reduced benzoquinone compound. In some embodiments, the reduced benzoquinone compound is hydroquinone.

[0054] In some embodiments, the ROS detection compound is a fluorescent probe or a chemiluminescent probe. In some embodiments, the ROS detection compound is a superoxide anion detection reagent or a hydrogen peroxide detection reagent. In some embodiments, the ROS detection compound is selected from coelenterate; ethidium dihydrofluorescein; 2',7'-dichlorodihydrofluorescein (H2DCF), luciferin, luminol, luciferin analogue (CLA), luciferin methoxy analogue (MCAL), methylthiazolium diphenyl-tetrazole bromide (MTT), p-nitrotetrazole blue (NBT), RedoxSensor Red CC-1, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazole-5-carboxyaniline (XTT), and their structural variants and analogues. In some embodiments, the ROS detection compound is 2',7'-dichlorodihydrofluorescein diacetate (H2DCF).

[0055] In some embodiments, the reducing agent and the ROS detection compound are the same compound. In other embodiments, the reducing agent and the ROS detection compound are different compounds. In one embodiment, the reducing agent is dihydroquinone and the ROS detection compound is 2',7'-dichlorodihydrofluorescein (H2DCF).

[0056] In some embodiments, the FXN protein is a conjugate protein (FXN). In some embodiments, the FXN protein is an FXN fusion protein comprising a full-length hFXN and a cell-penetrating peptide (CPP). In some embodiments, the CPP comprises a peptide selected from CPPs listed in the Cell-Penetrating Peptides CPPsite2.0 database. In some embodiments, the CPP comprises a peptide selected from HIV-TAT, glycopeptide, meliostepin, transport peptides, penetrantin, polyarginine, VP22, and variants or derivatives thereof.

[0057] In some embodiments, the CPP comprises HIV-TAT or its variants or derivatives. In some embodiments, the FXN fusion protein comprises SEQ ID NO: 12.

[0058] In some aspects, this disclosure also provides a composition comprising an FXN protein, wherein the activity of the FXN protein has been measured according to the methods of this disclosure.

[0059] In some aspects, this disclosure also provides a pharmaceutical composition comprising an FXN protein and a pharmaceutically acceptable excipient, wherein the activity of the FXN protein has been measured according to the methods of this disclosure.

[0060] In some aspects, this disclosure provides a method for preparing a pharmaceutical composition comprising an FXN protein, the method comprising measuring the activity of the FXN protein according to the method of this disclosure, and formulating the FXN protein to prepare the pharmaceutical composition.

[0061] In some embodiments, this disclosure provides a method for measuring the activity of a concordia protein (FXN), the method comprising mixing the FXN protein with a reducing agent and a reactive oxygen species (ROS) detection compound to produce an assay mixture, wherein the FXN protein promotes the oxidation of the reducing agent and the generation of ROS.

[0062] In some embodiments, the method further includes adding metal ions to the assay mixture. In some embodiments, the metal ions are selected from Mn. 2+ Fe 3+ Zn 2+ Cu 2+ and Mg 2+ In a further aspect, the metal ion is Fe. 2+ In another further aspect, the metal ion is Mn. 2+ .

[0063] In some embodiments, the method further includes incubating the assay mixture. In some embodiments, the method further includes measuring the amount of ROS generated. In some embodiments, the method further includes correlating the amount of ROS generated with the activity of the FXN protein, thereby determining the activity of the FXN protein.

[0064] In some aspects, the reducing agent is an organic compound. In some aspects, the reducing agent is a reduced quinone compound. In some aspects, the reduced quinone compound is selected from reduced benzoquinone compounds, reduced naphthoquinone compounds, and reduced anthraquinone compounds. In some embodiments, the reduced quinone compound is a reduced benzoquinone compound. In one embodiment, the reduced benzoquinone compound is hydroquinone.

[0065] In some aspects, the ROS detection compound is a fluorescent probe or a chemiluminescent probe. In some aspects, the ROS detection compound is a superoxide anion detection reagent or a hydrogen peroxide detection reagent. In some embodiments, the ROS detection compound is selected from coelenterate; ethidium dihydrogen fluorescein; 2',7'-dichlorodihydrofluorescein (H2DCF), luciferin, luminol, luciferin analogue (CLA), luciferin methoxy analogue (MCAL), methylthiazolium diphenyl-tetrazole bromide (MTT), p-nitrotetrazole blue (NBT), RedoxSensor Red CC-1, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazole-5-carboxyaniline (XTT), and structural variants and analogues thereof. In a particular embodiment, the ROS detection compound is 2',7'-dichlorodihydrofluorescein diacetate (H2DCF).

[0066] In some embodiments, the reducing agent and the ROS detection agent compound are the same compound. In other embodiments, the reducing agent and the ROS detection agent compound are different compounds.

[0067] In some implementations, the reducing agent is dihydroquinone and the ROS detection compound is 2',7'-dichlorodihydrofluorescein (H2DCF).

[0068] In some respects, FXN proteins are conjugate proteins (FXN). In other respects, FXN proteins are FXN fusion proteins comprising full-length hFXN and a cell-penetrating peptide (CPP).

[0069] In some embodiments, the CPP comprises a peptide selected from the CPPs listed in the Cell-Penetrating Peptides CPPsite 2.0 database. In some embodiments, the CPP contains a peptide selected from HIV-TAT, glycopeptide, melitoxin, transport peptide, penetrantin, polyarginine, VP22, and variants or derivatives thereof. In one aspect, the CPP contains HIV-TAT or its variants or derivatives.

[0070] In some implementations, the FXN fusion protein comprises or is composed of SEQ ID NO: 12.

[0071] In some embodiments, this disclosure provides a method for quality control of a sample containing a concordia protein (FXN), the method comprising measuring the activity of the FXN protein according to the method of this disclosure.

[0072] In some embodiments, the present invention provides a composition for measuring the activity of FXN protein, the composition comprising FXN protein, a reducing agent, and a reactive oxygen species (ROS) detection compound.

[0073] In some aspects, the composition further comprises metal ions. In some aspects, the metal ions are selected from Mn. 2+ Fe 3+ Zn 2+ Cu 2+ and Mg 2+ In a further aspect, the metal ion is Fe. 2+ In another further aspect, the metal ion is Mn. 2+ .

[0074] In some embodiments, the reducing agent is an organic compound. In some embodiments, the reducing agent is a reduced quinone compound. In some aspects, the reduced quinone compound is selected from reduced benzoquinone compounds, reduced naphthoquinone compounds, and reduced anthraquinone compounds.

[0075] In some respects, the reduced quinone compound is a reduced benzoquinone compound. In other respects, the reduced benzoquinone compound is a hydroquinone.

[0076] In one embodiment, the ROS detection compound is a fluorescent probe or a chemiluminescent probe. In one embodiment, the ROS detection compound is a superoxide anion detection reagent or a hydrogen peroxide detection reagent. In one embodiment, the ROS detection compound is selected from coelenterate; ethidium dihydrogen fluorescein; 2',7'-dichlorodihydrofluorescein (H2DCF), luciferin, luminol, luciferin analogue (CLA), luciferin methoxy analogue (MCAL), methylthiazolium diphenyl-tetrazole bromide (MTT), p-nitrotetrazole blue (NBT), RedoxSensor Red CC-1, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazole-5-carboxaniline (XTT), and structural variants and analogues thereof. In one embodiment, the ROS detection compound is 2',7'-dichlorodihydrofluorescein (H2DCF).

[0077] In some respects, the reducing agent and the ROS detection compound are the same compound. In other respects, the reducing agent and the ROS detection compound are different compounds. In one instance, the reducing agent is dihydroquinone and the ROS detection compound is 2',7'-dichlorodihydrofluorescein (H2DCF).

[0078] In some embodiments, the FXN protein is a conjugate protein (FXN). In some aspects, the FXN protein is an FXN fusion protein comprising a full-length hFXN and a cell-penetrating peptide (CPP). In some aspects, the CPP comprises a peptide selected from CPPs listed in the Cell-Penetrating Peptides CPPsite2.0 database. In some aspects, the CPP comprises a peptide selected from HIV-TAT, glycopeptide, meliantin, transport peptides, penetrantin, polyarginine, VP22, and variants or derivatives thereof. In one embodiment, the CPP comprises HIV-TAT or its variants or derivatives.

[0079] In one respect, the FXN fusion protein comprises or is composed of SEQ ID NO: 12.

[0080] In some embodiments, this disclosure provides compositions comprising FXN protein, wherein the FXN protein exhibits a specific activity for hydroquinone reduction in the range of about 1000 mU / mg FXN protein to about 6500 mU / mg FXN protein. In some embodiments, the specific activity for hydroquinone reduction is in the range of about 1200 mU / mg to about 6000 mU / mg. In some embodiments, the specific activity is in the range of about 1400 mU / mg to about 5900 mU / mg.

[0081] In some embodiments, this disclosure provides a method for identifying compounds capable of modulating the activity of a concordia protein (FXN), the method comprising the steps of: (i) combining the FXN protein with a test compound, a reducing agent, and a reactive oxygen species (ROS) detection compound to produce an assay mixture, wherein the FXN protein promotes the oxidation of the reducing agent and the generation of ROS; (ii) measuring the amount of ROS generated in the presence of the test compound; (iii) comparing the amount of ROS generated in the presence of the test compound with the amount of ROS generated in the absence of the test compound; and (iv) identifying the test compound as a compound capable of modulating the activity of the FXN protein when the amount of ROS generated in the presence of the test compound differs from the amount of ROS generated in the absence of the test compound.

[0082] In some aspects, the method further includes incubating the assay mixture. In some aspects, the method further includes correlating the amount of ROS generated with the activity of the FXN protein to determine the activity of the FXN protein.

[0083] In some embodiments, the reducing agent is an organic compound. In some embodiments, the reducing agent is a reduced quinone compound. In some embodiments, the reduced quinone compound is selected from reduced benzoquinone compounds, reduced naphthoquinone compounds, and reduced anthraquinone compounds. In some aspects, the reduced quinone compound is a reduced benzoquinone compound. In some aspects, the reduced benzoquinone compound is hydroquinone.

[0084] In one embodiment, the ROS detection compound is a fluorescent probe or a chemiluminescent probe. In one embodiment, the ROS detection compound is a superoxide anion detection reagent or a hydrogen peroxide detection reagent. In one embodiment, the ROS detection compound is selected from coelenterate; ethidium dihydrogen fluorescein; 2',7'-dichlorodihydrofluorescein (H2DCF), luciferin, luminol, luciferin analogue (CLA), luciferin methoxy analogue (MCAL), methylthiazolium diphenyl-tetrazole bromide (MTT), p-nitrotetrazole blue (NBT), RedoxSensor Red CC-1, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazole-5-carboxaniline (XTT), and structural variants and analogues thereof. In one embodiment, the ROS detection compound is 2',7'-dichlorodihydrofluorescein (H2DCF).

[0085] In some respects, the reducing agent and the ROS detection agent compound are the same compound. In other respects, the reducing agent and the ROS detection agent compound are different compounds.

[0086] In one particular implementation, the reducing agent is dihydroquinone and the ROS detection compound is 2',7'-dichlorodihydrofluorescein (H2DCF).

[0087] In some implementations, the FXN protein is a concordant protein (FXN).

[0088] In some aspects, this disclosure also provides a composition comprising an FXN protein, wherein the activity of the FXN protein has been measured according to the methods provided in this disclosure.

[0089] In some aspects, this disclosure also provides a pharmaceutical composition comprising an FXN protein and a pharmaceutically acceptable excipient, wherein the activity of the FXN protein has been measured according to the methods provided in this disclosure.

[0090] In some aspects, this disclosure also provides a method for preparing a pharmaceutical composition comprising an FXN protein, the method comprising measuring the activity of the FXN protein according to the method provided in this disclosure, and formulating the FXN protein to prepare the pharmaceutical composition. Brief description of the attached diagram

[0092] The following description, with reference to the accompanying drawings listed after this paragraph, illustrates non-limiting examples of embodiments of this disclosure. Identical features appearing in more than one drawing are generally labeled with the same reference numerals in all the drawings in which they appear. Labels in the drawings that indicate icons representing given features of embodiments of this disclosure may be used to refer to those given features. The dimensions of the features shown in the drawings are chosen for convenience and clarity and are not necessarily shown to scale.

