CN117241819A - Methods and compositions for protease reporter gene detection methods and protease reporter gene modulators - Google Patents

Abstract

The present application relates to a reporter gene for measuring OMA1 protease activity comprising a targeting sequence and a signal producing domain, wherein the targeting sequence is also a sequence recognized by OMA 1.

Description

Methods and compositions for protease reporter gene detection methods and protease reporter gene modulators

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional application US 63/061,156 filed on 8 th month 4 th 2020 according to 35u.s.c. ≡119 (e) and U.S. provisional application US 63/209,138 filed on 6 th month 10 of 2021 according to 35u.s.c. ≡119 (e), the entire contents of both of which are incorporated herein by reference.

The present application cites the disclosure of U.S. patent No. 10906931B2, entitled "method for treating diseases associated with mitochondrial stress", the inventors of which are also the inventors of the present application.

All documents cited above and all documents cited therein or cited during prosecution thereof ("patent citation documents") and all documents cited or referenced in patent citation documents ("patent citation documents") and all documents cited or referenced herein ("herein citation documents") are hereby incorporated by reference in their entirety, along with any manufacturer's instructions, product specifications, FDA labels and product specifications for any product mentioned herein or any document incorporated by reference, and may be used in the practice of the application. Citation or identification of any document in this application is not an admission that such document is available as prior art to the present application.

About sequence listing

Also submitted with the present application is a sequence listing in electronic form. The sequence listing is a 84,952 byte file created at 2021, 6, 10 and titled luke_seq_st25.txt. The information in the sequence listing in electronic form is incorporated herein by reference.

Preservation of biological samples

The biological sample referred to in the present application has been deposited at ATCC international deposit unit (10801University Boulevard,Manassas,Virginia20110, U.S.) according to budapest strip at about 4.7 of 2021 and deposit number PTA-127022, which deposit is incorporated herein by reference.

Federally sponsored

The present application is sponsored by government grant No. 1R43AG063642-01 from the national institutes of health. In the present application, the federal government may have certain rights in the application.

Technical Field

The present application relates to novel polypeptides, gene sequences and methods for combining gene sequences, whereby the encoded polypeptides have certain activities, which are particularly useful for identifying, selecting or improving compounds having OMA1 and/or OPA1 modulating properties for treating a subject in need of such treatment. The application also relates to these compounds, pharmaceutical compositions comprising these compounds, chemical processes for preparing these compounds, and the use of these compounds as pharmacological tools or in the treatment of diseases associated with OMA1 and/or OPA1 in cells, animals and specific humans. The present disclosure provides novel reporter genes that are particularly useful in identifying compounds having OMA1 and/or OPA1 modulating properties, methods for designing these reporter genes, and methods for using these reporter genes in drug screening assays. Also disclosed herein are methods of using the reporter gene to assess mitochondrial toxicity of a compound and/or to predict adverse events of a subject compound.

Disclosure of Invention

The present invention provides a reporter gene that is capable of detecting OMA1 protease activity in vivo after expression in a host. These synthetic genes are constructed in modular form and are operably combined with the following individual components: (a) a targeting signal, (b) an entire enzyme group or protein domain or an "N" fragment of an enzyme group or protein domain, (C) a "C" fragment of an entire enzyme group or protein domain or an enzyme group or protein structure corresponding to the "N" fragment, and (d) a hydrolyzable sequence motif recognizable by an OMA1 protease, whereby the complementary action of N and C produces a detectable signal. The present invention provides further synthetic mitochondrial import signals that can target polypeptides or reporter genes to the inner mitochondrial membrane.

The present invention solves the problem of monitoring a specific OMA1 protease by targeting a reporter gene to the inner mitochondrial membrane, wherein the OMA1 protease recognizes the reporter gene and modulates the activity of the reporter gene. These new and innovative targeting-based units in vivo protease assays have been shown to be inversely related to OMA1 protease activity, thereby overcoming some of the limitations in current assays for OMA1 activity. The detection methods disclosed herein can be used in vivo, as demonstrated by the inventors of the present invention, which are effective and suitable for high throughput drug screening.

OMA1 protease is a very desirable drug target with a variety of disease characterizations supported by epidemiological and genetic data in human and animal disease models. Examples of such diseases are disclosed in the form of a non-limiting list in another U.S. patent No. 10906931B2 to the inventors. The reporter genes disclosed herein are used in high throughput drug screening. The inventors describe the screening mode in the examples provided herein and in the pending paper manuscript "extension OMA1 protease activation by kinase inhibitors" for the pending list. The manuscript of the article of the pending list is incorporated herein by reference in its entirety, along with all data.

Disclosed herein are medicaments having OMA1 modulating properties. These drugs have been approved by regulatory authorities for use in humans to treat certain malignant diseases. In light of the teachings provided herein, U.S. patent No. 10906931B2 and other references incorporated herein provide teachings that a person of ordinary skill in the art can use the medicaments disclosed herein to treat a subject suffering from a disease or pathological condition that can benefit from such treatment. It should be appreciated that those skilled in the art are also able to identify subjects who cannot benefit from treatment with a drug having OMA 1-modulating properties or to identify when treatment with a drug having OMA 1-modulating properties is stopped due to an increased risk of adverse events.

Drawings

FIG. 1 shows an overview of the OMA1 reporter gene of the invention and its general function.

FIG. 1A shows the different fragments encoded by the genes of the OMA1 reporter gene.

FIG. 1B shows a Western blot of reporter genes that can be hydrolyzed by OMA 1.

FIG. 1C shows OMA1 reporter gene detection. The OMA1 reporter gene produced a signal that was inversely related to OMA1 protease activity (average.+ -. SD; n=80 &48; T-test: p < 0.001).

FIG. 2 shows a vector suitable for expression of a reporter gene of the invention.

FIG. 3 compares two reporter genes Rep#01 and Rep#15 expressed in Hek293T cells incubated for 30 minutes in the absence or presence of 10. Mu.M CCCP followed by detection of the signal. Rep #15 shows much better performance than Rep #01 (average ± SD; n=4; note that the Y-axis has two different dimensions).

FIG. 4 compares Rep#01 and Rep#15 in western immunoblots and establishes OMA1 specificity.

FIG. 4A shows a Western blot of 25 μg reporter cell lysates separated on 12% PAGE using reporter specific antibodies (nanoLuc) markers. Rep#15 shows a much higher abundance than Rep#01.

Fig. 4B shows detection of Rep #15. To establish OMA1 specificity, reporter cells were pre-incubated with 500. Mu.M phenanthroline (OMA 1 inhibitor) for 1 hour, followed by an additional 30 min incubation with 10. Mu.M CCCP (average.+ -. SD; n=4; one-way ANOVA: p < 0.001).

Fig. 5 shows temporal performance of Rep #01 and Rep #15 assays.

Fig. 5A shows the signal of rep#01 at the indicated time point after t=0 addition of luciferase substrate. Rep#01Hek293T cells were exposed to medium without or with 10. Mu.M CCCP for 30 min (mean.+ -. SD; n=4) before detection.

Fig. 5B shows the signal of rep#15 at the indicated time point after t=0 addition of luciferase substrate. Rep#15Hek293T cells were exposed to medium without or with 10. Mu.M CCCP for 30 min (mean.+ -. SD; n=4) prior to detection.

Fig. 5C shows the Z' values of rep#01 and rep#15 calculated at different times.

FIG. 6 compares Rep#15 with two different recognition sequences. Rep#15-S1 incorporates the cleavage site of OPA 1S 1. Wherein the TEV cleavage site replaces the S1 site in Rep#15-TEV as the recognition sequence. Both reporter genes were stably expressed in Neuro2A cells, both of which were exposed to increased concentrations of valinomycin for 30 minutes, and then their signals were detected. Both reporter genes showed comparable valinomycin dose dependency (mean ± SD; n=2).

FIG. 7 shows two different reporter genes and how they combine different functional fragments.

FIG. 8 compares the different Hek293 reporter cells in the western blot with and without CCCP.

FIG. 8A shows hydrolysis of Rep#01 (indicated by the arrow) in CCCP-treated cells. The cleavage products are also recognized by antibodies (indicated by asterisks).

FIG. 8B shows Rep#04 (indicated by the arrow) in untreated cells and CCCP-treated cells.

FIG. 8C shows Rep#08 (indicated by the arrow) in untreated cells and CCCP-treated cells.

FIG. 8D shows Rep#10 (indicated by the arrow) in untreated cells and CCCP-treated cells.

FIG. 8E shows hydrolysis of Rep#15 (indicated by the arrow) in CCCP-treated cells. The cleavage products are also recognized by antibodies (indicated by asterisks).

Fig. 9 provides an example of how to evaluate mitochondrial toxicity using Rep #01 or Rep # 15.

Figure 9A shows a significant decrease in bioluminescence of Rep #01 cells exposed to the indicated molecule for 30 minutes (mean ± SD; n=4; one-way ANOVA: p < 0.001).