[0093] Figure 1 This diagram illustrates the kinetics of hydroquinone (HQ) reduction in the presence of the exemplary FXN fusion protein, measured by the amount of reactive oxygen species (ROS) generated over time. The amount of ROS is expressed as fluorescence and measured in arbitrary fluorescence units (AFU). Figure 1 Figure A illustrates an embodiment according to this disclosure, in the presence or absence of the FXN fusion protein, the generation of superoxide (O2·) through the oxidation of hydroquinone (HQ). - A diagram of the dynamics of ). Figure 1 Figure B shows an embodiment according to this disclosure, in the presence of the FXN fusion protein and metal ions such as Mn. 2+ and Fe 3+ A diagram illustrating the kinetics of superoxide formation under certain conditions. Figure 1 Figure C shows the presence of FXN fusion protein and Fe 3+ In the case of (gray square); in the presence of FXN fusion protein and the absence of Fe 3+ In the case of (gray circle); in the presence of FXN fusion protein, Fe 3+ Graphs of superoxide production kinetics in the presence of deferoxamine (black circles) and in the absence of FXN fusion protein (small black rectangles).

[0094] Figure 2 This is a standard curve of 2',7'-dichlorofluorescein (DCF), the oxidation product of the ROS detection compound 2',7'-dichlorofluorescein diacetate (H2DCFDA). According to embodiments of this disclosure, the standard curve is generated by plotting the fluorescence (AFU) exhibited by dichlorofluorescein (DCF) as a function of DCF concentration (in μM).

[0095] Figure 3 This shows the specific activity of different batches of exemplary FXN fusion protein, wherein the different batches were treated with different concentrations of FXN fusion protein and with NADH or NADPH. Figure 3Figure A is a bar chart showing the specific activity (in milliunits / mg) of four different batches of FXN fusion protein (batches 05, 18, 28 and 32) at different concentrations (0.03 μM, 0.1 μM, 0.3 μM and 1 μM) according to embodiments of the present disclosure. Figure 3 Figure B shows an implementation scheme according to this disclosure, in the presence of Fe 3+ Fe 3+ and NADH and Fe 3+ Bar graph showing the specific activity (mUnits / mg) of the FXN fusion protein in the case of NADPH.

[0096] Figure 4 The effect of storage temperature on the HQ-dependent superoxide generation activity of an exemplary FXN fusion protein is shown. Figure 4 Figure A is a graph showing the kinetics of superoxide generation in the presence of FXN fusion protein that has been stored at -60°C, 2-4°C and 25°C, according to an embodiment of the present disclosure, compared with a drug substance (DS) control. Figure 4 Figure B is a bar chart showing the maximum initial rate (Vi) of superoxide generation in the presence of FXN fusion protein that has been stored at -60°C, 2-4°C and 25°C, according to an embodiment of the present disclosure, compared with a drug substance (DS) control.

[0097] Figure 5 This demonstrates the effect of chemical treatment on the activity of an exemplary FXN fusion protein. Specifically, Figure 5 This is a bar graph showing the maximum initial rate (Vi) of superoxide generation in the presence of an oxidant and at elevated temperatures, in the presence of FXN fusion protein already stored at low and high pH, ​​according to an embodiment of this disclosure.

[0098] Figure 6 This is a graph showing the kinetics of HQ-dependent superoxide generation activity of an exemplary FXN fusion protein in the presence of different metal ions.

[0099] Figure 7 This is a graph showing the activity of the FXN fusion protein as an example, as a function of pH.

[0100] Figure 8 This is a graph showing experimental results demonstrating the performance of the FXN activity assay designed to validate the contents of this disclosure under conditions used for high-throughput screening (HTS). Specifically, Figure 8The figure shows the Vi values ​​of plates 1 and 2 determined for each test sample containing 10 μM FXN fusion protein (high control), 5 μM FXN fusion protein (low control), and 0 μM FXN fusion protein (blank control).

[0101] Figure 9 It is a flowchart showing the three steps involved in the HTS experiment and the related results.

[0102] Figure 10 This is a box plot showing the HTS initial screening results of compounds in DMSO, including compounds that increase FXN activity by more than 30%, compounds that decrease FXN activity by more than 30%, and compounds that cause changes in FXN activity of less than 30%.

[0103] Figure 11 This is a bar chart showing exemplary results of the confirmation screening of compound 1 and compound 2, wherein compound 1 passed the reverse screening due to lack of activity in the absence of the FXN fusion protein; and compound 2 failed to pass the reverse screening due to activity in the absence of the FXN fusion protein. Invention Details

[0105] Methods and compositions for measuring the activity of FXN proteins

[0106] This disclosure provides methods and compositions for measuring the activity of FXN proteins. This disclosure is based on the surprising finding that FXN proteins, such as FXN fusion proteins, including exemplary FXN fusion proteins having the amino acid sequence SEQ ID NO: 12, are capable of catalyzing reactive oxygen species (ROS), particularly superoxide (O2·4O3), in the presence of a reducing agent. - The production of ROS (e.g., superoxide) by FXN proteins is also based on the surprising finding that the kinetics of ROS production from FXN proteins in the presence of a reducing agent exhibit an enzymatic reaction. Therefore, without being bound by any particular theory, it is believed that the biological activity of FXN proteins involves enzymatically catalyzed ROS production.

[0107] Given previous reports in the art that FXN can inhibit ROS production, the discovery that FXN proteins can catalyze the production of ROS (e.g., superoxide) is particularly surprising. Specifically, Vyas et al., Human Molecular Genetics 2012, 21(6): 1230-1247 (hereinafter “Vyas”) described the FXN fusion protein TAT-FXN, which, when mixed with ferrous sulfate and hydroquinone (HQ), inhibits superoxide production by binding free ferrous and ferric ions and preventing them from participating in the active redox cycle that produces superoxide. However, the assay described in Vyas is a static endpoint assay that does not measure enzyme activity and therefore does not measure the enzymatic peroxidase activity of the FXN protein. Instead, in some embodiments, the methods of this disclosure for measuring the activity of the FXN protein involve kinetic measurements of ROS production. In some embodiments, the methods of this disclosure for measuring the activity of the FXN protein further include identifying enzyme parameters associated with the ROS-producing activity of the FXN protein.

[0108] Therefore, in some embodiments, this disclosure provides a method for activating FXN proteins, comprising combining the FXN protein with a reducing agent and a reactive oxygen species (ROS) detection compound to produce an assay mixture, wherein the FXN protein promotes the oxidation of the reducing agent and the generation of ROS, such that the amount of ROS generated is higher than the amount of ROS generated in an assay mixture containing the reducing agent and the ROS detection compound but not the FXN protein.

[0109] In some embodiments, this disclosure also provides a method for measuring the activity of an FXN protein, comprising combining the FXN protein with a reducing agent and a ROS detection agent compound to produce an assay mixture; and measuring the amount of ROS generated in the assay mixture over time, wherein the FXN protein promotes the oxidation of the reducing agent and the generation of ROS such that the amount of ROS generated is higher than the amount of ROS generated in an assay mixture containing the reducing agent and the ROS detection agent compound but without the FXN protein.

[0110] In some embodiments, this disclosure also provides a method for measuring the activity of an FXN protein, comprising combining the FXN protein with a reducing agent and a ROS detection agent compound to produce an assay mixture; and determining the maximum initial rate (Vi) of the FXN protein. As used herein, the term “maximum initial rate” or “Vi” refers to the fastest rate of change of any fluorescence unit (AFU) over time.

[0111] In some embodiments, the method of this disclosure further includes determining the time to reach the maximum initial velocity (Ti), i.e., the time taken for the measured mixture to reach the maximum initial velocity.

[0112] In some embodiments, the method of this disclosure further includes determining the unit activity of the FXN protein, such as the milli-unit activity of the FXN protein. As used herein, the term "milli-unit activity of the FXN protein" is defined as the amount of FXN protein required to catalyze the production of 1 micromolar of 2',7'-dichlorofluorescein (DCF) per minute at a pH greater than about 7.4 (e.g., about 7.9).

[0113] The determination described in Vyas requires Fe 2+ The presence of metal ions is not required for the ROS generation activity of FXN proteins. Conversely, the method for measuring FXN protein activity provided in this disclosure can be performed in the absence or presence of metal ions. It has been surprisingly found that the ROS generation activity of FXN proteins does not require the presence of metal ions, but metal ions (e.g., Fe) can be present. 3+ and Mn 2+ Enhancing the ROS-generating activity of FXN proteins. Without being bound by any specific theory, it is believed that metal ions can bind to FXN proteins and stabilize their conformation, thereby leading to enhanced ROS-generating activity.

[0114] Therefore, in some embodiments, this disclosure provides a method for measuring the activity of an FXN protein, comprising combining the FXN protein with a reducing agent and a reactive oxygen species (ROS) detection compound in the absence of metal ions to produce an assay mixture, wherein the FXN protein promotes the oxidation of the reducing agent and the generation of ROS, such that the amount of ROS generated is higher than the amount of ROS generated in an assay mixture containing the reducing agent and the ROS detection compound but without the FXN protein. In some embodiments, this disclosure also provides a method for measuring the activity of an FXN protein, comprising combining the FXN protein with a reducing agent and a reactive oxygen species (ROS) detection compound in the absence of iron ions to produce an assay mixture, wherein the FXN protein promotes the oxidation of the reducing agent and the generation of ROS, such that the amount of ROS generated is higher than the amount of ROS generated in an assay mixture containing the reducing agent and the ROS detection compound but without the FXN protein. In some embodiments, the iron ion may be Fe. 2+ and / or Fe 3+ .

[0115] Therefore, in some embodiments, this disclosure provides a method for measuring the activity of an FXN protein, comprising combining the FXN protein with a reducing agent and a reactive oxygen species (ROS) detection compound to produce an assay mixture, wherein the FXN protein promotes the oxidation of the reducing agent and the generation of ROS, such that the amount of ROS generated is higher than the amount of ROS generated in an assay mixture containing the reducing agent and the ROS detection compound but without the FXN protein, and wherein the oxidation of the reducing agent and the generation of ROS can be carried out in the absence of metal ions. Therefore, in some embodiments, this disclosure also provides a method for measuring the activity of an FXN protein, comprising combining the FXN protein with a reducing agent and a reactive oxygen species (ROS) detection compound to produce an assay mixture, wherein the FXN protein promotes the oxidation of the reducing agent and the generation of ROS, such that the amount of ROS generated is higher than the amount of ROS generated in an assay mixture containing the reducing agent and the ROS detection compound but without the FXN protein, and wherein the oxidation of the reducing agent and the generation of ROS can be carried out in the absence of iron ions. In some embodiments, the iron ions may be Fe. 2+ and / or Fe 3+ .

[0116] As used herein, the term "in the absence of metal ions" refers to the absence of the addition of metal ions, such as Fe, to the test mixture. 3+ or Mg 2+ The activity of FXN proteins is measured under certain conditions. In some embodiments, this term includes measuring the activity of FXN proteins in a assay mixture that does not contain added metal ions but may still contain trace amounts of metal ions. In some embodiments, the metal ion may be Mn. 2+ Fe 3+ Fe 2+ Zn 2+ Cu 2+ and Mg 2+ One or more of them.

[0117] As used herein, the term "in the absence of iron ions" refers to the absence of the addition of iron ions, such as Fe, to the test mixture. 2+ Fe 3+ or Mn 2+ The activity of FXN proteins is measured under certain conditions. In some embodiments, this term includes measuring the activity of FXN proteins in an assay mixture that does not contain added iron ions but may still contain trace amounts of iron ions. In some embodiments, the assay mixture may contain trace amounts of iron ions, such as Fe. 2+ and / or Fe 3+ Ions and / or Mn2 + In some embodiments, the term "trace metal ions" or "trace metal ions" refers to very small amounts of metal ions, such as less than about 0.2 parts per million parts (ppm), for example less than about 0.1 ppm, less than about 0.05 ppm, or less than about 0.01 ppm, or less than about 0.05-0.2 ppm. In some embodiments, the term "trace metal ions" or "trace metal ions" refers to metal ions present in a concentration of less than about 100 μg per liter of a measured mixture. In some embodiments, the metal ion may be Mn. 2+ Fe 3+ Fe 2 + Zn 2+ Cu 2+ and Mg 2+ One or more of them.

[0118] In some embodiments, this disclosure also provides compositions for measuring the activity of FXN proteins, said compositions comprising FXN protein, a reducing agent, and a reactive oxygen species (ROS) detection compound, wherein said compositions are substantially free of metal ions. In some embodiments, this disclosure provides compositions for measuring the activity of FXN proteins, comprising FXN protein, a reducing agent, and a reactive oxygen species (ROS) detection compound, wherein said compositions are substantially free of iron ions.

[0119] As used herein, the term "substantially free of metal ions" refers to a composition in which no metal ions have been added. In some embodiments, a substantially metal-ion-free composition may contain trace amounts of metal ions. Similarly, as used herein, the term "substantially free of iron ions" refers to a composition in which no iron ions have been added. In some embodiments, a substantially metal-ion-free composition may contain trace amounts of iron ions, such as Fe. 2+ and / or Fe 3+ .

[0120] The assay described in Vyas is performed in PBS, which typically has a pH of approximately 7.4. Contrary to the assay described in Vyas, it has been surprisingly found that the activity of the FXN protein, as measured using the method of this disclosure, is low or absent at a pH of approximately 7.4. As described in Example 7 and... Figure 7 As shown, the activity of the FXN protein is pH-dependent, increasing from a low level or absence at approximately 7.4 to a peak at approximately 8.5. Without being bound by any particular theory, it is believed that the increased activity of the FXN protein at pH greater than approximately 7.4, compared to its activity at approximately 7.4, reflects the fact that FXN is a mitochondrial protein that functions within the mitochondrial matrix at approximately 7.8 pH.

[0121] Therefore, in some embodiments, this disclosure provides a method for measuring the activity of a synergist protein (FXN), the method comprising combining the FXN protein with a reducing agent and a reactive oxygen species (ROS) detection compound at a pH greater than about 7.4 to produce an assay mixture, wherein the FXN protein promotes the oxidation of the reducing agent and the generation of ROS, such that the amount of ROS generated is higher than the amount of ROS generated in an assay mixture containing the reducing agent and the ROS detection compound but not the FXN protein. In some embodiments, the FXN protein may be combined with the reducing agent and the ROS detection compound at a pH greater than about 7.5, greater than about 7.6, greater than about 7.7, greater than about 7.8, greater than about 7.9, greater than about 8.0, greater than about 8.1, greater than about 8.2, greater than about 8.3, greater than about 8.4, greater than about 8.5, greater than about 8.6, greater than about 8.7, greater than about 8.8, greater than about 8.9, or greater than about 9.0. In one embodiment, the pH may be greater than about 7.9.

[0122] In some embodiments, the FXN protein can be combined with a reducing agent and a ROS detection agent compound at a pH of about 7.5 or higher, about 7.6 or higher, about 7.7 or higher, about 7.8 or higher, about 7.9 or higher, about 8.0 or higher, about 8.1 or higher, about 8.2 or higher, about 8.3 or higher, about 8.4 or higher, about 8.5 or higher, about 8.6 or higher, about 8.7 or higher, about 8.8 or higher, about 8.9 or higher, or about 9.0 or higher. In one embodiment, the pH may be about 7.9 or higher.

[0123] In some embodiments, the FXN protein can be combined with reducing agents and ROS detection compounds at pH values ​​of about 7.4 to about 8.0, about 7.6 to about 8.3, about 7.7 to about 8.4, about 7.9 to about 8.7, about 8.0 to about 8.5, about 8.2 to 8.7, about 8 to about 9, or about 8.5 to about 9.

[0124] In some embodiments, this disclosure also provides compositions for measuring the activity of FXN proteins, comprising FXN proteins, a reducing agent, and a reactive oxygen species (ROS) detection compound, wherein the pH of said composition is greater than about 7.4, for example greater than about 7.5, greater than about 7.6, greater than about 7.7, greater than about 7.8, greater than about 7.9, greater than about 8.0, greater than about 8.1, greater than about 8.2, greater than about 8.3, greater than about 8.4, greater than about 8.5, greater than about 8.6, greater than about 8.7, greater than about 8.8, greater than about 8.9, or greater than about 9.0. In one embodiment, the pH may be greater than about 7.9.

[0125] In some embodiments, this disclosure provides compositions for measuring the activity of FXN proteins, comprising FXN protein, a reducing agent, and a reactive oxygen species (ROS) detection compound, wherein the pH of said composition is about 7.4 or higher, such as about 7.5 or higher, about 7.6 or higher, about 7.7 or higher, about 7.8 or higher, about 7.9 or higher, about 8.0 or higher, about 8.1 or higher, about 8.2 or higher, about 8.3 or higher, about 8.4 or higher, about 8.5 or higher, about 8.6 or higher, about 8.7 or higher, about 8.8 or higher, about 8.9 or higher, or about 9.0 or higher. In one embodiment, the pH may be about 7.9 or higher.

[0126] In some embodiments, this disclosure provides compositions for measuring the activity of FXN proteins, comprising FXN proteins, a reducing agent, and a reactive oxygen species (ROS) detection compound, wherein the pH of the composition is about 7.4 to about 8.0, about 7.6 to about 8.3, about 7.7 to about 8.4, about 7.9 to about 8.7, about 8.0 to about 8.5, about 8.2 to about 8.7, about 8 to about 9, or about 8.5 to about 9.

[0127] In some embodiments, the method for measuring the activity of FXN proteins provided in this disclosure includes combining the FXN protein with a reducing agent and a reactive oxygen species (ROS) detection compound to produce an assay mixture. In this mixture, the FXN protein promotes the oxidation of the reducing agent and the generation of ROS. The method may further include incubating the assay mixture for a period of time to allow ROS generation. The method may further include measuring the amount of ROS generated after incubation, for example, by detecting a signal generated by the ROS detection compound.

[0128] In some instances, the method for measuring the activity of FXN proteins provided in this disclosure includes adding metal ions to the assay mixture. The metal ions may be selected from Mn. 2+ Fe 3+ Zn 2+ Cu 2+ and Mg 2+ In a specific instance, the metal ion could be Fe. 3+ In another specific instance, the metal ion could be Mn. 2+ In some embodiments, the method of this disclosure may include adding more than one metal ion to the assay mixture. In one example, according to an embodiment of this disclosure, Fe can be added... 3+ and Mn 2+ All were added to the mixture to be measured.

[0129] In some instances, the FXN protein exhibits a specific activity for hydroquinone reduction ranging from about 1000 mU / mg FXN protein to about 6500 mU / mg FXN protein. In one embodiment, the FXN protein exhibits a specific activity for hydroquinone reduction ranging from about 1200 mU / mg to about 6000 mU / mg, and from about 1400 mU / mg to about 5900 mU / mg. The milliunits (mU) of the FXN protein are defined as the rate of initial assay achieved, V. i =1 The amount of FXN protein required.

[0130] FXN protein

[0131] This disclosure provides a method for measuring the activity of FXN proteins. As used herein, the terms "FXN protein" or "FXN protein" refer to any polypeptide containing FXN or a functional analogue, derivative, or fragment of FXN. FXN is an essential and phylogenetically conserved protein found in cells throughout the body, with the highest levels in the heart, spinal cord, liver, pancreas, and skeletal muscle. FXN is encoded in the cell nucleus, expressed in the cytoplasm, and imported into the mitochondria, where it is processed into its mature form.

[0132] In the context of this disclosure, the FXN included in "FXN protein" can be derived from any species, such as mammalian species like mice, cynomolgus monkeys, or humans. In one aspect, the FXN protein includes human FXN (hFXN). In humans, hFXN is a full-length hFXN of 210 amino acids (hFXN1-210, 23.1 kDa) containing a typical mitochondrial targeting sequence (MTS) at its N-terminus, which is processed in two steps by matrix mitochondrial processing peptidase (MPP) when introduced into the mitochondrial matrix. The resulting protein is a mature hFXN protein of 130 amino acids and 14.2 kDa (hFXN). 81-210 The sequences of full-length hFXN and mature hFXN are shown in Table 1 below.

[0133] Table 1. Sequences of full-length hFXN and mature hFXN.

[0134]

[0135] The full-length hFXN (SEQ ID NO: 1) contains mature hFXN (SEQ ID NO: 2) and a mitochondrial targeting sequence (MTS) with the amino acid sequence MWTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMNLRK (SEQ ID NO: 3).

[0136] In some instances, the FXN protein comprises hFXN, such as full-length hFXN (SEQ ID NO: 1) or mature hFXN (SEQ ID NO: 2). In some instances, the FXN protein comprises a functional fragment of hFXN. In some instances, the FXN protein comprises a derivative of hFXN. In some instances, the FXN protein comprises a functional analogue of hFXN.

[0137] In some instances, an FXN protein may be an FXN fusion protein. As used herein, the term "FXN fusion protein" refers to an artificial polypeptide containing FXN, such as full-length hFXN (SEQ ID NO: 1) or mature hFXN (SEQ ID NO: 2), or a functional analogue, derivative, or fragment of FXN, as well as other portions. Other portions that may be included in an FXN fusion protein may be proteins different from FXN, such as full-length proteins or protein fragments. In some instances, an FXN fusion protein may contain FXN, such as full-length hFXN (SEQ ID NO: 1) or mature hFXN (SEQ ID NO: 2), and a cell-penetrating peptide (CPP) as an additional portion.

[0138] As used herein, the term "cell-penetrating peptide" or "CPP" refers to a short peptide sequence typically between 5 and 30 amino acids in length that facilitates cellular uptake of various molecular cargoes such as proteins. In the context of this disclosure, the CPP present in the FXN fusion protein facilitates the delivery of the FXN fusion protein to cells, such as recipient cells. Once inside the cell, the FXN fusion protein can be processed by cellular machinery to remove the CPP and the FXN fusion protein itself.

[0139] CPPs can be polycationic, meaning they have an amino acid composition containing high relative abundance of positively charged amino acids such as lysine or arginine. CPPs can also be amphoteric, meaning they have a sequence containing alternating patterns of polar / charged amino acids and nonpolar, hydrophobic amino acids. CPPs can also be hydrophobic, meaning they contain only nonpolar residues with low net charge, or hydrophobic amino acid groups that are crucial for cellular uptake.

[0140] The CPP that can be included in the FXN fusion protein useful in the context of this invention can be any CPP known to those skilled in the art. For example, the CPP can be any CPP listed in the Cell-Penetrating Peptides CPPsite2.0 database, the entire contents of which are incorporated herein by reference. For example, the CPP used in this invention can be a cell-penetrating peptide derived from HIV trans-activators (HIV-TAT), glycopeptide, melipotyl venom peptide, transport peptide, penetrantin, polyarginine, or VP22. In some embodiments, the CPP can include a TAT protein domain comprising amino acids 47-57 of the full-length 86-amino acid HIV-TAT protein (of which the 11-amino acid peptide may also be referred to herein as "HIV-TAT"; SEQ ID NO: 4). In one embodiment, the CPP consists of HIV-TAT (SEQ ID NO: 4). In some embodiments, the CPP comprises amino acids 47-57 of the full-length HIV-TAT protein, consisting of 86 amino acids, with a methionine residue added at the amino terminus for initiation (12AA; "HIV-TAT+M"): MYGRKKRRQRRR (SEQ ID NO: 5). Table 2 below lists the amino acid sequences of exemplary CPPs.

[0141] Table 2. Exemplary CPPs and Corresponding Sequences

[0142]

[0143] In some respects, the FXN fusion protein may comprise a full-length hFXN, such as SEQ ID NO: 1, or an amino acid sequence having at least 85%, such as at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the full-length hFXN sequence (such as SEQ ID NO: 1 listed in Table 1).

[0144] In some respects, the FXN fusion protein may comprise a mature hFXN, such as SEQ ID NO: 2, or an amino acid sequence having at least 85%, such as at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 92%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 200% sequence identity with the sequence of the mature hFXN (such as SEQ ID NO: 2 listed in Table 1).

[0145] In some respects, the FXN fusion protein may contain an MTS, such as SEQ ID NO: 3, or an amino acid sequence having at least 85%, such as at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence of the MTS (e.g., SEQ ID NO: 3).