Figure 9B shows a significant decrease in bioluminescence of Rep #15 cells exposed to the indicated molecule for 30 minutes (mean ± SD; n=4; one-way ANOVA: p < 0.001).

Figure 10 shows the dose response relationship of telanavir and kava factor in the Rep #15 assay.

Fig. 10A shows that Hek293T rep#15 cells were exposed to increased concentration of telanavir for 60 minutes followed by measurement of their bioluminescence (mean ± SD; n=4 ec ₅₀ :9μM)。

FIG. 10B shows that Hek293T Rep#15/Luke-S1 cells were exposed to elevated concentrations of kava for 60 minutes and their bioluminescence was measured (average.+ -. SD; n=2 EC ₅₀ :14μM)。

FIG. 11 shows the properties of luciferase-based OMA1 protease reporter.

FIG. 11A shows the design of the Luke-S1 reporter gene (i.e., rep#15) and the Luke-TEV reporter gene in which the S1 site is a TEV site substitution.

FIG. 11B shows the enzymatic kinetics of Luke-S1 in stably transfected Hek293T cells.

FIG. 11C shows the enzymatic kinetics of Luke-TEV in stably transfected Hek293T cells.

FIG. 11D shows the enzymatic kinetics of the unmodified native luciferase "Luke".

FIG. 12 shows that Luke-S1 and Luke-TEV are hydrolyzed under conditions where OPA1 is hydrolyzed and demonstrate mitochondrial migration.

FIG. 12A shows CCCP-dependent OPA1 hydrolysis in Hek293T cells after 30 minutes of exposure. In the Western blot, 3. Mu.M CCCP resulted in complete L-OPA1 proteolysis.

Fig. 12B shows valomycin-dependent OPA1 hydrolysis in Hek293T cells after 30 minutes exposure. In the Western blot, 0.1. Mu.M valinomycin resulted in complete L-OPA1 proteolysis.

FIG. 12C shows that 3. Mu.M CCCP and 0.1. Mu.M valinomycin (vmc) induced cleavage of Luke-S1 and Luke-TEV in Hek293T reporter cells.

FIG. 12D shows that Luke-S1 and Luke-TEV co-migrate with OPA1 and OMA1 in mitochondrial rich fragments (P) in a Western blot after cell debris by differential centrifugation. P, small particles; s, supernatant; vmc, valinomycin.

Figure 13 shows corresponding non-limiting and illustrative examples of different Hek293T reporter cells for CCCP and valinomycin after 30 minutes of incubation.

FIG. 13A shows the CCCP dose response curve of Luke-S1 cells.

FIG. 13B shows valinomycin dose response curves for Luke-S1 cells.

FIG. 13C shows the CCCP dose response curve of Luke-TEV cells.

FIG. 13D shows valinomycin dose response curves for Luke-TEV cells.

Fig. 13E shows CCCP dose response curves for Luke cells.

Fig. 13F shows valinomycin dose response curves for Luke cells.

Figure 14 provides additional data regarding the specificity and dynamic behavior of the reporter gene.

FIG. 14A demonstrates that knock-down of OMA1 prevents valinomycin-induced signal reduction in Luke-S1 assays. Valinomycin (vmc) resulted in a significant decrease in signal in Luke-S1 cells treated with control siRNA, but no decrease in signal occurred in Luke-S1 cells treated with OMA1 siRNA (n=3; one-way ANOVA: p=0.003).

FIG. 14B shows Western blot images of Luke-S1 cells treated with control siRNA (cntrl) or with the indicated antibody-tagged OMA1 siRNA. OMA1 levels were reduced by about 70%.

FIG. 14C shows the dynamic behavior of the Luke-S1 reporter gene. Bioluminescence was recorded at 20 second intervals over a period of 25 minutes after addition of luciferase substrate (either without or with 100nM valinomycin) to the cells. Note that in this assay, signal decay is a complex of the reporter enzyme inactivation over time and substrate consumption over time.

FIG. 14D shows the dynamic behavior of the Luke-TEV reporter gene.

Figure 14E shows the dynamic behavior of the Luke reporter gene.

FIG. 15 provides an illustrative example of drug screening for OMA1 activators, wherein Hek293TLuke-S1 cells were exposed to test compounds for 1 to 2 hours and signal density was compared to 100nM valinomycin treated cells.

FIG. 15A shows a representative legend for Luke-S1 cells with valinomycin treatment in columns #2 and #23 as positive control.

FIG. 15B compares untreated Luke-S1 cells with valinomycin treated Luke-S1 cells.

FIG. 15C shows that 1,280 chemically distinct molecules are ordered according to signal intensity. The signal was normalized to the average of 128 valinomycin treated samples, which was defined as 100%. Molecules with signals within three standard errors (SD, dashed line) of valinomycin treated cells are considered potent OMA1 activators.

FIG. 15D shows the signal distribution of 128 valinomycin treated samples and the signal distribution of 1,280 test molecules, the critical value (3 XSD) being shown by the dashed line.

FIG. 16 provides an illustrative example of drug screening for OMA1 inhibitors, wherein Luke-S1 cells were pre-incubated in test compound for 60 minutes followed by an additional 30 to 60 minutes incubation with 100nM valinomycin. The signal intensity was compared to untreated Luke-S1 cells.

FIG. 16A shows an exemplary graphical representation of untreated Luke-S1 cells in columns #2 and #23 as positive controls.

FIG. 16B shows the ordering of 3,520 chemically distinct molecules according to signal intensity. The signal was normalized to the average of 352 untreated samples, which was defined as 100%. Molecules with signals within three standard errors (SD, dashed line) of untreated cells are considered potent inhibitors of OMA 1.

Fig. 16C shows the signal distribution of 352 untreated samples and the signal distribution of 3,520 test molecules, with the critical value (3 x SD) shown by the dashed line.

FIG. 16D shows a six-point dose response curve of Luke-S1 cells for only one molecule crossing the threshold.

Figure 17 shows a list of FDA approved drugs that significantly reduced Luke-S1 bioluminescence by more than 37.5% in 166 cancer drugs screened.

FIG. 18 shows a non-limiting and exemplary example of a dose response curve for Hek293T Luke-S1 cells exposed to FDA approved drugs for 1 hour.

FIG. 18A shows the Pecibutatinib dose response curve for Luke-S1 cells.

Fig. 18B shows the exendin dose response curve of Luke-S1 cells.

FIG. 18C shows the Geranitinib dose response curve for Luke-S1 cells.

FIG. 18D shows the actinomycin D dose response curve of Luke-S1 cells.

FIG. 18E shows the plug Li Nisuo (Selinesor) dose response curve for Luke-S1 cells.

FIG. 18F shows the Lagrantinib dose response curve for Luke-S1 cells.

FIG. 18G shows the Orientinib dose response curve for Luke-S1 cells.

FIG. 18H shows the Rabosinib dose response curve for Luke-S1 cells.

FIG. 19 shows a non-limiting and illustrative example of a dose response curve for Hek293T Luke-S1 cells exposed to FDA approved drugs for 1 hour.

FIG. 19A shows daunorubicin hydrochloride dose response curves for Luke-S1 cells.

FIG. 19B shows the dapafinib mesylate dose response curve for Luke-S1 cells.

FIG. 19C shows the dose response curve of the graphically depicted calitinib for Luke-S1 cells.

FIG. 19D shows the bosutinib dose response curve of Luke-S1 cells.

FIG. 19E shows the Venetutock dose response curve for Luke-S1 cells.

FIG. 19F shows the mitotane dose response curve for Luke-S1 cells.

FIG. 19G shows the emtrictinib dose response curve for Luke-S1 cells.

FIG. 19H shows the ceritinib dose response curve for Luke-S1 cells.

FIG. 20 shows a non-limiting and exemplary example of a dose response curve for Hek293T Luke-S1 cells exposed to FDA approved drugs for 1 hour.

FIG. 20A shows the regorafenib dose response curve for Luke-S1 cells.

FIG. 20B shows ibrutinib dose response curves for Luke-S1 cells.

FIG. 20C shows the doxorubicin hydrochloride dose response curve for Luke-S1 cells.

FIG. 20D shows the cabozantinib dose response curve for Luke-S1 cells.

Fig. 20E shows tamoxifen citrate dose response curves for Luke-S1 cells.

FIG. 20F shows the dose response curve of the trametinib of Luke-S1 cells.

FIG. 20G shows the valrubicin dose response curve of Luke-S1 cells.

FIG. 20H shows the idarubicin hydrochloride dose response curve of Luke-S1 cells.

FIG. 21 shows a non-limiting and illustrative example of a dose response curve for Hek293T Luke-S1 cells exposed to FDA approved drugs for 1 hour.

Figure 21A shows the celecoxib dose response curve for Luke-S1 cells.

FIG. 21B shows the Pazopanib hydrochloride dose response curve for Luke-S1 cells.

FIG. 21C shows the imatinib dose response curve for Luke-S1 cells.