[0146] In some respects, the FXN fusion protein may contain HIV-TAT as a CPP, such as SEQ ID NO: 4, or an amino acid sequence having at least 85%, such as at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with HIV-TAT (e.g., SEQ ID NO: 4). In some respects, the FXN fusion protein may contain HIV-TAT as a CPP, wherein a methionine is added at the amino terminus for initiation, such as SEQ ID NO: 5, or an amino acid sequence having at least 85%, such as at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 5.

[0147] In some instances, the CPP contained in the FXN fusion protein is HIV-TAT (SEQ ID NO: 4). In some embodiments, the FXN fusion protein may comprise a full-length FXN, such as SEQ ID NO: 1, and HIV-TAT, such as SEQ ID NO: 4, as the CPP. In some embodiments, the FXN fusion protein may comprise a mature FXN (e.g., SEQ ID NO: 2), an MTS (e.g., SEQ ID NO: 3), and HIV-TAT (e.g., SEQ ID NO: 4) as the CPP.

[0148] In some embodiments, in the FXN fusion protein of this disclosure, the CPP may be fused to an FXN (e.g., a full-length FXN) or to an MT via a linker to form a single polypeptide chain. In some embodiments, the linker may contain the amino acid sequence GG.

[0149] In some implementations, the FXN fusion protein comprises the following amino acid sequence (224 amino acids):

[0150] MYGRKKRRQRRRGGMWTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMNLRKSGTLGHPGSLDETTYERLAEETLDSLAEFFEDLADKPYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSPSSGPKRYDWTGKNWVYSHDGVSLHELLAAELTKALKTKLDLSSLAYSGKDA (SEQ ID NO: 12). In some embodiments, the FXN fusion protein consists of the amino acid sequence of SEQ ID NO: 12. FXN fusion proteins comprising or consisting of SEQ ID NO: 12 are further described in U.S. Provisional Patent Application No. 62 / 891,029 and U.S. Patent Application Serial No. 16 / 942,276, the entire contents of which are incorporated herein by reference.

[0151] In some embodiments, the methods of this disclosure can be used to measure the activity of FXN proteins (e.g., FXN fusion proteins), which may comprise FXN or functional analogs, derivatives, or fragments of FXN. As used herein, the term "derivative" encompasses an amino acid sequence (peptide) that differs from a peptide (e.g., SEQ ID NO. 1-3) specifically defined in this disclosure by the insertion, deletion, substitution, or modification of amino acids that substantially does not alter the activity of the original peptide. It should be understood that, as used herein, the terms "insertion," "deletion," or "substitution" respectively encompass 1 to 50 amino acid residues of the peptide, such as 1 to 5 amino acid residues, 1 to 10 amino acid residues, 5 to 15 amino acid residues, 10 to 20 amino acid residues, 25 to 40 amino acid residues, or 30 to 50 amino acid residues. More specifically, an insertion, deletion, or substitution can be any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. It should be noted that insertion, deletion, or substitution can occur at any position of the modified peptide, and either at its N' or C' terminus.

[0152] In some embodiments, the amino acid sequence of the FXN protein within the context of this disclosure may differ from the amino acid sequence of naturally occurring FXN (e.g., hFXN). For example, the FXN protein may include conserved amino acid substitutions, i.e., substitutions with structurally similar amino acids. For example, structurally similar amino acids include: (isoleucine (I), leucine (L), and valine (V)); (phenylalanine (F) and tyrosine (Y)); (lysine (K) and arginine (R)); (glutamine (Q) and asparagine (N)); (aspartic acid (D) and glutamic acid (E)); and (glycine (G) and alanine (A)).

[0153] As used herein, the term "derivative" encompasses homologs, variants, and analogs of the original polypeptide (e.g., FXN), as well as covalent modifications of the original polypeptide. Derivatives, variants, and analogs of FXN will have substantially the same biological activity as their natural form.

[0154] reducing agent

[0155] The methods provided in this disclosure include combining FXN proteins with reducing agents and ROS detection reagent compounds to produce an assay mixture. As used herein, the term "reducing agent" refers to any reducing agent that, when combined with FXN proteins, results in the generation of ROS (e.g., superoxide). In some instances, the reducing agent may be an organic compound. In further instances, the organic compound may be a reduced quinone compound.

[0156] As used herein, the term "quinone compound" refers to a compound having a fully conjugated cyclic diketone structure derived from aromatic compounds (including polycyclic and heterocyclic analogs) by converting an even number of -CH= groups to -C(=O)- groups, while the double bonds undergo any necessary double bond rearrangement. For example, a quinone compound may contain an aromatic group having an even number of -C(=O)- groups. In some aspects, the aromatic group of a quinone compound may contain portions having one or more, such as two, three, four, five, or six or more conjugated rings. For example, a quinone compound may be a benzoquinone compound, i.e., a compound containing a benzoquinone having a single aromatic ring. In another example, a quinone compound may be a naphthoquinone compound, i.e., a compound containing a naphthoquinone having two conjugated aromatic rings. In yet another example, a quinone compound may be an anthraquinone compound, i.e., a compound containing anthraquinone having three aromatic rings.

[0157] Quinone compounds containing a -C(=O)- group are in their oxidized form. In some embodiments, the quinone compound may be a reduced quinone compound, wherein the -C(=O)- group has been reduced to a -C-OH group. Exemplary quinone compounds in oxidized and reduced forms are shown in Table 3 below.

[0158] Table 3. Exemplary quinone compounds

[0159]

[0160] In one instance, the reducing agent used in the methods of this disclosure is a reduced quinone compound, such as a reduced benzoquinone compound, a reduced naphthoquinone compound, or a reduced anthraquinone compound. In another instance, the reducing agent is a reduced benzoquinone compound, such as hydroquinone having the following structure:

[0161] Hydroquinone.

[0162] ROS detection reagent compounds

[0163] The method provided in this disclosure includes mixing FXN protein with a reducing agent and a ROS detection compound to produce an assay mixture. As used herein, the term "ROS detection compound" refers to any compound capable of generating a detectable signal in response to ROS (e.g., superoxide) generated in the presence of FXN protein and the reducing agent. In some instances, the ROS detection compound may be a fluorescent probe or a chemiluminescent probe. In some instances, the ROS detection compound may be a superoxide detection reagent, i.e., a reagent capable of generating a signal in response to superoxide, or a hydrogen peroxide detection reagent, i.e., a reagent capable of generating a signal in response to hydrogen peroxide. Exemplary ROS detection compounds, such as superoxide detection reagents or hydrogen peroxide detection reagents, can be found, for example, in the ThermoFisher Scientific catalogue. In some embodiments, the superoxide anion detection reagent may be selected from the reagents shown in Table 4 below and their structural analogs or variants.

[0164] Table 4. Exemplary ROS detection reagent compounds

[0165]

[0166]

[0167]

[0168] In one particular embodiment, the ROS detection compound is 2',7'-dichlorodihydrofluorescein (H2DCF). H2DCF can be obtained, for example, by treating commercially available 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) with an alkali to remove the acetyl group (see, for example, Example 1 of this disclosure). In the presence of ROS, H2DCF is oxidized to 2'-7'-dichlorofluorescein (DCF), which is highly fluorescent and can be used for the quantification of ROS. The reporting wavelengths used to measure the fluorescence of DCF are 498 nm (excitation) and 522 nm (emission).

[0169] In some instances, the ROS detection compound can be an antioxidant compound or a ROS scavenger compound. The ROS detection compound can also be a hydrogen peroxide probe. For example, certain fluorescent substrates produce a strong fluorescent product in the presence of hydrogen peroxide (H₂O₂) and horseradish peroxidase (HRP), which can be measured. Such fluorescent substrates can also be used in the methods of this disclosure to measure the amount of ROS generated due to FXN protein activity.

[0170] In one embodiment, the ROS detection compound can be a hydrogen peroxide probe, such as peroxide green 1 (PG1) with the structure 9-(4-methoxy-2-methylphenyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)-3H-xanthone-3-one, as shown below:

[0171] Peroxy Green 1 (PG1).

[0172] In another embodiment, the ROS detection compound can be a hydrogen peroxide probe, such as peroxide deep red 1 (PC1) of the structure 7-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)-3H-phenoxazine-3-one, as shown below:

[0173] Peroxy-Crimson 1 (PC1).

[0174] PG1 and PC1 are borate-based hydrogen peroxide probes with visible excitation and emission wavelengths. Reaction with hydrogen peroxide resulted in a 10-fold and 40-fold increase in fluorescence for PG1 and PC1, respectively. PG1 exhibited an excitation wavelength of 460 nm and a maximum emission wavelength of 510 nm, while PC1 showed improved properties with a redshift excitation and a larger Stokes shift that reduced autofluorescence (excitation: 480 nm; emission: 584 nm).

[0175] In some instances, the ROS detection compound can be a hydrogen peroxide probe, such as homovanillic acid, which has the following structure:

[0176] High vanillic acid.

[0177] Homovanillic acid dimerizes when oxidized by horseradish peroxidase. As a monomer, homovanillic acid is non-fluorescent, but as a dimer, it has a peak excitation wavelength of 315 nm and an emission wavelength of 425 nm.

[0178] In some instances, ROS detection compound can be a protein, such as an engineered protein containing a redox-reactive domain capable of inducing conformational changes in response to ROS. For example, fluorescent proteins can be engineered to contain a redox-reactive domain and to change conformation in response to the redox activity of the redox-reactive domain. One such exemplary protein, called HyPer, consists of a yellow fluorescent protein (cpYFP) with a cyclic rearrangement inserted into the regulatory domain of the prokaryotic H2O2-sensor protein OxyR.

[0179] In some instances, the ROS detection compound may contain a marker, which may be an integral part of the ROS detection compound, such as in a DCF. Alternatively, the marker may be covalently linked to the ROS detection compound.

[0180] For example, the markers contained in the ROS detection reagent compound can be fluorescent markers, colorimetric markers, or luminescence-based markers. Fluorescence can be measured using a standard fluorometer; luminescence can be measured using a standard photometer; and colorimetric markers can be measured using a standard colorimeter.

[0181] In some instances, the reducing agent and the ROS detection compound can be the same compound. Such a compound can be oxidized by FXN proteins to produce ROS, such as superoxide, while simultaneously emitting a detectable signal in its oxidized form. One such exemplary compound is H2DCF.

[0182] In other instances, the reducing agent and the ROS detection compound can be different compounds. For example, the reducing agent can be a reduced quinone compound, such as hydroquinone, and the ROS detection compound can be H2DCF.

[0183] In this specification, unless otherwise stated, adjectives such as “substantially” and “about” that modify one or more features of embodiments of this disclosure are understood to mean that the condition or feature is limited to an operational tolerance acceptable for the intended application. Unless otherwise stated, the word “or” in the specification and claims is considered inclusive (having the meaning of and / or) rather than exclusive and indicates at least one or any combination of items combined therewith.

[0184] In the specification and claims of this application, each verb “comprising,” “including,” and “having,” and its variations, is used to indicate that one or more objects of the verb are not necessarily a complete list of components, elements, or parts of one or more subjects of the verb.

[0185] Kit for measuring FXN protein activity

[0186] This disclosure also provides kits for determining the activity of FXN proteins. Such kits may comprise a reducing agent, a ROS detection compound, and instructions for use. For example, the instructions for use may include instructions for combining the reducing agent, the ROS detection compound, and the FXN protein to produce an assay mixture. The instructions for use may further include instructions for determining Vi of the FXN protein in the assay mixture.

[0187] The kits provided in this disclosure can be used to determine the activity of any FXN protein as described herein. In one embodiment, the FXN protein may be a human FXN protein, such as comprising SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the FXN protein may be an FXN fusion protein, such as comprising or consisting of SEQ ID NO: 12.

[0188] In some embodiments, the kit may further comprise a metal ion compound, such as a salt. As used herein, the term "metal ion compound" refers to a compound that, when dissolved in aqueous solution, can be a source of metal ions. In some embodiments, the metal ion may be selected from Mn. 2+ Fe 3+ Zn 2+ Cu 2+ and Mg 2+ In one implementation, the metal ion is Fe. 3+ In another embodiment, the metal ion is Mn. 2+ In another embodiment, the metal ion is Zn. 2+ In another embodiment, the metal ion is Cu. 2+ In another embodiment, the metal ion is Mg. 2+ In one embodiment, the metal ion compound may be MgCl2.