FIG. 21D shows the sorafenib dose response curve for Luke-S1 cells.

Figure 21E shows the raloxifene dose response curve for Luke-S1 cells.

FIG. 21F shows the sunitinib dose response curve for Luke-S1 cells.

Detailed Description

Before the present invention is described in detail, it is to be understood that this invention is not limited to particular formulations or process parameters, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting of the invention. Preferred materials and methods are described herein, as are various aspects and materials similar or equivalent to those described herein, which can also be used in the practice of the present invention.

The compositions and methods provided herein relating to reporter genes and assays are useful in a variety of different fields, including basic research, drug research, molecular diagnostics, and the like, although the reporter genes and assays described herein are limited to any particular application and any useful application should be considered as being within the scope of the invention, drug development being one example of the utility of the invention.

Mitochondria are a dynamic organelle that form an interconnected tubular network, and mitochondria maintain homeostasis by continuing fusion and differentiation. Thus, fragmented mitochondria are more prone to cause apoptosis, while fused mitochondria show tolerance to stress.

OPA1 is an essential fusion protein which in principle exists in both the larger L-OPA1 isoform, which anchors to the mitochondrial inner membrane, and the smaller S-OPA1 isoform, which does not have a transmembrane domain. S-OPA1 is derived from the proteolytic cleavage of L-OPA1 by OMA1 protease and YME1L1 protease, also known as i-AAA protease. L-OPA1 is recognized as essential for mitochondrial fusion. On the other hand, S-OPA1 is thought to play a role in permeabilization of the outer membrane of mitochondria and release of cytochrome c, since cleavage of L-OPA1 is highly correlated with programmed cell death.

OMA1 is a mitochondrial inner membrane protease of the MEROPS M48 family of zinc-metal endopeptidases (see Rawlings ND, et al, nucleic Acids Res (2014) 42 (Database issue): D503-D509). OMA1 cleaves substrates (e.g., DELE 1) involved in the signal pathway that conducts signals to the integrated stress response. OMA1 protease cleaves the OPA1 fusion protein, thereby producing S-OPA1 under broad cellular stress conditions, and thus, OMA1 activation favors outer membrane permeabilization and cytochrome c release, which ultimately leads to apoptosis.

It is well known in the art that mitochondrial dysfunction (or a corresponding mitochondrial disease or disorder) is associated with a decrease in L-OPA 1. However, in this context, additional possible OPA1 isoforms or other proteins (e.g., delete 1, PGAM5 and PINK 1) may be altered.

In this context, it should be understood that the OPA1 isoform is merely indicative of protease activity, in particular of OMA1 protease activity. Thus, the invention is not limited to modulation of OPA1 isoforms, but also includes any and every other indicator of modulation of OMA1 activity, including other OMA1 substrates, e.g., del 1, PGAM5 or PINK1. Those skilled in the art can also infer other amino acid chains/polypeptides as substrates for (artificial) OMA1, which are also within the scope of the invention.

In contrast, the compounds disclosed herein may modulate the proportion of the OPA1 isoform by directly or indirectly interacting with an OMA1 protease, e.g., by interacting with a protein complex comprising OMA1, or by interacting with cleavable OPA1 and/or other proteases of OMA1 (e.g., i-AAA proteases), or by interacting with an OMA 1-modulating enzyme (e.g., m-AAA protease or antiproliferative protein) (see Alavi m.v. biochem Biophys Acta Proteins protein 2020oct 29: 14058).

OMA1 protease is a very desirable drug target with a variety of disease characterizations supported by epidemiological and genetic data in human and animal disease models. Moreover, there is no specific OMA1 inhibitor or activator currently available, let alone any drug targeting OMA1 protease, other than the drugs disclosed herein by the inventors of the present invention and in U.S. Pat. No. 10906931B 2.

The problems encountered in developing OMA1 modulators are two problems (dual problems): (1) OMA1 protease is quite cluttered in its substrate recognition and there is currently no clear consensus for cleavage sites. This makes it almost impossible to rationally design an OMA1 inhibitor based on the substrate recognition site; (2) OMA1 protease undergoes autoproteolysis once activated, which makes it almost impossible to isolate functional proteins for in vitro enzymatic detection. Thus, there is currently no specific OMA1 modulator that can be used.

Most protease assays are based on Fluorescence Resonance Energy Transfer (FRET) from a donor fluorophore to a quencher at the opposite end of a short peptide chain containing a potential cleavage site (please see Knight CG, methods in enzymol. (1995) 248:18-34). Proteolysis separates the fluorophore and quencher, thereby allowing the luminescence intensity of the donor fluorophore to be increased.

This general principle can also be used to design OMA1 protease assays using the "S1" protease cleavage site of the rat OPA1 protein, which has the amino acid sequence: ala-Phe-Arg-/-Ala-Thr-Asp-His-Gly (A-F-R-/-A-T-D-H-G), wherein "-/-" represents a scissile bond cleaved by OMA 1. Hydrolysis of the scissile bond between the donor/acceptor pair of the FRET peptide produces so-called fluorescence that allows measurement of OMA1 protease activity.

Such FRET-based assays are known in the art (see U.S. patent No. 10739331B 2). However, such FRET-based detection is not useful for measuring the activity of OMA 1. The problems are that: the linker sequence Ala-Phe-Arg-Ala-Thr-Asp-His-Gly is recognized by a variety of proteases including the intracellular proteases Arg-C, intracellular proteases Asp-N, chymotrypsin, clostripain, pepsin, proteinase K, thermolysin and trypsin. Thus, FRET-based assays exhibit very little to no specificity when used with natural cell debris. Highly purified functional OMA1 protease is an essential component for achieving specificity in such in vitro assays. As already mentioned above, the isolation of functional OMA1 enzymes for in vitro detection is inherently challenging, since OMA1 proteases will self-digest after activation (see Baker MJ et al, EMBO Journal (2014) 33 (6): 578-593;Zhang K et al, EMBO Reports (2014) 15 (5): 576-585). FRET-based assays are also limited to in vitro use due to the specific nature of the fluorophore and quencher. Fluorophores cannot be gene-encoded and therefore cannot be used in vivo, which can increase specificity by, for example, localizing the reporter gene to mitochondria.

Another limitation of FRET-based in vitro detection is that the detection generates a signal after OMA1 activation. This means that higher OMA1 activity produces higher fluorescence. In other words, there is a positive correlation between OMA1 protease activity and the detected signal. This makes this type of detection less desirable for drug screening because a higher false positive rate is generated in screening for OMA1 inhibitors. All interfering signal compounds will be considered potential OMA1 inhibitors, whether these selected compounds inhibit OMA1 or simply quench the FRET donor. Moreover, in vitro assays have only limited predictive value for in vivo pharmacodynamics of a particular selected compound, as, for example, cell permeability is not within the considerations of such assays.

The present invention solves the problem in the detection of a specific OMA1 protease by targeting a reporter gene or enzyme to the inner mitochondrial membrane, wherein the reporter gene or enzyme is recognized by the OMA1 protease and the activity of the reporter gene or enzyme is altered by the OMA1 protease. These new and innovative target-based in vivo protease assays have been shown to be inversely related to OMA1 protease activity, thereby overcoming some of the limitations of current assays for OMA1 activity. The assays disclosed herein can be used in vivo, which are very effective and suitable for high throughput drug screening (as exemplified in the examples).

Targeting the OMA1 reporter gene to mitochondria may appear to be obvious. However, it has not been achieved, and many of those skilled in the art have attempted this but have not been successful. The problem here is that OPA1 introduced sequences (which may be relatively obvious to try) cannot be used by themselves to migrate the OMA1 reporter gene to the inner mitochondrial membrane. The invention discovers the construction of OMA1 reporter gene. The reporter gene of the present invention is characterized by the absence of the mitochondrial import sequence of OPA1, which is a departure from all teachings of the prior art (see US10739331B 2). Unexpectedly, the reporter migrates to the mitochondrial inner membrane with higher efficiency, wherein when the first 80 to 90 amino acids of the OPA1 amino acid terminus are deleted, the reporter is recognized by OMA1 protease and cleaved.

The present invention provides a novel reporter gene that is capable of detecting OMA1 protease activity in vivo after expression in a host. These synthetic genes are constructed in modular fashion and operatively incorporate the following individual components: (a) a targeting signal; (b) An entire enzyme group or protein domain or an N fragment of an enzyme group or protein domain; (c) An entire enzyme group or protein domain or a C fragment of an enzyme group or protein domain corresponding to the N fragment; and (d) a hydrolyzable sequence motif recognizable by OMA1 protease, whereby the complementation of N and C results in a signal that can be measured (see the accompanying figures). The present invention provides further synthesized mitochondrial import signals that can target polypeptides or reporter genes to the inner mitochondrial membrane.