[0189] In some embodiments, the reducing agent may be as described herein. In one embodiment, the reducing agent is hydroquinone.

[0190] In some embodiments, the ROS detection compound may be as described herein. In one embodiment, the ROS detection compound is 2',7'-dichlorodihydrofluorescein (H2DCF).

[0191] Methods for identifying compounds that can modulate FXN activity

[0192] This disclosure also provides a method for identifying compounds capable of modulating the activity of FXN proteins. The method includes measuring the activity of the FXN protein in the presence of a test compound, thereby identifying compounds capable of modulating the activity of the FXN protein.

[0193] In some embodiments, this disclosure provides a method for identifying compounds capable of modulating the activity of a FXN protein, comprising combining the FXN protein with a test compound, a reducing agent, and a reactive oxygen species (ROS) detection compound to produce an assay mixture, wherein the FXN protein promotes the oxidation of the reducing agent and the generation of ROS; and measuring the amount of ROS generated in the presence of the test compound. In some embodiments, the method further comprises determining the Vi of the FXN protein. In some embodiments, the method further comprises: comparing the Vi of the FXN protein in the presence of the test compound with the Vi of the FXN protein in the absence of the test compound; and identifying the test compound as a compound capable of modulating the activity of the FXN protein when the Vi of the FXN protein in the presence of the test compound differs from the Vi of the FXN protein in the absence of the test compound.

[0194] In some embodiments, the method further includes: comparing the Vi of the FXN protein in the presence of the test compound with the Vi of the FXN protein in the absence of the test compound, and identifying the test compound as a compound capable of increasing the activity of the FXN protein when the Vi of the FXN protein in the presence of the test compound is greater than the Vi of the FXN protein in the absence of the test compound.

[0195] In some embodiments, the method further includes: comparing the Vi of the FXN protein in the presence of the test compound with the Vi of the FXN protein in the absence of the test compound, and identifying the test compound as a compound capable of reducing the activity of the FXN protein when the Vi of the FXN protein in the presence of the test compound is lower than the Vi of the FXN protein in the absence of the test compound.

[0196] In some embodiments, the method further includes adjusting the pH of the assay mixture to a pH greater than or equal to about 7.4. In some embodiments, the method includes adjusting the pH of the assay mixture to a pH greater than or equal to about 7.9. In one embodiment, the method includes adjusting the pH of the assay mixture to about 8.0. In one embodiment, the method includes adjusting the pH of the assay mixture to a pH of about 7.4 to about 8.0, about 7.6 to about 8.3, about 7.7 to about 8.4, about 7.9 to about 8.7, about 8.0 to about 8.5, about 8.2 to about 8.7, about 8 to about 9, or about 8.5 to about 9.

[0197] In some embodiments, the method further includes adding metal ions, such as those selected from Mn, to the assay mixture. 2+ Fe 3+ Zn 2+ Cu 2+ and Mg 2+ The metal ion. In one embodiment, the metal ion is Mn. 2+ In another embodiment, the metal ion is Fe. 3+ In another embodiment, the metal ion is Zn. 2+ In another embodiment, the metal ion is Cu. 2+ In another embodiment, the metal ion is Mg. 2+ .

[0198] In some embodiments, the reducing agent may be as described herein. In one embodiment, the reducing agent is hydroquinone.

[0199] In some embodiments, the ROS detection compound may be as described herein. In one embodiment, the ROS detection compound is 2',7'-dichlorodihydrofluorescein (H2DCF).

[0200] The FXN protein can be any FXN protein as described herein. In one embodiment, the FXN protein can be a human FXN protein, such as comprising SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the FXN protein can be an FXN fusion protein, such as comprising or consisting of SEQ ID NO: 12.

[0201] As used herein, the term "compound capable of modulating the activity of the FXN protein" refers to a compound that, as measured by the methods of this disclosure, can cause an increase or decrease in the activity of the FXN protein. In one embodiment, the compound capable of modulating the activity of the FXN protein can cause an increase in the activity of the FXN protein. In another embodiment, the compound capable of modulating the activity of the FXN protein can cause a decrease in the activity of the FXN protein.

[0202] Exemplary methods for identifying compounds that can modulate the activity of FXN proteins may include, for example, high-throughput screening (HTS) of a compound library as described in Example 8 herein.

[0203] As used herein, the term “about” can allow for a certain degree of variation in a value or range (e.g., pH value) such that the value or range may be, for example, within 10%, 5%, 1%, or 0.1% of the limits of the value or range.

[0204] The description of embodiments of this disclosure in this application is provided by way of example and is not intended to limit the scope of this disclosure. The described embodiments include different features, and not all features are required in all embodiments. Some embodiments utilize only some features or possible combinations of features. Variations of the described embodiments of this disclosure, as well as embodiments including different combinations of features mentioned in the described embodiments, will be apparent to those skilled in the art. Example

[0205] Example 1. Scheme for hydroquinone (HQ) reduction assay

[0206] 1. Activation of H2DCFDA to H2DCF

[0207] a. Add 1 mL of 1 mM H2DCFDA (in DMSO) to 20 mL of 0.01 M NaOH working solution to generate an activation solution. Stir the solution in the dark at room temperature for 30 minutes.

[0208] b. Add 79 ml of 33 mM NaH2PO4 solution to the activation solution to obtain a final concentration of 10 μM H2DCF.

[0209] c. Activated H2DCFDA can be stored in 10 mL aliquots in the dark at 4°C for up to 2 weeks.

[0210] 2. Fluorescence reading

[0211] a. Before each reading, shake the porous plate containing the reaction mixture described in sections 3 to 5 below in a linear oscillator for three (3) seconds at a frequency of 731 cpm (2 mm).

[0212] b. Read the excitation and emission wavelengths of the plate every minute or as needed using a spectrometer with the following parameters set: excitation at 485 / 20°, emission at 528 / 20°, light from the top, reading height of 1 mm, and gain of 50.

[0213] 3. Preparation of HQ test plate

[0214] a. A 96-well polystyrene plate (black, flat-bottomed, with a non-bonding surface) is recommended for the assay. Equivalent alternatives may also be used.

[0215] Add to each hole:

[0216] i. 10 μL of 20X hydroquinone (to a final concentration of 20 μM)

[0217] ii. 10 μL of 20X Mn 2+ (Final concentration of 5 μM)

[0218] b. Add Tris buffer:

[0219] i.FXN protein sample well: 168μL

[0220] ii. DCF standard curve orifice: 170μL

[0221] Examples of HQ test plates are detailed in Table 5 below.

[0222] Table 5. Test plates used for HQ determination

[0223] Test plate FXN protein comparison Testing FXN protein batches DCF Standard Curve A 3μM 3μM 1μM B 1μM 1μM 0.5μM C 0.3μM 0.3μM 0.25μM D 0.1μM 0.1μM 0.125μM E 0.03μM 0.03μM 0.0625μM F 0.01μM 0.01μM 0.0313μM G 0 (blank) control 0 (blank) control 0.0156μM H 0 (blank) control 0 (blank) control 0μM (blank)

[0224] 4. DCF standard dilution plate

[0225] a. Use 96-well plates (natural, 0.5 mL, V-bottom, sterile, single-pack) as dilution plates.

[0226] b. Preparation of the 20X DCF standard curve

[0227] i. Dilute 500 μL of 20 μM DCF solution according to the plate map illustrated in Table 6 below.

[0228] Table 6. Compound (DCF) dilution plates

[0229]

[0230]

[0231] c. Preparation of control and testing FXN protein samples

[0232] i. Perform serial dilutions of the 100×FXN protein according to the plate plots shown in the example in Table 7 below.

[0233] Table 7. FXN Protein Dilution Plates

[0234]

[0235] 5. Preparation of the final HQ test plate

[0236] a. Based on the plate plot shown in Table 5, transfer 10 μL of the 20×DCF standard curve from the dilution plate (shown in Table 6) to the HQ assay plate.

[0237] b. Add 10 μL of H2DCF (10 μM working solution) to each well (in addition to the DCF standard curve) to achieve a final concentration of 0.5 μM H2DCF.

[0238] c. According to the plate diagram shown in Table 5, quickly add 2 μL of FXN protein from the FXN protein dilution plate to the HQ assay plate (Table 6).

[0239] d. Quickly place the plate in the plate reader and begin dynamic readings immediately.

[0240] e. Steps (c) and (d) are time-sensitive and executed quickly.

[0241] 6. Data Analysis

[0242] a. Dynamic analysis

[0243] i. Comparison of multiple batches - scatter plot

[0244] 1. Plot the FXN protein at each concentration on a separate graph.

[0245] 2. Any fluorescence unit (AFU) will be on the y-axis, and time [HH: MM or minutes] will be on the x-axis.

[0246] 3. The average value of the two copies plus or minus the standard deviation is used as the error bar.

[0247] b. Maximum initial velocity (Vi) analysis

[0248] i. A maximum velocity dynamics analysis should be performed:

[0249] 1. From 2 to 20 minutes;

[0250] 2. Calculate the speed at 5 points;

[0251] 3. Relative fluorescence units (RFU) / minute.

[0252] ii. Plot Vi generated from the fluorescence scanner on a scatter plot.

[0253] 1. Subtract the background from the reference hole.

[0254] 2. The Y-axis represents Vi units.

[0255] 3. The XX axis represents the concentration in [μM].

[0256] 4. The average value of the two copies plus or minus the standard deviation is used as the error bar.

[0257] c. Milli-unit analysis

[0258] i. Subtract the background of [0]DCF and plot the DCF standard curve. The line should pass through 0,0, and the slope should be recorded. The slope must be converted from Y = mX → X = Y / m.

[0259] ii. Subtract the background from Vi using the [0μM] control wells specified as rows G and H in Table 7.

[0260] iii. Divide the value by the slope and provide the unit (U).

[0261] iv. Multiply the unit by 1,000 to milliunits (mU).

[0262] v. The graphs presented in Table 8 are used to determine the mg of protein. Divide mU by mg of protein to obtain mU / mg.

[0263] Table 8. Conversion of μM FXN protein to mg / well

[0264]

[0265]

[0266] vi. Calculate the average of two copies and record the standard deviation as the error.

[0267] vii. The μM concentration of FXN protein that appears most linearly in the Vi scatter plot should be selected.

[0268] viii. Different batches can be plotted on a bar chart to compare activity.

[0269] Example 2. FXN fusion protein as a catalyst for hydroquinone oxidation

[0270] The FXN fusion protein having the amino acid sequence SEQ ID NO: 12 comprises the cell-penetrating peptide HIV-TAT (SEQ ID NO: 4) and a full-length hFXN (SEQ ID NO: 1) linked via a linker at the N-terminus of hFXN. The therapeutic properties of this FXN fusion protein have been demonstrated in vitro and in vivo. In vitro, the genomic footprint of FXN-deficient cell lines before and after treatment with the FXN fusion protein has been characterized, and significant differences in the expression of a set of genes have been identified (data not shown). In vivo, treatment of an FDRA mouse model with the FXN fusion protein showed measurable improvements in cardiomyopathy phenotypes (e.g., myocardial contractility and other symptoms) (data not shown).

[0271] Because coagulant replacement therapy (i.e., administration of FXN fusion protein) has demonstrated biological activity in vitro and in vivo, and can be used as a therapeutic agent for conditions associated with FXN deficiency, an activity assay was developed to compare the activity of FXN protein in different batches (e.g., FXN fusion protein having the amino acid sequence of SEQ ID NO: 12). This assay utilizes the previously unidentified redox properties of FXN protein.

[0272] The hydroquinone (HQ) reduction assay was performed using the FXN fusion protein of SEQ ID NO: 12 and the basic protocol described in Example 1. The reduction of HQ in the presence of the FXN protein can be represented by the following protocol 1:

[0273]

[0274] H2Q→Q 2- +2H +

[0275] Q 2- -2ē+2O2→Q+2O2· -

[0276] The generation of ROS (i.e., superoxide) is detected using ROS detection reagent compounds (e.g., 2',7'-dichlorodihydrofluorescein (H2DCF)) and kinetic tracking is performed. Figure 1 As shown in Figure A.