The embodiments described herein may be used for drug screening and/or drug development. For example, interactions of a small molecule drug or entire library of small molecules with a target protein of interest (e.g., therapeutic target) are monitored under one or more relevant conditions (e.g., physiological conditions, disease conditions, etc.). In other embodiments, the ability of a small molecule drug or an entire library of small molecules to promote or inhibit interactions between two groups is tested. In some embodiments, drug screening applications are performed in a high throughput format to allow detection of binding of tens of thousands of different molecules to a target, or to detect the role of those molecules in binding to other groups.

In some embodiments, the invention provides for detecting molecular interactions in organisms (e.g., bacteria, yeast, eukaryotic cells, mammals, primates, humans, etc.) and/or cells. In some embodiments, a fusion protein comprising a signal and an interaction (target) polypeptide are co-expressed in a cell or whole organism, the signal is detected and the signal is correlated with the formation of an interaction complex. In some embodiments, the cells are transiently and/or stably transformed or transfected with a vector encoding a non-fluorescent moiety, an interactive moiety, a fusion protein (e.g., comprising a signal and an interactive moiety), or the like. In some embodiments, transgenic organisms are produced to encode a desired reporter gene for use in performing the assays described herein. In other embodiments, the vector is injected into the whole organism. In some embodiments, transgenic animals or cells (e.g., expression reporter genes) are used to monitor or measure mitochondrial toxicity of small molecules or biomolecules.

In a particular embodiment of the invention, reporter #15 combines (a) a synthetic polypeptide sufficient for and necessary for mitochondrial induction, (b) an N-terminal domain and (C) a C-terminal domain of NanoLuc luciferase, and (d) exon 5 of OPA1, which is capable of emitting a light signal upon incorporation, exon 5 of OPA1 encoding a polypeptide that is recognized by OMA1 protease. Thus, the OPA1 exon 5 is located inside the reordered NanoLuc enzyme, such that the OPA1 exon 5 operably links the C-terminal domain to the N-terminal domain (see, US9757478B2, US10107800B2, US9339561B2 and US10077433B 2). Activation of OMA1 separates the two domains, thereby inactivating the NanoLuc enzyme. In a non-limiting example, reporter gene #15 is expressed in Hek293T cells under the control of a CMV promoter and functions as desired.

In another embodiment of the invention, reporter gene #01 combines (a) a portion of the amino terminal domain of OPA1 sufficient for mitochondrial induction, (b) NanoLuc C-terminus, (C) exon 5 of OPA1, and (d) NanoLuc N-terminus. Likewise, exon 5 links the reordered NanoLuc sequences in such a way that activation of OMA1 would inactivate the NanoLuc luciferase. In a non-limiting example, reporter gene #01 is expressed in Hek293T cells, particularly under the control of the CMV promoter, and appears to function as desired.

The disclosure also provides a method of using a reporter gene in a cellular assay for screening for potential OMA1 modulators. In a non-limiting example, reporter #15 is used to screen for potential OMA1 inhibitors. For this purpose, reporter #15 was transiently expressed in Hek293T cells, which resulted in a stronger bioluminescence signal. OMA1 enzyme was dormant in these cells under physiological cell culture conditions, but OMA1 enzyme could be readily activated by the addition of CCCP (carbonyl cyanide m-chlorophenylhydrazone) to the cell culture medium. Incubation of transfected cells with 10 μm CCCP in cell culture medium for 30 min resulted in a significant reduction or substantial disappearance of luciferase activity relative to untreated control cells. The OMA1 inhibitor phenanthroline is capable of antagonizing the effects of CCCP on cells. Pre-incubation with 500. Mu.M phenanthroline for 1 hour prior to treatment with CCCP prevents inactivation of reporter #15 and maintains bioluminescence signal. This demonstrates that the detection methods disclosed herein are very useful for screening for OMA1 inhibitors.

In addition, the present invention provides a method for detecting compounds with potential mitochondrial toxicity. Some drugs exhibit mitochondrial toxicity, which can produce undesirable side effects in patients, limiting the use of these drugs. It is known in the art that toxic drugs such as sorafenib can act through the OMA1 pathway (Zhao X, et al, laboratory Investigation (2013) 93 (1): 8-19). In a non-limiting example, sorafenib inactivated reporter #15, which resulted in a significant decrease in signal in the assay, the effect of which was comparable to that observed in CCCP. Sorafenib also has a comparable effect in the detection performed by reporter gene # 01.

Moreover, other drugs intended for different markers and known to have mitochondrial toxicity may activate the OMA1 pathway. Mitochondrial toxicity can ultimately lead to complications in patients. Telanavir is a drug known in the art to cause mitochondrial toxicity, which is also listed as a side effect in the FDA label of Aptivus (telanavir). In another non-limiting example, telanavir inactivates reporter gene #15 and reporter gene #01 in the assays of the present invention, thereby exhibiting mitochondrial toxicity. This provides proof of principle to demonstrate the utility of the detection method of the present invention in identifying potential mitochondrial toxicity. Other examples are provided in example 6 and in the claims.

Also provided herein are methods of detection in a scalable microtiter format suitable for high throughput screening of compound libraries. In another non-limiting example, we demonstrate that our assay method is very effective in high throughput screening of compound libraries and can be modified for use in high throughput screening of compound libraries.

The invention also discloses a medicine with OMA1 regulating performance. These drugs have been approved by regulatory authorities for use in humans to treat some malignant diseases and have been identified by the OMA1 detection method of the invention. Methods of chemical synthesis of pharmaceutically active ingredients of the medicaments of the present invention and processes for preparing pharmaceutical compositions comprising said pharmaceutically active ingredients are well known in the art. The FDA labels for these drugs and the FDA labels for each and all other drugs described herein are incorporated by reference and may be used in the practice of the present invention. Based on the teachings provided herein, the documents incorporated herein and the teachings provided in US10906931B2, one of skill in the art can use the medicaments disclosed herein to treat patients suffering from diseases characterized by altered levels or activity of OMA 1. Conversely, it should be appreciated that those skilled in the art, based on the disclosure herein and the examples provided herein, are also able to identify those subjects who are unable to benefit from treatment with a drug having OMA 1-modulating properties or when to discontinue treatment with a drug having OMA 1-modulating properties due to an increased risk of adverse events. In this context, it is very clear that the detection methods disclosed herein impose certain limitations on side effects for the design and development of improved drugs or therapies that can avoid the activation of OMA 1. Moreover, the detection methods disclosed herein are very useful in developing drugs that activate OMA1, e.g., activate OMA1 in cancer cells, thereby inhibiting tumor growth.

Paraphrasing meaning

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any method or material similar or equivalent to the methods or materials disclosed herein can be used in the practice of the present invention. For the purposes of the present invention, the following terms are defined below.

Reference herein to an aspect, an embodiment, an exemplary embodiment, etc., means that the aspect described may include a particular feature, structure, or characteristic, but every aspect may not necessarily include the particular feature, structure, or characteristic. Moreover, some phrases are not necessarily referring to the same aspect. Further, when a particular feature, structure, or characteristic is described in connection with an aspect, it is submitted that it is within the knowledge of one skilled in the art to effect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.

The indefinite articles "a," "an," and "the" as used herein and in the claims include a plurality of meanings unless specifically indicated otherwise. For example, "cell" also refers to a plurality of cells, and so forth.

The term "and/or" as used herein and in the claims means that the phrases preceding and following the term can be considered to be either or a combination of both.

The term "OPA1" as used herein refers to a mitochondrial motility-like protein (mitochondrial dynamin-like protein) encoded by the OPA1 gene in eukaryotic cells. OPA1 is defined as broadly as possible and will include all natural and non-natural variants and homologues thereof from any and every species.

The term "OMA1" as used herein is known in the art and refers to an inner mitochondrial membrane protease encoded by the OMA1 gene within eukaryotic cells. OMA1 is defined as broadly as possible and will include all natural and non-natural variants and their homologs from any and every species.

The term "YME1L1" as used herein is known in the art as a component of the mitochondrial inner membrane i-AAA protease and is encoded by the YME1L1 gene within eukaryotic cells. YME1L1 is defined as broadly as possible and will include all natural and non-natural variants and homologs thereof from any and every species.

The terms "compound," "molecule," "chemical," "agent," "reagent," "modulator," and the like refer to any substance, chemical, composition, or extract that has a positive or negative biological effect on a cell, tissue, body fluid, or within any biological system or within any detection system.

The term "nucleic acid molecule" or "polynucleotide" as used herein refers to ribonucleic acid or deoxyribonucleic acid in single-or double-stranded form, and unless otherwise explicitly indicated, the term "nucleic acid molecule" or "polynucleotide" encompasses polynucleotides comprising known analogs of naturally occurring nucleotides that can function in a manner analogous to naturally occurring nucleotides. It will be appreciated that when a nucleic acid molecule is represented by a DNA sequence, it also includes RNA molecules having a corresponding RNA sequence in which U (uracil) replaces T (thymine).