[0277] Figure 1 This diagram illustrates the kinetics of hydroquinone (HQ) reduction in the presence of the exemplary FXN fusion protein, measured by the amount of reactive oxygen species (ROS) generated over time. The amount of ROS is expressed as fluorescence, measured in arbitrary fluorescence units (AFU). Figure 1 Figure A shows the production of superoxide (O2·) through the oxidation of hydroquinone (HQ) in the presence or absence of the exemplary FXN fusion protein. -The dynamics of ).

[0278] Reaction efficiency for metal ions, especially Fe 3+ and / or Mn 2+ Sensitivity to the presence of ions was demonstrated. The effects of two metal ions were compared in assays using 4.5 μM FXN fusion protein from two different batches. Table 9 provides an illustration of the different samples tested in the experiments (including different combinations of reagents). In Table 9, checkmarks (√) indicate the presence of a given component in the assay mixture.

[0279] Table 9. Test results for the mixtures used in the experiment

[0280]

[0281] Representative results from the experiment are provided in Figure 1 In Figure B, the results confirm that Mn... 2+ and Fe 3+ Both act as enhancers of the FXN fusion protein or cofactor in the HQ oxidation reaction. Furthermore, Mn exists alone... 2+ Under these conditions, the reaction rate and fluorescence output were significantly higher than those of Fe alone. 3+ In the presence of Mn. Furthermore, with... 2+ And does not contain Fe 3+ Compared with the determination method, for Mn-containing 2+ Fe was added in the determination method 3+ It will not have an incremental effect.

[0282] In another experiment, Fe was studied. 3+ The effect of the presence of ions on the activity of an exemplary FXN fusion protein. In this experiment, the kinetics of ROS generation over time were measured in an assay mixture containing 10 μM of the exemplary FXN fusion protein, 10 μM HQ, and 5 μM H2DCF. One assay mixture also contained 1 mM of the iron chelating agent deferoxamine. Figure 1 Figure C shows the presence of an exemplary FXN fusion protein and Fe. 3+ In the case of (gray square); where an exemplary FXN fusion protein is present and Fe is absent. 3+ In the case of (gray circle); in the presence of exemplary FXN fusion protein, Fe 3+ Graphs showing the kinetics of superoxide generation in the presence of deferoxamine (black circle) and in the absence of the exemplary FXN fusion protein (small black rectangle).

[0283] Figure 1 The data shown in Figure C confirms that, under the test conditions, the presence and absence of Fe... 3+ROS is generated in all cases, however, in the absence of Fe 3+ In this case, the delay time caused by ROS was observed to be approximately 2 hours.

[0284] Figure 1 The data shown confirms that the exemplary FXN fusion protein, as measured by ROS over time, possesses enzymatic activity. This enzymatic activity is evident in both the presence and absence of metal ions; however, it is less pronounced in the presence of metal ions such as Fe. 3+ and / or Mn 2+ In the presence of metal ions, enzyme activity is enhanced. Without being bound by any specific theory, it is believed that the presence of metal ions can induce conformational changes in FXN proteins (e.g., FXN fusion proteins), thereby enhancing their activity.

[0285] The activity assay of the FXN fusion protein described in this embodiment follows the basic protocol provided in Example 1, and the method is described in more detail below.

[0286] A scheme for measuring the redox activity of an exemplary FXN fusion protein against hydroquinone in the presence of metal ions.

[0287] 1. Hole loading in columns 1-6:

[0288] i. 10 μl of 100 μM HCl (5 μM final concentration);

[0289] ii. 10 μl of 100 μM ferric sulfate (III) or 10 μl of 100 μM manganese chloride (II) (both with a final concentration of 5 μM).

[0290] The holes in columns 1-4 are further loaded:

[0291] iii. 160 μl of Tris-hydrochloride (THCl); and

[0292] The holes in columns 5-6 are further loaded:

[0293] IV. 170 μl THCl.

[0294] 2. Prepare serial dilutions of the exemplary FXN fusion protein (SEQ ID NO. 12) for standard and test samples as described in Table 7, wherein the initial 1:3 dilution (6 μL diluted to 12 μL of 50 mM Tris-HCl, pH 8.0) is further diluted 1:10 (1.5 μL diluted to 13.5 μL).

[0295] 3. Prepare a series of DCF diluents as described in Table 6, wherein 10 μM DCF is diluted 1:1 (200 μL diluted in 200 μL of 0.04% DMSO aqueous solution).

[0296] 4. Transfer 10 μL from the DCF standard curve plate (Table 6) to columns 5-6 of the 96-well HQ test plate (Table 5).

[0297] 5. Add 10 μL of standard FXN protein or test FXN protein from the dilution plate shown in Table 7 to columns 1-2 and 3-4 of the 96-well HQ assay plate, respectively (Table 5).

[0298] As described in the basic scheme of Example 1 above, the steps of adding 10 μL of activated H2DCF solution to each well in columns 1-4 (final H2DCF concentration of 0.5 μM) and transferring the plate to a plate reader for kinetic reading are performed very quickly.

[0299] In the presence of Fe and / or Mn 2+ In this case, the exemplary FXN fusion protein was incubated with HQ and H2DCF as a ROS detection compound. Fluorescence was measured in a fluorescence plate reader, with the excitation filter in the range of 485 nm (485 / 20) and the emission filter in the range of 530 nm (528 / 20), and the optical system having a gain of 50 from top to bottom.

[0300] Titration of the commercially available oxidation product of DCF, 2',7'-dichlorofluorescein, enables the establishment of assay conditions that demonstrate the linearity between the amount of product in the reaction and the fluorescence reading. The titration provides a basis for defining "exemplary units of FXN fusion protein" for use in different batches of the exemplary FXN fusion protein.

[0301] Figure 2 This is the standard curve for 2',7'-dichlorofluorescein (DCF), generated by plotting the fluorescence (AFU) exhibited by DCF as a function of DCF concentration (μM). In the DCF standard curve, ΔAFU = 66681 corresponds to 1 μM DCF. Based on the following formula, the amount of FXN fusion protein required to catalyze the production of 1 micromolar (μM) DCF per minute is defined as the amount of FXN fusion protein extrapolated from the DCF standard curve to provide one milliunit of FXN fusion protein:

[0302] V i =ΔAFU / min = ΔμM / min.

[0303] Therefore, the amount of FXN protein required to obtain the maximum initial velocity Vi = 1 is defined as milliunits of FXN protein.

[0304] The results of this invention demonstrate for the first time that FXN proteins (e.g., FXN fusion proteins) act as redox-sensitive enzymes and can catalyze the oxidation of hydroquinone while generating superoxide. The redox activity of FXN fusion proteins is enhanced in the presence of metal ions such as Mn. 2+ and Fe 3+ The effects were enhanced under these conditions. These experiments also enabled the inventors to define the activity of the enzymatic units of the FXN fusion protein.

[0305] Example 3. Comparison of activity in different batches of FXN fusion protein and in the presence of NADH or NADP.

[0306] Once the activity of the enzyme units of the exemplary FXN fusion protein was defined, the HQ assay was used to compare the specific activity (defined as mUnits / mg of FXN fusion protein) of four different batches of SEQ ID NO: 12 FXN fusion protein. The experimental results are presented in… Figure 3 In Figure A. Specifically, Figure 3 Figure A is a bar chart showing the specific activity (in milliunits / mg) of four different batches of FXN fusion protein (batches 05, 18, 28 and 32) at different concentrations of FXN fusion protein (0.03 μM, 0.1 μM, 0.3 μM and 1 μM). Figure 3 The results presented in Figure A indicate that the specific activity of each test batch of FXN fusion protein remained relatively constant across the four different concentrations of FXN fusion protein. Furthermore, batch 32 exhibited the lowest activity with an average activity of 1582 mUnits / mg, while batch 05 showed the highest activity with an average activity of 5790 mUnits / mg.

[0307] The redox activity of the FXN fusion protein was also measured in the presence of NADPH and NADH. Figure 3 Figure B shows the presence of Fe. 3+ Fe 3+ and NADH and Fe 3+ A bar chart showing the specific activity (mUnits / mg) of the FXN fusion protein under NADPH conditions. Figure 3 The results shown in Figure B indicate that the addition of NADPH to the assay significantly reduced ROS production and inhibited the redox activity of the FXN fusion protein, while the addition of NADH did not affect the redox activity of the FXN fusion protein.

[0308] Example 4: The activity of the FXN fusion protein is sensitive to high-temperature storage conditions.

[0309] The objective of this experiment was to evaluate the thermal stability of an exemplary FXN fusion protein by assessing its activity after short-term storage (one month) at different temperatures. In this experiment, the FXN fusion protein of SEQ ID NO: 12 was used. The storage conditions tested in the study and the corresponding results are summarized in Table 10 below.

[0310] Table 10. Temperature stability study conditions for exemplary FXN fusion proteins ( Figure 4 (Figure A)

[0311]

[0312] The experimental results are also presented in Figure 4 In particular, Figure 4 Figure A is a graph showing the kinetics of superoxide production in the presence of exemplary FXN fusion proteins that have been stored at -60°C (SPL1), 2-4°C (SPL2), and 25°C (SPL3), compared to the DS control. Figure 4 Figure B is a bar chart showing the maximum initial rate (Vi) of superoxide production in the presence of exemplary FXN fusion proteins that have been stored at -60°C (SPL1), 2-4°C (SPL2), and 25°C (SPL3), compared to the DS control.

[0313] Figure 4 The results presented in Table 10 confirm that storing the exemplary FXN fusion protein for one month under conditions other than -60°C resulted in a decrease in the activity of the exemplary FXN fusion protein in the HQ assay. This also confirms that incubating the exemplary FXN fusion protein at temperatures above -60°C reduced its activity in the HQ assay.

[0314] Example 5. The activity of an exemplary FXN fusion protein is sensitive to chemical stress.

[0315] The objective of this experiment was to evaluate the stability of an exemplary FXN fusion protein to chemical stress by assessing its activity after exposure to chemical stressors that can induce protein denaturation and / or degradation of the FXN fusion protein. In this experiment, the FXN fusion protein of SEQ ID NO: 12 was used. The storage conditions tested in the study and the corresponding results are shown in [data missing]. Figure 5 This is also outlined in Table 11 below.

[0316] Table 11. Chemical stability study conditions for exemplary FXN fusion proteins

[0317]

[0318] In short, the study controls were stored at 2-8°C during the study. Each treatment was performed as follows:

[0319] i. Acid treatment: Add 1N HCl to the solution containing the FXN fusion protein until the pH reaches ≤4. Maintain the sample at 37°C for 2 days, then replace the buffer with the buffer from the DS formulation. For the remainder of the study, store the sample at 2–8°C.

[0320] ii. Alkali treatment: Add 1M Tris base to the solution containing the FXN fusion protein until the pH reaches ≥10. Maintain the sample at 37°C for 2 days, then replace the buffer with the buffer from the DS formulation. Store the sample at 2–8°C.

[0321] iii. Acid and base control: Store the solution containing the FXN fusion protein at 37°C for 2 days, and then store at 2-8°C.

[0322] iv. Oxidative stress treatment: The exemplary FXN fusion protein was incubated at 37°C for 2 hours in the presence of 0.05% hydrogen peroxide, and then stored at 2–8°C.

[0323] v. Oxidation control: The exemplary FXN fusion protein was incubated at 37°C for 2 hours and then stored at 2–8°C.

[0324] vi. Heat stress treatment: Incubate the exemplary FXN fusion protein at 50°C for 2 hours, and then store it at 2-8°C.

[0325] All samples were analyzed for appearance, pH, protein quantification (via A280), size exclusion high-performance liquid chromatography (SE-UPLC), reversed-phase high-performance liquid chromatography (RP-HPLC), ion-exchange high-performance liquid chromatography (IE-HPLC), reducing capillary electrophoresis with sodium dodecyl sulfate (rCE-SDS), non-reducing capillary electrophoresis with sodium dodecyl sulfate (nrCE-SDS), and free thiol groups (data not shown). The concentration of the FXN fusion protein was determined by Western blotting, and redox activity was determined using the HQ assay as described above.