The term "recombinant nucleic acid molecule" as used herein refers to a non-naturally occurring nucleic acid molecule comprising two or more polynucleotide sequences. Recombinant nucleic acid molecules can be prepared by recombinant methods, in particular by genetic engineering techniques, or can be prepared by chemical synthesis methods. The recombinant nucleic acid molecule may encode a fusion protein, e.g., a reporter protein of the invention linked to a polypeptide of interest.

The term "recombinant host" or "host" as used herein refers to a cell comprising a recombinant nucleic acid molecule. Thus, recombinant host cells can express polypeptides from "genes" that are not found in natural cells (non-recombinant cells). The recombinant host cell may be transiently transfected or stably transformed or transfected with one or more vectors encoding a nucleic acid molecule (e.g., a reporter gene) for recombination. It should be understood that the recombinant host may be produced by any method, which may also include methods not specifically mentioned herein.

As used herein, a polynucleotide "encodes" a polypeptide refers to the production of the polypeptide after transcription of the polynucleotide and translation of the mRNA produced thereby. The encoded polynucleotide is considered to include the coding strand and its complementary strand, which has a nucleotide sequence equivalent to that of mRNA. Polynucleotides so encoded are considered to include degenerate nucleotide sequences which encode identical amino acid residues. The nucleotide sequence encoding the polypeptide may include a polynucleotide comprising an intron and encoding an exon.

The term "expression control sequence" as used herein refers to a nucleotide sequence that modulates the transcription or translation of a polynucleotide or the position of a polypeptide to which it is operably linked. An expression control sequence is "operably linked" to a particular chamber of a cell when it controls or modulates the transcription of the position or nucleotide sequence of the encoded polypeptide and the translation (i.e., transcription or translation of the regulatory element) as appropriate. Thus, an expression control sequence may be a promoter, an enhancer, a transcription terminator, an initiation codon (ATG), a splicing signal for intronic excision and maintenance of the correct reading frame, a stop codon, a ribosome binding site or a sequence which targets a polypeptide to a particular location (e.g., a particular cell compartment).

The terms "targeting signal" or "targeting peptide" or "targeting sequence" and the like as used herein refer to a polypeptide that can be targeted to the cytosol, nucleus, plasma membrane, endoplasmic reticulum, mitochondrial outer membrane, mitochondrial inner membrane, space or matrix between mitochondrial membranes, chloroplast outer membrane or thylakoid membrane, space or lumen between membranes, medial trans-Golgi cisternae, or lysosomes or endosomes. Cellular compartment domains are well known in the art and include, for example, peptides containing 1 to 81 amino acid residues of human type II membrane anchored protein galactose transferase, or peptides containing 1 to 12 amino acid residues of the pro sequence of subunit IV of cytochrome oxidase (see Hancock et al, EMBO J. (1991) 10:4033-4039; buss et al, mol. Cell. Biol. (1988) 8:3960-3963; U.S. Pat. No. 5,776,689, both of which are incorporated herein by reference).

The term "mitochondrial targeting signal" or "mitochondrial signal transduction peptide" or "mitochondrial import sequence" and the like as used herein refers to a peptide or polypeptide that can target a polypeptide to mitochondria. It will be appreciated that such mitochondrial targeting sequences may direct polypeptides or proteins to the outer or inner mitochondrial membrane or the space between mitochondrial membranes or to the mitochondrial matrix, depending on the nature of the mitochondrial targeting sequence. The mitochondrial targeting signal may be a naturally occurring, recombinant, or mutated sequence located anywhere within the polypeptide or protein. An example of a naturally occurring mitochondrial targeting signal is the N-terminal region comprising amino acid residues 1 to 87 of the human OPA1 protein (NCBI Reference Sequence: NP-056375.2 from 11-Jul-2020). Other mitochondrial inlet sequences are known in the art (see, e.g., WO2006117250A2; US9540421B2; US6316652B1; US20110245146A1; US9932377, all of which are incorporated herein by reference). The invention discloses a synthetic mitochondrial import sequence.

The terms "operably linked" or "operably linked" and the like, as used herein in reference to a synthetic polypeptide or chimeric protein, refer to a sequence of polypeptides in a position such that they are in physical and functional relationship with one another. In a most preferred embodiment, the functional activity of the polypeptide component of the chimeric molecule is unchanged relative to the functional activity of the isolated portion. For example, the synthetic mitochondrial import signal of the invention may be incorporated into a polypeptide of interest. In this case, it is preferable that the fusion molecule maintains the function of the mitochondrial import signal and migrates the fusion protein to the mitochondria, and the target polypeptide maintains its original biological activity. In some embodiments of the invention, the activity of the mitochondrial import signal or protein of interest may be reduced relative to its activity in the isolated state. These fusions can also be used in the present invention.

The term "polypeptide" or "protein" as used herein refers to a polymer of four or more amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding naturally occurring amino acid as well as naturally occurring amino acid polymers. The term "recombinant protein" refers to a protein produced by expression of a nucleotide sequence encoding an amino acid sequence of a protein from a recombinant DNA molecule.

The terms "peptide" and "polypeptide" as used herein refer to a polymeric compound of two or more amino acids linked by peptide amino bonds (-C (O) NH-) through the backbone, unless specifically indicated otherwise. The term "peptide" generally refers to a short amino acid polymer (e.g., having a chain of less than 25 amino acids), while the term "polypeptide" generally refers to a longer amino acid polymer (e.g., having a chain of more than 25 amino acids).

The terms "wild-type", "naturally occurring", and the like, as used herein, refer to naturally occurring proteins, nucleic acid molecules, cells, or other materials. For example, a polypeptide or polynucleotide sequence that is present in an organism, including viruses. Naturally occurring materials may be in a form in which they exist in nature, and may be artificially modified, e.g., in an isolated form.

The terms "synthetic," "artificial," "non-naturally occurring," and the like as used herein refer to polypeptides, proteins, nucleic acid molecules, cells, or other materials that are not found in nature. For example, the mitochondrial import signals and fusion proteins provided herein are non-naturally occurring in that they are composed of fragments of OPA1 protein or variants thereof, and are not found isolated from the remainder of the naturally occurring protein.

The term "identical" as used herein when applied to two or more nucleotide sequences or two or more polypeptide sequences refers to the residues in the sequences being identical when the sequences are aligned for maximum correspondence. When describing polypeptides using percentages of sequence identity, it is understood that non-equivalent one or more residue positions may differ by conservative amino acid substitutions, wherein one amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., similar charge or hydrophobicity or hydrophilicity), and thus the functional properties of the polypeptide are not altered. In the event that the sequence of the polypeptide changes in conservative substitutions, the percent sequence identity may be up-regulated to correct the nature of the conservative substitution. Such adjustments can be made using methods known in the art, for example, scoring conservative substitutions as partial mismatches, rather than complete mismatches, thereby increasing the percent sequence identity. Thus, for example, where an equivalent amino acid is scored as 1 and a non-conservative substitution is scored as 0, the conservative substitution is scored as between 0 and 1. Scoring of conservative substitutions may be calculated using any known algorithm (see, e.g., meyers and Miller, comp. Appl. Biol. Sci. (1988) 4:11-17;Smith and Waterman,Adv.Appl.Math. (1981) 2:482;Needleman and Wunsch,J.Mol.Biol. (1970) 48:443;Pearson and Lipman,Proc.Natl.Acad.Sci., (1988) 85:2444;Higgins and Sharp,Gene (1988) 73:237-244;Higgins and Sharp,CABIOS (1989) 5:151-153; corpet et al, nucleic acids Res. (1988) 16:10881-10890;Huang,et al., (Comp. Appl. Biol. Sci. (1992) 8:155-165,1992;Pearson et al., (1994) 24:307-331). Alignment can also be performed by simple visual inspection and manual sequence alignment.

The term "sequence identity" as used herein refers to the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have the same sequential composition of monomer subunits. The term "sequence similarity" refers to the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have similar polymer sequences. For example, similar amino acids are those that share the same biophysical properties and can be grouped into families, e.g., acidic amino acids (e.g., aspartate, glutamate), basic amino acids (e.g., lysine, arginine, histidine), nonpolar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar amino acids (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). "percent sequence identity" (or "percent sequence similarity") is calculated as follows: (1) comparing two optimally aligned sequences over a comparison window (e.g., length of longer sequence, length of shorter sequence, length of specific window), (2) determining the number of positions containing identical (or similar) monomers (e.g., identical amino acids occur in both sequences, similar amino acids occur in both sequences) to obtain the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., length of longer sequence, length of shorter sequence, length of specific window), and (4) multiplying the result by 100 to obtain the percent sequence identity or percent sequence similarity. For example, if peptide a and peptide B are each 20 amino acids in length and have identical amino acids, the amino acids at only one position are not identical, then the sequence identity of peptide a and peptide B is 95%. If the amino acids at non-equivalent positions share the same biophysical properties (e.g., are both acidic amino acids), then peptide A and peptide B have 100% sequence similarity. As another example, if peptide C is 20 amino acids in length, peptide D is 15 amino acids in length, and 14 of the 15 amino acids of peptide D are identical to the amino acids of this portion of peptide C, then peptide C and peptide D have 70% sequence identity, but peptide D has 93.3% sequence identity to the optimal window of comparison of peptide C. For purposes of calculating the "percent sequence identity (or percent sequence similarity)" herein, any interval in aligned sequences is considered a mismatch at that position.

When the term "conservatively modified alterations" is used herein to describe a particular polynucleotide sequence, it refers to a different polynucleotide sequence encoding the same or substantially the same amino acid sequence, or when the polynucleotide does not encode an amino acid sequence, it refers to a substantially the same sequence. Because of the degeneracy of the genetic code, a large number of functionally equivalent polynucleotides encode any given polypeptide. For example, codons CGU, CGC, CGA, CGG, AGA and AGG both encode the amino acid arginine. Thus, at each position where arginine is specified by a codon, the codon can be changed to any of the corresponding codons described above without changing the encoded polypeptide. Such a change in nucleotide sequence is a "silent change," which may be considered a "conservatively modified change. Thus, it is to be understood that each polynucleotide sequence disclosed herein as encoding a reporter protein variant also describes each possible silent change. It will also be appreciated that each codon in the polynucleotide (except AUG, which is typically the only codon for methionine, and UUG, which is typically the only codon for tryptophan) can be modified by standard techniques to a functionally equivalent molecule. Thus, each silent change in a polynucleotide that does not alter the sequence of the encoded polypeptide is implicitly described herein. Moreover, it should be understood that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage (typically less than 5%, often less than 1%) of amino acids in the encoded sequence may be considered conservative modifications, as long as the change results in the substitution of an amino acid by a chemically similar amino acid.

Conservative amino acid substitutions that provide functionally similar amino acids are known in the art. Different groupings of amino acids may be considered conservative substitutions of each other based on the function of the particular amino acid, i.e., catalytically important function, structurally important function, spatially important function. The following list provides groupings of amino acids that are considered conservatively substituted based on their charge and polarity: (1) H, R and K; (2) D and E; (3) C, T, S, G, N, Q and Y; (4) A, P, M, L, I, V, F and W. The following list provides groupings of amino acids based on which hydrophobicity of the amino acids is considered a conservative substitution: (1) D, E, N, K, Q and R; (2) C, S, T, P, G, H and Y; (3) A, M, I, L, V, F and W. The following list provides groupings of amino acids whose surface exposure/structural properties are considered conservatively substituted based on amino acids: (1) D, E, N, K, H, Q and R;

(2) C, S, T, P, A, G, W and Y; (3) M, I, L, V and F. The following list provides groupings of amino acids based on their secondary structural propensity to be considered conservatively substituted: (1) A, E, Q, H, K, M, L and R; (2) C, T, I, V, F, Y and W; (3) S, G, P, D and N. The following list provides groupings of amino acids that are considered conservatively substituted based on the evolutionary conservation of amino acids: (1) D and E; (2) H, K and R; (3) N and Q; (4) S and T; (5) L, I and V; (6) F, Y and W; (7) A and G; (8) M and C.

Two or more amino acid sequences or two or more nucleotide sequences are considered substantially identical or substantially similar if they share at least 80% sequence identity with each other or with a reference sequence over a given comparison window. Thus, substantially similar sequences include those having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity. In some embodiments, substantially similar sequences have at least 75%,76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%, or 99% sequence identity.

A target nucleotide sequence may be considered to be substantially complementary to a reference nucleotide sequence if the target nucleotide sequence is substantially identical to the reference nucleotide sequence. The term "stringent conditions" refers to the temperature and ionic conditions used in nucleic acid hybridization reactions. Stringent conditions are sequence-dependent and will be different under different environmental parameters. In general, stringent conditions are selected to be about 5℃to 20℃below the defined ionic strength and thermal melting point (Tm) for the specific sequence under pH conditions. Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched label under defined ionic strength and pH conditions.

The term "variant" as used herein refers to the genetic variation morphology (polymorphic forms) of a gene at a particular genetic locus, as well as cdnas from mRNA transcripts of the gene and polypeptides encoded thereby. The term "preferred mammalian codon" refers to a subset of codons in a codon set encoding an amino acid most commonly used in a protein expressed in a mammalian cell, the codon set selected from the group consisting of: gly (GGC, GGG); glu (GAG); asp (GAC); val (GUG, GUC); ala (GCC, GCU); ser (AGC, UCC); lys (AAG); asn (AAC); met (AUG); ile (AUC); thr (ACC); trp (UGG); cys (UGC); tyr (UAU, UAC); leu (CUG); phe (UUC); arg (CGC, AGG, AGA); gln (CAG); h is (CAC); and Pro (CCC).

The term "substantially" as used herein means that the recited characteristics, parameters, and/or values do not require complete implementation, but that the resulting deviations or variables (including, for example, tolerances, measurement errors, measurement accuracy limitations and other factors known to those of skill in the art) do not materially affect the intended effect of the recited characteristics. The substantially non-existent property or characteristic (e.g., substantially non-luminescent) may be a property or characteristic within noise, below the detection limit of the detection used, or a fraction (e.g., <1%, <0.1%, <0.01%, <0.001%, <0.00001%, < 0.0000001%) of a significant property (e.g., luminescent intensity of a bioluminescent protein or bioluminescent complex).

The term "complementary" or "complementary" as used herein refers to the nature of two or more structural components (e.g., peptides, polypeptides, nucleic acids, small molecules, etc.) that are capable of undergoing hybridization, dimerization, or other reactions that form a complex with each other. For example, a "complementary peptide and polypeptide" can together form a complex. Complementary components may be required to assist in forming the complex (e.g., by interacting components), e.g., by placing components in a suitable conformation for complementation, by co-locating complementary components, by reducing interaction energy for complementation, etc. The complementary components may spontaneously form complexes within a suitable proximity of each other.

The term "complex" as used herein refers to aggregated or pooled molecules (e.g., peptides, polypeptides, small molecules, etc.) that are in direct and/or indirect contact with each other. In one aspect, "contact" or more specifically "direct contact" refers to the two or more molecules being sufficiently close that attractive non-covalent interactions, such as van der Waals forces, hydrogen bonding forces, ionic interactions, hydrophobic interactions, and the like, become the dominant forces for interactions between the molecules. In this aspect, the complex of molecules is formed under detection conditions whereby the complex is thermodynamically favored (e.g., unagglomerated or uncomplexed relative to the state of its constituent molecules). The term "complex" as used herein refers to the aggregation of two or more molecules (e.g., peptides, polypeptides, small molecules, or any combination thereof), unless otherwise specified.

The term "bioluminescence" as used herein refers to the production and emission of light by or capable of being catalyzed by an enzyme, protein complex, or other biological molecule (e.g., bioluminescent complex). Examples of such enzymes (bioluminescent enzymes) include Oplophorus luciferase, firefly luciferase, beetle luciferase, renilla luciferase, jellyfish photoprotein, obelin photoprotein, and the like. In typical embodiments, the substrate of the bioluminescent enzyme is converted and emits light in the form of bioluminescence.

The terms "light emitting enzyme", "bioluminescent enzyme" or "luciferase" are used interchangeably herein to refer to a class of oxidase enzymes used in bioluminescence wherein the enzyme produces and emits light upon a given substrate. The luciferase may be naturally occurring, recombinant, or mutant using a luciferase substrate. The luciferase substrate may be luciferin, a luciferin derivative or analogue, a pre-luciferin (pre-luciferin) derivative or analogue, coelenterazine, or a derivative or analogue of coelenterazine. If the luminescent enzyme is naturally occurring, it can be readily obtained from an organism by a person skilled in the art. Those skilled in the art are able to further adjust, modify or alter the properties of the bioluminescent enzyme to further enhance bioluminescence or other properties in the present invention (see for example US 10202584). If the luminescent enzyme is a naturally occurring or recombinant or mutant luminescent enzyme (e.g., an enzyme that retains activity in the luciferase-coelenterazine reaction or the luciferase-luciferin reaction of a naturally occurring luminescent enzyme), it may be readily active by bacterial culture, yeast culture, mammalian cell culture, insect cell culture, plant cell culture, and the like, and may be transformed to express a nucleic acid encoding the luminescent enzyme. Further, the recombinant or mutated light emitting enzyme may be derived from an in vitro cell-free system using a nucleic acid or variant, recombinant or mutant thereof.

The term "non-luminescent" as used herein refers to an entity (e.g., peptide, polypeptide, complex, protein, etc.) that exhibits properties that do not emit a detectable amount of light in the visible spectrum (e.g., in the presence of a substrate). For example, an entity may be said to be non-luminescent if the entity does not exhibit detectable luminescence in a given detection. The term "non-luminescent" as used herein is synonymous with the term "substantially non-luminescent". For example, a non-luminescent polypeptide (NLpoly) is substantially non-luminescent, exhibiting, for example, a 10-fold or more decrease in fluorescence (e.g., 100-fold, 200-fold, 500-fold, 1X 103-fold, 1X 104-fold, 1X 105-fold, 1X 106-fold, 1X 107-fold, etc.) relative to a complex of NLpoly with its non-luminescent complementary peptide. In some embodiments, a group is "non-luminescent" if any light emission is small enough not to interfere with the background of a particular detection.