[0326] The results of HQ activity are presented Figure 5 In particular, Figure 5 This is a bar graph showing the maximum initial rate (Vi) of superoxide production in all tested samples. The results indicate that the FXN fusion protein is largely resistant to heat stress after 2 hours of application. The results also show that the FXN fusion protein is sensitive to alkaline and oxidative stress, and partially sensitive to acid stress.

[0327] Example 6. Activity of exemplary FXN fusion proteins in the presence of different metal ions

[0328] The objective of this experiment was to evaluate the HQ-dependent superoxide production activity of an exemplary FXN fusion protein in the presence of different metal ions. In this experiment, the FXN fusion protein of SEQ ID NO: 12 was used. The kinetics of superoxide production were evaluated in a reaction mixture containing 20 μM HQ, 5 μM metal, 5 μM FXN fusion protein, and 0.25 μM H2DCF in 50 mM Tris-HCl buffer at pH 8.0. Metal ions tested in the experiment included Mn. 2+ Fe 3+ Zn 2+ Cu 2+ and Mg 2 + .

[0329] Figure 6 This is a graph showing the kinetics of HQ-dependent superoxide generation activity of an exemplary FXN fusion protein in the presence of different metal ions. Figure 6 The data presented confirms that the FXN fusion protein exists in the presence of Mn 2+ It exhibits the highest activity under certain conditions.

[0330] Example 7. pH dependence of exemplary FXN fusion protein activity

[0331] The objective of this experiment was to evaluate the pH dependence of the HQ-dependent superoxide generation activity of the exemplary FXN fusion protein. In this experiment, the FXN fusion protein of SEQ ID NO: 12 was used. Specifically, the superoxide generation kinetics of the exemplary FXN fusion protein were evaluated at pH values ​​ranging from 7.0 to 9.0. Each reaction mixture contained 5 μM HQ, 5 μM ferric(III) sulfate, 0.05 μM H2DCF, and 10 μM of the exemplary FXN fusion protein in 50 mM Tris-HCl at pH values ​​of 7.0, 7.4, 7.8, 8.0, 8.2, 8.5, and 9.0.

[0332] The assay plate was placed in the plate reader, and kinetic readings were initiated immediately after all assay components were mixed together. The plate reader was set as follows: 2-hour kinetic scan, fluorescence readings every 5 minutes (excitation 485 / 20, emission 528 / 25, gain 50), with a 10-second shake between readings. The maximum initial velocity (Vi) for each well was determined by measuring the fastest change in arbitrary fluorescence units (AFU) at 5 data points.

[0333] Figure 7 This is a graph showing Vi as a function of pH, illustrating the activity measurement results of an FXN fusion protein as an example. Figure 7The data presented confirm that the superoxide production activity of the exemplary FXN fusion protein is pH-dependent. Specifically, the FXN fusion protein exhibits almost no activity at pH 7.4 or lower. As the pH increases above 7.4, the activity of the exemplary FXN fusion protein increases, peaking at approximately 8.5. The optimal pH range for the exemplary FXN fusion protein overlaps with the pH of the mitochondrial matrix at approximately 7.8, which is higher than the pH of the cytosol at approximately 7.4.

[0334] Example 8. High-throughput screening (HTS) for identifying compounds that can affect FXN activity.

[0335] The objective of this experiment was to identify compounds capable of modulating the activity of the FXN protein using the HQ assay and high-throughput screening (HTS) of this disclosure. In this experiment, the FXN fusion protein of SEQ ID NO: 12 was used. The experiment consisted of two parts. In the first part of the experiment, the performance of the HQ assay was verified under the conditions used for HTS. In the second part of the assay, a series of screenings of a library of 770 compounds was performed to identify compounds capable of enhancing the activity of the exemplary FXN fusion protein.

[0336] Evaluation of the performance of the assay method

[0337] The first part of the experiment aimed to verify the performance of the HQ assay under conditions suitable for HTS. To this end, the HQ assay was characterized by measuring statistical parameters such as the signal-to-background (S / B) ratio, the Z' factor (a measure of the statistical robustness of the assay readings), and intra- and inter-plate variability. For this purpose, two 96-well plates were each divided into nine sections: four sections in each plate contained 10 μM of the exemplary FXN fusion protein (high control); four sections contained 0.5 μM of the exemplary FXN fusion protein (low control); and one section contained only the assay mixture without the FXN fusion protein (blank control).

[0338] H2DCF was activated by incubating 1 mL of 1 mM H2DCF-DA in 20 mL of 0.01 M NaOH in the dark for 30 minutes with stirring. Subsequently, 75 mL of 33 mM NaH2PO4 was added to the activation mixture to bring the final H2DCF concentration to 10 μM. This solution was stored at 4 °C and protected from light until use. Each well contained 50 mM Tris-HCl (pH 8.0), 50 μM HCl, 1 μM MnCl2, 0.5 μM activated H2DCF, and exemplary FXN fusion protein at concentrations of 10 μM (high control), 0.5 μM (low control), and 0 μM (blank control). Immediately after adding the assay mixture to the plate, the plate was placed in a plate reader, and kinetic readings were initiated. The plate reader settings were as follows: 45-minute kinetic scans, fluorescence readings per minute (excitation 482 / 20, emission 530 / 25, gain 50), with a 3-second shake between readings. The maximum initial velocity (Vi) for each well was determined by measuring the fastest change in arbitrary fluorescence units (AFU) at 5 data points. Statistical parameters, such as the signal-to-background (S / B) ratio, Z' factor, and intra- and inter-plate variability, were calculated to characterize the assay performance.

[0339] The results show Figure 8 And in Table 12 below. Figure 8 This is a graph showing the Vi values ​​measured for each test sample containing 10 μM FXN fusion protein (high FXN control), 5 μM FXN fusion protein (low FXN control), and 0 μM FXN fusion protein (blank control) for plates 1 and 2. Based on this data, the Z' factor for the high FXN control sample was calculated to be greater than 0.67 and the corresponding S / B ratio was calculated to be greater than 7.8, while the Z' factor for the low FXN control sample was calculated to be greater than 0.85 and the S / B ratio was calculated to be greater than 6.18.

[0340] Table 12 presents data used to evaluate interplate variability in the HQ assay.

[0341] Table 12. Vi values ​​and related statistical parameters for each test condition

[0342]

[0343] Figure 8 The results shown in Table 12 confirm that the HQ assay is suitable for HTS experiments under the test conditions.

[0344] High-throughput screening (HTS)

[0345] The objective of this experimental section is to perform high-throughput screening of a drug library using the HQ assay described in this disclosure to identify compounds that regulate the activity of the FXN protein in this assay. SCREEN, purchased from Enzo Life Sciences, was used. HTS was performed on the FDA-approved drug library V2 (product number BML-2843-0100). This library contains over 770 compounds that are FDA-approved and have known and well-characterized biological activities, safety profiles, and bioavailability. The library also avoids irrelevant compounds such as herbicides, pesticides, sunscreens, and cytotoxic agents. Compounds in the FDA-approved drug library V2 are screened for their ability to enhance the activity of the exemplary FXN fusion protein.

[0346] A summary of the HTS experiments conducted is in Figure 9 As shown in the image. Specifically, Figure 9 This is a flowchart showing the three steps involved in the HTS experiment and the related results. The first step includes preliminary screening, where... Compounds in the FDA-approved drug library V2 were screened for enhanced FXN activity. In this screening, 17 hits were identified, exhibiting an enhancement of greater than 30% in FXN activity. A second step included a confirmatory screening, in which the 17 compounds identified in the initial screening were rescreened, and 16 compounds were confirmed to enhance FXN activity by more than twice the standard deviation of the low-FXN control wells included in each assay plate. A third step included a reverse screening, in which the 16 compounds identified in the confirmatory screening were screened in the absence of the exemplary FXN fusion protein. In this screening, two of the 16 compounds were identified whose FXN-enhancing activity was strictly dependent on the presence of FXN.

[0347] Preliminary screening was performed using the following procedure. A complete assay mixture was freshly prepared just before being added to the assay plate, comprising 50 mM Tris-HCl (pH 8.0), 50 μM HQ, 1 μM MnCl2, 0.5 μM activated H2DCF, 0.5 μM of the exemplary FXN fusion protein, and a final concentration of 10 μM of the test compound. Additionally, each plate included an internal control containing the exemplary FXN fusion protein at concentrations of 10 μM (upper limit control), 0.5 μM (lower limit control), or 0 μM (medium for blank control) in 1% DMSO and water. Controls were included in both DMSO and water because the library compounds are soluble in either. Immediately after mixing all components of the assay, the plate was placed in a plate reader and kinetic readings were initiated. The plate reader was set as follows: 45-minute kinetic scans, fluorescence readings per minute (excitation 482 / 20, emission 530 / 25, gain 50), with 3-second shaking between readings.

[0348] The maximum initial velocity (Vi) for each well was determined by measuring the fastest change in arbitrary fluorescence units (AFU) at five data points anywhere on the curve. The average Vi value of the control wells containing 0.5 μM of FXN fusion protein was calculated, and the window above 30% of the plate average was determined as the cutoff value for hitting the compound.

[0349] Figure 10 This is a box plot showing the results of the initial HTS screening of compounds in DMSO, including compounds that increased FXN activity by more than 30%, compounds that decreased FXN activity by more than 30%, and compounds that caused changes in FXN activity of less than 30%. Any compound with a Vi value higher than the cutoff value was eligible for secondary screening. As described above, in the initial screening, 17 hits were identified, which demonstrated an enhancement of FXN activity of more than 30%.

[0350] A confirmatory screening was performed using a procedure similar to that used for the initial screening. In the confirmatory screening, 16 compounds were identified as enhancing FXN activity by more than twice the standard deviation of the low FXN control wells included in each plate containing 0.5 μM of FXN fusion protein.

[0351] Compounds exhibiting FXN-enhancing activity, dependent on the presence of the FXN fusion protein, were identified through reverse screening. In this screening, compounds whose FXN activity deviated from that shown by the internal mediator control by no more than two standard deviations were selected. Figure 11 This is a bar chart showing exemplary results of the confirmation screening of compound 1 and compound 2. Figure 11The results shown indicate that compound 1 passed the reverse screening because it showed no activity in the absence of the FXN fusion protein; while compound 2 was disqualified because it showed activity in the absence of the FXN fusion protein. In the reverse screening, two of the 16 compounds from the confirmatory screening were identified as having FXN-enhancing activity in the presence of FXN.

Claims

1. A method for measuring the enzymatic activity of a Frataxin (FXN) protein, wherein the enzymatic activity comprises enzymatically catalyzed production of reactive oxygen species (ROS), the method comprising: (a) combining the FXN protein with a reducing agent and a ROS detector compound at a pH greater than 7.4, thereby producing an assay mixture; (b) measuring the amount of ROS produced in the assay mixture over time; and (c) determining an enzymatic parameter related to the ROS production activity of the FXN protein.

2. The method of claim 1, comprising combining the FXN protein with a reducing agent and a ROS detector compound at a pH of 7.9 or higher.

3. A method for measuring the enzymatic activity of a Frataxin (FXN) protein, wherein the enzymatic activity comprises enzymatically catalyzed production of reactive oxygen species (ROS), the method comprising: (a) combining the FXN protein with a reducing agent and a ROS detector compound at a pH greater than 7.4 in the absence of metal ions, thereby producing an assay mixture; (b) measuring the amount of ROS produced in the assay mixture over time; and (c) determining an enzymatic parameter related to the ROS production activity of the FXN protein.

4. A method for measuring the enzymatic activity of a Frataxin (FXN) protein, wherein the enzymatic activity comprises enzymatically catalyzed production of reactive oxygen species (ROS), the method comprising: (a) combining the FXN protein with a reducing agent and a ROS detector compound at a pH greater than 7.4 in the absence of iron ions, thereby producing an assay mixture; (b) measuring the amount of ROS produced in the assay mixture over time; and (c) determining an enzymatic parameter related to the ROS production activity of the FXN protein.

5. The method of claim 3 or 4, further comprising adjusting the pH of the assay mixture to a pH of 7.9 or higher.

6. The method of claim 1 or 2, further comprising adding metal ions to the assay mixture.

7. The method of claim 6, wherein the metal ion is not Fe 2+ .

8. The method of claim 6, wherein the metal ion is selected from the group consisting of Mn 2+ , Fe 3+ , Zn 2+ , Cu 2+ , and Mg 2+ .