The terms "non-luminescent peptide" and "non-luminescent polypeptide" as used herein refer to the following peptides and polypeptides: which exhibits substantially no luminescence (e.g., substantially no luminescence in the presence of a substrate) or an amount of luminescence that is 10-fold or more (e.g., 100-fold, 200-fold, 500-fold, 1 x 103-fold, 1 x 104-fold, 1 x 105-fold, 1 x 106-fold, 1 x 107-fold, etc.) lower than that of a significant signal (e.g., a luminescent complex) under standard conditions (e.g., physiological conditions, detection conditions, etc.) and under conditions using typical instruments (e.g., photometers, etc.). In some embodiments, such non-luminescent peptides and polypeptides are assembled according to the standards described herein to form bioluminescent complexes. The term "non-luminescent element" as used herein is a non-luminescent peptide or a non-luminescent polypeptide. The term "bioluminescent complex" refers to a complex of two or more non-luminescent peptides and/or polypeptides that are assembled. The bioluminescent complex catalyzes or is capable of converting a substrate of the bioluminescent complex into an unstable situation, which then emits light. When no recombination is performed, the two non-light emitting elements forming the bioluminescent complex may be referred to as a "non-light emitting pair". If the bioluminescent complex is formed from three or more non-luminescent peptides and/or non-luminescent polypeptides, the uncomplexed component of the bioluminescent complex may be referred to as a "non-luminescent group".

The term "fluorescent protein" as used herein refers to any protein that fluoresces when excited by suitable electromagnetic radiation, except chemically labeled proteins, wherein fluorescence due to chemical labeling is not considered to be the fluorescent protein of interest in the present invention. In general, fluorescent proteins for use in the methods of the invention are proteins that are derived from an automatically formed chromophore. Fluorescent proteins may comprise naturally occurring amino acid sequences or amino acid sequences that have been engineered (e.g., variant or mutant). When referring to fluorescent proteins, the term "mutation" or "variant" is used to refer to a protein that is different from the reference protein. For example, spectral variants of the multiple tube jellyfish GFP can be derived from naturally occurring GFP by engineering mutations such as amino acid substitutions of a reference GFP protein. Another non-limiting example of a fluorescent protein according to the present disclosure is a fluorescent protein derived from the Japanese eel fluorescent protein Unag and variants thereof (US 2016/0009771), both of which are incorporated herein by reference. Another example of a fluorescent protein according to the invention is cyan-excitable orange-red fluorescent protein (CyOFP) and variants thereof, which are derived from mNeptun (US 9908918) by mutagenesis, and which fluorescent protein and all variants thereof are incorporated herein by reference.

The term "derivative" or "derived from" refers to any suitable modification of the native polypeptide of interest, or of a fragment of the native polypeptide, or of an individual analog of the native polypeptide, provided that the desired biological or fluorescent properties or bioluminescence properties of the native polypeptide are retained, e.g., glycosylation, phosphorylation, polymer coupling (e.g., with polyethylene glycol), or addition of other groups. Methods for preparing polypeptide fragments, analogs and derivatives are generally known in the art.

The terms "bioluminescent fusion protein" and "bioluminescent fusion polypeptide" as used herein refer to a fusion protein comprising at least one fluorescent protein linked to at least one luciferase enzyme, wherein the fluorescent protein is operably linked to the luciferase enzyme such that Bioluminescence Resonance Energy Transfer (BRET) is allowed to occur between the fluorescent protein as a fluorescent BRET acceptor and the luciferase reaction product as a bioluminescence BRET donor after the fluorescent protein has reacted with a chemiluminescent substrate at the active site of the luciferase enzyme.

The term "linker" as used herein refers to a group that facilitates the attachment of a pair of non-luminescent elements or non-luminescent groups to form a bioluminescent complex. In typical embodiments, the linker is attached to a pair of non-luminescent elements (e.g., non-luminescent peptide/polypeptide pairs), and the attractive interaction between the two interacting elements facilitates the formation of a bioluminescent complex, although the present invention is not limited to such a mechanism and an understanding of such a mechanism is not necessary to practice the present invention. The linker may facilitate the formation of the bioluminescent complex by any suitable mechanism (e.g., bringing the non-luminescent pairs/groups in close proximity, bringing the non-luminescent pairs/groups in a suitable conformation for stable interaction, lowering the activation energy for complex formation, or a combination thereof, etc.). The linker may be a protein, polypeptide, peptide, small molecule, cofactor, nucleic acid, lipid, sugar, antibody, or the like. The linker may have additional functional properties, e.g., binding to other proteins, enzymes, etc. In some embodiments, the linker is recognizable by the OMA1 protein. Such a linker may be referred to as a "recognition peptide". The recognition peptide may be further hydrolyzed or its function may be abolished or altered in any other way such that the operable complex of elements linked by the linker alters its mode of action. For example, in one embodiment, a recognition peptide links a fluorescent BRET acceptor and a bioluminescent BRET donor, and an OMA1 protease hydrolyzes the recognition peptide, thereby altering the fluorescence spectrum of the BRET complex.

The term "pre-existing protein" as used herein refers to an amino acid sequence that is physically present prior to some event or date. A "peptide that is not a fragment of a pre-existing protein" is a short amino acid chain that is not a fragment or subsequence of a protein (e.g., synthetic or naturally occurring) that is physically present prior to design and/or synthesis of the peptide.

The term "fragment" as used herein, unless otherwise indicated, refers to a peptide or polypeptide that results from the isolation or "fragmentation" of a larger entire entity (e.g., protein, polypeptide, enzyme, etc.), or a peptide or polypeptide that is prepared to have the same sequence as that. Thus, a fragment is a subsequence of the entire entity (e.g., protein, polypeptide, enzyme, etc.) from which the fragment was made and/or designed. Peptides or polypeptides that are not subsequences of the pre-existing whole protein are not fragments (e.g., are not fragments of the pre-existing protein). A peptide or polypeptide that is not a fragment of a pre-existing bioluminescent protein is an amino acid chain that is not a subsequence of a protein (e.g., natural or synthetic) as follows: (1) Physically present prior to designing and/or synthesizing the peptide or polypeptide, and (2) exhibit significant bioluminescence activity. Fragments may include a C-terminal deletion, an N-terminal deletion, and/or an internal deletion of the polypeptide. Active fragments of a particular protein or polypeptide generally include at least about 5-10 contiguous amino acid residues of the full-length molecule, preferably at least about 15-25 contiguous amino acid residues of the full-length molecule, and most preferably at least about 20-50 or more contiguous amino acid residues of the full-length molecule, or any integer between 4 amino acids to the full-length sequence, so long as the target fragment retains biological activity, e.g., catalytic activity, ligand binding activity, regulatory activity, or fluorescent or bioluminescent properties, as described herein.

The term "subsequence" as used herein refers to a peptide or polypeptide that has 100% sequence identity to another larger peptide or polypeptide. Subsequences are sequences that match perfectly with a portion of a larger amino acid chain.

The term "physiological condition" as used herein includes any condition that is compatible with living cells, e.g., temperature, pH, salinity, chemical composition, etc., of the vast majority of aqueous conditions, which is compatible with living cells.

The term "sample" as used herein is used in its broadest sense. In one sense, it is meant to include specimens or cultures obtained from any source, as well as biological samples and environmental samples. Biological samples are available from animals (including humans) and include liquids, solids, tissues and gases. Biological samples include blood products, e.g., plasma, serum, and the like. A sample may also refer to a cell lysate or purified form of a peptide and/or polypeptide described herein. Cell lysates may include cells that have been lysed by a lysing agent or lysates such as rabbit reticulocyte lysate or wheat germ lysate. The sample may also include a cell-free expression system. Environmental samples include environmental samples such as surface materials, soil, water, crystals, and industrial samples. However, these samples should not be construed as limiting the type of sample that is applied to the present invention.

Aspects of the invention

The present invention has been made based on the findings, inter alia, that: the various parts of OPA1 can still be introduced into mitochondria without the need for mitochondrial introduction sequences. The naturally occurring mitochondrial import sequence of OPA1 comprises amino acids 1 to 87 (see NCBI reference sequence: NP 056375.2 at 7/11 of 2020), and therefore it was not foreseeable and surprising to see that fragments of the import sequence of OPA1 or even upstream fragments of the OMA1 import sequence could deliver polypeptides into mitochondria. This finding is a departure from the teachings of the prior art.

(1) The inventors have created new mitochondrial targeting sequences that are capable of stabilizing proteins.