9. The method of claim 8, wherein the metal ion is Mn 2+ .

10. The method of claim 8, wherein the metal is Fe 3+ .

11. The method of any one of claims 1-4, further comprising incubating the assay mixture.

12. The method of any one of claims 1-4, comprising measuring the amount of ROS produced at regular time intervals.

13. The method of claim 12, comprising measuring the amount of ROS produced every 0.1 seconds to every 10 minutes after initiating incubation of the assay mixture.

14. The method of any one of claims 1-4, comprising determining the maximum initial velocity (Vi) of ROS production in the assay mixture.

15. The method of any one of claims 1-4, comprising determining the time to reach the maximum initial velocity (Ti).

16. The method of claim 15, further comprising determining the unit activity of the FXN protein at a pH greater than 7.

4.

17. The method of any one of claims 1-4, further comprising determining the specific activity of the FXN protein.

18. The method of any one of claims 1-4, wherein the reducing agent is an organic compound.

19. The method of claim 18, wherein the reducing agent is a reduced quinone compound.

20. The method of claim 19, wherein the reduced quinone compound is selected from the group consisting of a reduced benzoquinone compound, a reduced naphthoquinone compound, and a reduced anthraquinone compound.

21. The method of claim 20, wherein the reduced quinone compound is a reduced benzoquinone compound.

22. The method of claim 21, wherein the reduced benzoquinone compound is hydroquinone.

23. The method of any one of claims 1-4, wherein the ROS detector compound is a fluorescent probe or a chemiluminescent probe.

24. The method of any one of claims 1-4, wherein the ROS detector compound is a superoxide anion detection reagent or a hydrogen peroxide detection reagent.

25. The method of any one of claims 1-4, wherein the ROS detector compound is selected from the group consisting of coelenterazine; dihydroethidium; 2',7'-dichlorodihydrofluorescein (H2DCF), lucigenin, luminol, luciferin analogs (CLA), luciferin methoxy analog (MCAL), methylthiazol diphenyl-tetrazolium bromide (MTT), nitroblue tetrazolium (NBT), Redox Sensor Red CC-1, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), and structural variants and analogs thereof.

26. The method of claim 25, wherein the ROS detector compound is 2',7'-dichlorodihydrofluorescein (H2DCF).

27. The method of any one of claims 1-4, wherein the reducing agent and the ROS detector compound are the same compound.

28. The method of any one of claims 1-4, wherein the reducing agent and the ROS detector compound are different compounds.

29. The method of claim 28, wherein the reducing agent is dihydroquinone and the ROS detector compound is 2',7'-dichlorodihydrofluorescein (H2DCF).

30. The method of any one of claims 1-4, wherein the FXN protein is frataxin (FXN).

31. The method of any one of claims 1-4, wherein the FXN protein is a FXN fusion protein comprising full-length hFXN and a cell-penetrating peptide (CPP).

32. The method of claim 31, wherein the CPP comprises a peptide selected from the group consisting of CPPs listed in the Cell-Penetrating Peptides CPPsite 2.0 database.

33. The method of claim 31, wherein the CPP comprises a peptide selected from the group consisting of HIV-TAT, glycosaminopeptide, melittin, transportan, penetratin, polyarginine, VP22, and variants or derivatives thereof.

34. The fusion protein of claim 33, wherein the CPP comprises HIV-TAT or a variant or derivative thereof.

35. The method of claim 31, wherein the FXN fusion protein comprises SEQ ID NO:

12.

36. A method of quality control of a sample comprising Frataxin (FXN) protein, the method comprising measuring the enzymatic activity of the FXN protein according to the method of any one of claims 1-35.

37. Use of a kit for determining the enzymatic activity of a FXN protein, the kit comprising a reducing agent, a ROS detector compound, instructions for combining the reducing agent, the ROS detector compound, and a FXN protein at a pH greater than 7.4 to produce an assay mixture; and instructions for determining the enzymatic parameter of ROS production activity of the FXN protein in the assay mixture.

38. The use of claim 37, wherein the kit further comprises a metal ion compound.

39. The use of claim 38, wherein the metal ion compound comprises ions selected from the group consisting of Mn 2+ , Fe 3+ , Zn 2+ , Cu 2+ , and Mg 2+ .

40. The use of claim 39, wherein the metal ion compound comprises Fe 3+ .

41. The use of claim 39, wherein the metal ion compound comprises Mn 2+ .

42. The use of claim 37, wherein the reducing agent is an organic compound.

43. The use of claim 42, wherein the reducing agent is a reduced quinone compound.

44. The use of claim 43, wherein the reduced quinone compound is selected from the group consisting of a reduced benzoquinone compound, a reduced naphthoquinone compound, and a reduced anthraquinone compound.

45. The use of claim 44, wherein the reduced quinone compound is a reduced benzoquinone compound.

46. The use of claim 45, wherein the reduced benzoquinone compound is hydroquinone.

47. The use of claim 37, wherein the ROS detector compound is a fluorescent probe or a chemiluminescent probe.

48. The use of claim 37, wherein the ROS detector compound is a superoxide anion detection reagent or a hydrogen peroxide detection reagent.

49. The use of claim 47 or 48, wherein the ROS detector compound is selected from the group consisting of coelenterazine; dihydroethidium; 2',7'-dichlorodihydrofluorescein (H2DCF), lucigenin, luminol, luciferin analogs (CLA), luciferin methoxy analog (MCAL), methylthiazol diphenyl-tetrazolium bromide (MTT), nitroblue tetrazolium (NBT), RedoxSensor Red CC-1, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), and structural variants and analogs thereof.

50. The use of claim 49, wherein the ROS detector compound is 2',7'-dichlorodihydrofluorescein (H2DCF).

51. The use of claim 37, wherein the reducing agent and the ROS detector compound are the same compound.

52. The use of claim 37, wherein the reducing agent and the ROS detector compound are different compounds.

53. The use of claim 52, wherein the reducing agent is dihydroquinone and the ROS detector compound is 2',7'-dichlorodihydrofluorescein (H2DCF).

54. A method for identifying a compound capable of modulating the enzymatic activity of a Frataxin (FXN) protein, the method comprising the steps of: (i) combining the FXN protein with a test compound, a reducing agent, and a reactive oxygen species (ROS) detector compound at a pH greater than 7.4, thereby producing an assay mixture; (ii) measuring the enzymatic activity of the FXN protein in the assay mixture; and (iii) comparing the enzymatic activity of the FXN protein in the assay mixture to the enzymatic activity of the FXN protein in the absence of the test compound. (ii) measuring the amount of ROS produced in the assay mixture over time in the presence of the test compound; (iii) determining an enzyme parameter related to the ROS producing activity of the FXN protein in the presence of the test compound; (iv) comparing the enzyme parameter related to the ROS producing activity of the FXN protein in the presence of the test compound with the enzyme parameter related to the ROS producing activity of the FXN protein in the absence of the test compound; and (v) identifying the test compound as a compound capable of modulating the enzymatic activity of the FXN protein when the enzyme parameter related to the ROS producing activity of the FXN protein in the presence of the test compound is different from the enzyme parameter related to the ROS producing activity of the FXN protein in the absence of the test compound.

55. The method of claim 54, further comprising incubating the assay mixture.

56. The method of claim 54, wherein step (ii) comprises measuring the amount of ROS produced at regular time intervals.

57. The method of claim 56, wherein step (ii) comprises measuring the amount of ROS produced every 0.1 seconds to every 10 minutes.

58. The method of claim 54, comprising determining the maximum initial velocity (Vi) of ROS production in the assay mixture.

59. The method of claim 58, further comprising determining the time to reach the maximum initial velocity (Ti).

60. The method of claim 59, further comprising determining the unit activity of the FXN protein.

61. The method of claim 59, further comprising determining the specific activity of FXN protein.

62. The method of claim 58, further comprising the steps of: comparing the Vi of the ROS producing activity of the FXN protein in the presence of the test compound with the Vi of the ROS producing activity of the FXN protein in the absence of the test compound; and identifying the test compound as a compound capable of modulating the enzymatic activity of the FXN protein when the Vi of the ROS producing activity of the FXN protein in the presence of the test compound is different from the Vi of the ROS producing activity of the FXN protein in the absence of the test compound.

63. A method for identifying a compound capable of modulating the enzymatic activity of a Frataxin (FXN) protein, the method comprising the steps of: (i) combining the FXN protein with a test compound, a reducing agent, and a reactive oxygen species (ROS) detector compound at a pH greater than 7.4, thereby producing an assay mixture; (ii) measuring the amount of ROS produced in the assay mixture over time in the presence of the test compound and determining the Vi of the ROS producing activity of the FXN protein; (iii) comparing the Vi of the ROS-producing activity of the FXN protein in the presence of the test compound to the Vi of the ROS-producing activity of the FXN protein in the absence of the test compound; and (iv) identifying the test compound as a compound capable of modulating the enzymatic activity of the FXN protein when the Vi of the ROS-producing activity of the FXN protein in the presence of the test compound is different from the Vi of the ROS-producing activity of the FXN protein in the absence of the test compound.

64. The method of claim 63, further comprising adjusting the pH of the assay mixture to a pH of 7.9 or higher.

65. The method of claim 54 or 63, further comprising adding a metal ion to the assay mixture.

66. The method of claim 65, wherein the metal ion is selected from the group consisting of Mn 2+ , Fe 3+ , Zn 2+ , Cu 2+ , and Mg 2+ .

67. The method of claim 66, wherein the metal ion is Mn 2+ .

68. The method of claim 66, wherein the metal ion is Fe 3+ .

69. The method of claim 54 or 63, wherein the reducing agent is an organic compound.

70. The method of claim 69, wherein the reducing agent is a reduced quinone compound.

71. The method of claim 70, wherein the reduced quinone compound is selected from the group consisting of a reduced benzoquinone compound, a reduced naphthoquinone compound, and a reduced anthraquinone compound.

72. The method of claim 71, wherein the reduced quinone compound is a reduced benzoquinone compound.

73. The method of claim 72, wherein the reduced benzoquinone compound is hydroquinone.

74. The method of claim 54 or 63, wherein the ROS detector compound is a fluorescent probe or a chemiluminescent probe.

75. The method of claim 54 or 63, wherein the ROS detector compound is a superoxide anion detection reagent or a hydrogen peroxide detection reagent.

76. The method of claim 54 or 63, wherein the ROS detector compound is selected from the group consisting of coelenterazine; dihydroethidium; 2',7'-dichlorodihydrofluorescein (H2DCF), lucigenin, luminol, luciferin analogs (CLA), luciferin methoxy analog (MCAL), methylthiazol diphenyl-tetrazolium bromide (MTT), nitroblue tetrazolium (NBT), RedoxSensor Red CC-1, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT), and structural variants and analogs thereof.

77. The method of claim 76, wherein the ROS detector compound is 2',7'-dichlorodihydrofluorescein (H2DCF).

78. The method of claim 54 or 63, wherein the reducing agent and the ROS detector compound are the same compound.

79. The method of claim 54 or 63, wherein the reducing agent and the ROS detector compound are different compounds.

80. The method of claim 79, wherein the reducing agent is dihydroquinone and the ROS detector compound is 2',7'-dichlorodihydrofluorescein (H2DCF).

81. The method of claim 54 or 63, wherein the FXN protein is frataxin (FXN).

82. The method of claim 54 or 63, wherein the FXN protein is a FXN fusion protein comprising full-length hFXN and a cell-penetrating peptide (CPP).

83. The method of claim 82, wherein the CPP comprises a peptide selected from the CPPs listed in the Cell-Penetrating Peptides CPPsite 2.0 database.

84. The method of claim 82, wherein the CPP comprises a peptide selected from the group consisting of HIV-TAT, Galanin, Melittin, Transportan, Penetratin, Polyarginine, VP22, and variants or derivatives thereof.

85. The method of claim 84, wherein the CPP comprises HIV-TAT or a variant or derivative thereof.

86. The method of claim 85, wherein the FXN fusion protein comprises SEQ ID NO:

12.

87. A method of making a pharmaceutical composition comprising a FXN protein, the method comprising measuring the enzymatic activity of the FXN protein according to the method of any one of claims 1-36, and formulating the protein, thereby making a pharmaceutical composition.

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