(2) The inventors have subsequently engineered novel genes encoding reporter proteins that operably bind such synthetic targeting signals and enzyme functions or reporter peptides.

(3) To this end, the inventors have further engineered novel synthetases and reporter peptides operably binding at least two elements and a recognition peptide such that the reporter or enzyme function is eliminated after the recognition peptide is cleaved.

(4) The inventors have synthesized recognition peptides that are recognized by OMA1 protease.

(5) The inventors have invented new processes and methods for measuring the activity of OMA1 protease using these new reporter genes. For a further understanding of the present invention, a more detailed discussion of the reporter compositions of the present invention and methods of using the same will be provided below.

In one aspect, the present invention relates to a novel composition. Provided herein is a novel reporter gene that is capable of measuring OMA1 activity in vivo after expression in a suitable host. These synthetic genes are constructed in a modular fashion and operably bind four different functional elements: (a) a targeting signal; (b) An entire enzyme group or protein domain or an "N" fragment thereof; (c) An entire enzyme group or protein domain or a "C" fragment thereof corresponding to an "N" fragment; and (d) a sequence motif that is recognized by OMA1 protease, whereby the complementation of N and C results in a signal that can be measured. In some embodiments, the targeting sequence and recognition engine may also be a single entity. The term segment used above is merely an illustration to emphasize that the functions of the two elements or entities are complementary and not to be limited to segments of the same entity. In some embodiments of the invention, the reporter gene is operably linked to two different polypeptides or enzymes or proteins to achieve the desired effect, and in other embodiments of the invention, the reporter gene is operably linked to two fragments of the enzyme or polypeptide or protein.

Non-limiting examples of reporter genes and their use are described in the examples section. It will be appreciated that in some embodiments, the N-terminal and C-terminal fragments may be arranged in the same order as found in naturally occurring enzymes or polypeptides or proteins. In some embodiments, the N-terminal fragment and the C-terminal fragment may be arranged in an order that is opposite (e.g., in an order that is changed) to the order found in a naturally occurring enzyme or polypeptide or protein. In other embodiments, the operably linked sequence is non-naturally occurring.

In some embodiments, the reporter gene operably binds to a mitochondrial targeting peptide and a bioluminescent enzyme, which may be comprised of two or more non-luminescent peptides and/or non-luminescent polypeptides operably bound in such a way that upon binding they become bioluminescent. In some embodiments, the non-luminescent peptide is a fragment of a bioluminescent enzyme. In some embodiments, the bioluminescent enzyme is a NanoLuc luciferase (e.g., WO 2014/151736). In some embodiments, the reporter gene further comprises a recognition peptide that operably binds to the non-luminescent peptide and/or the non-luminescent polypeptide, and the non-luminescent peptide and/or the non-luminescent polypeptide may be a recognition peptide that is hydrolyzable by the OMA1 protease.

In some embodiments, the reporter gene is operable to bind the mitochondrial targeting peptide and two or more non-luminescent peptides and/or non-luminescent polypeptides and the recognition peptide, wherein the non-luminescent peptides and/or non-luminescent polypeptides assemble to form a bioluminescent enzyme after the recognition peptide is hydrolyzed. In some embodiments, the non-luminescent peptide is a fragment of a luciferase.

In some embodiments, the reporter gene operably binds to a mitochondrial targeting peptide and a fluorescent protein, which may be comprised of two or more non-fluorescent peptides and/or non-fluorescent polypeptides operably bound in such a way that upon binding, they become fluorescent. In some embodiments, the non-luminescent peptide is a fragment of a fluorescent protein. In some embodiments, the fluorescent protein is a UnaG protein. In some embodiments, the reporter gene further comprises a recognition peptide that operably binds to a non-fluorescent peptide and/or a non-fluorescent polypeptide, and the non-fluorescent peptide and/or the non-fluorescent polypeptide may be a recognition peptide that is hydrolyzable by an OMA1 protease.

In some embodiments, the reporter gene is operably bound to a mitochondrial targeting peptide and a bioluminescent fusion protein, which may include at least one fluorescent protein linked to at least one luciferase, wherein the fluorescent protein is operably linked to the luciferase to allow Bioluminescence Resonance Energy Transfer (BRET) to occur between the fluorescent protein as a fluorescent BRET acceptor and a luciferase reaction product as a bioluminescence BRET donor after the fluorescent protein reacts with a chemiluminescent substrate at the active site of the luciferase. In some embodiments, the reporter gene further comprises a recognition peptide that operably binds to the fluorescent BRET acceptor and the bioluminescent BRET donor. In some embodiments, the recognition peptide may be hydrolyzed by an OMA1 protease. An exemplary bioluminescent fusion protein includes a NanoLuc luciferase linked to at least one CyOFP. In the presence of a bioluminescent substrate (e.g., coelenterazine or coelenterazine analog, e.g., furimazine), the bioluminescent fusion protein emits light orange color due to bioluminescence resonance energy transfer of the luciferase reaction product to the CyOFP fluorophore.

In general, the mitochondrial targeting peptide consists of 30 or more amino acids, preferably 80 amino acids, but no more than 160 amino acids. In one embodiment, the mitochondrial targeting peptide consists of 50 to 150 amino acids. In another embodiment, the mitochondrial targeting peptide consists of 50 to 140 amino acids. In another embodiment, the mitochondrial targeting peptide consists of 50 to 130 amino acids. In another embodiment, the mitochondrial targeting peptide consists of 50 to 120 amino acids. In another embodiment, the mitochondrial targeting peptide consists of 50 to 110 amino acids. In another embodiment, the mitochondrial targeting peptide consists of 50 to 100 amino acids. In another embodiment, the mitochondrial targeting peptide consists of 50 to 90 amino acids. In another embodiment, the mitochondrial targeting peptide consists of 60 to 100 amino acids. In another embodiment, the mitochondrial targeting peptide consists of 70 to 100 amino acids. In another embodiment, the mitochondrial targeting peptide consists of 80 to 100 amino acids. In another embodiment, the mitochondrial targeting peptide consists of 80 to 90 amino acids. In another embodiment, the mitochondrial targeting peptide consists of 86 amino acids. In other embodiments, the mitochondrial targeting peptide consists of 50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149, or 150 amino acids.

In general, the recognition peptide is composed of four or more amino acids, preferably 23 amino acids, but not more than 50 amino acids. In one embodiment, the recognition peptide consists of 10 to 50 amino acids. In another embodiment, the recognition peptide consists of 10 to 40 amino acids. In another embodiment, the recognition peptide consists of 10 to 30 amino acids. In another embodiment, the recognition peptide consists of 10 to 20 amino acids. In another embodiment, the recognition peptide consists of 23 amino acids. In other embodiments, the recognition peptide is comprised of 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49, or 50 amino acids.

In some embodiments, the mitochondrial targeting peptide of the reporter gene may comprise the amino acid sequence of SEQ ID NO. 17 or SEQ ID NO. 19 or SEQ ID NO. 21 or SEQ ID NO. 23 or SEQ ID NO. 25 or SEQ ID NO. 27, or a variant or combination thereof.

In some embodiments, the recognition peptide of the reporter gene may comprise the amino acid sequence of SEQ ID NO. 33 or SEQ ID NO. 35 or SEQ ID NO. 37 or SEQ ID NO. 39 or SEQ ID NO. 41 or SEQ ID NO. 43 or SEQ ID NO. 45 or SEQ ID NO. 47, or a variant or combination thereof.

In some embodiments, the fragment "N" of the reporter gene may comprise the amino acid sequence of SEQ ID NO. 49 or SEQ ID NO. 51 or SEQ ID NO. 53 or SEQ ID NO. 55 or SEQ ID NO. 57 or SEQ ID NO. 59, or a variant or combination thereof.

In some embodiments, fragment "C" of a reporter gene may comprise the amino acid sequence of SEQ ID NO:61 or SEQ ID NO:63 or SEQ ID NO:65 or SEQ ID NO:67 or SEQ ID NO:69 or SEQ ID NO:71 or SEQ ID NO:73, or a variant or combination thereof.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 49 and SEQ ID NO. 61.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 51 and SEQ ID NO. 63.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 51 and SEQ ID NO. 65.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 53 and SEQ ID NO. 67.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 55 and SEQ ID NO. 69.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 57 and SEQ ID NO. 71.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO 59 and SEQ ID NO 73.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 17 and SEQ ID NO. 33.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 19 and SEQ ID NO. 35.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 19 and SEQ ID NO. 37.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 21 and SEQ ID NO. 39.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 23 and SEQ ID NO. 41.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 25 and SEQ ID NO. 43.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 27 and SEQ ID NO. 45.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 27 and SEQ ID NO. 33.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 33, SEQ ID NO. 49 and SEQ ID NO. 61.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO:35, SEQ ID NO:51 and SEQ ID NO: 63.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO:35, SEQ ID NO:51, and SEQ ID NO: 65.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 37, SEQ ID NO. 53 and SEQ ID NO. 67.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO:39, SEQ ID NO:55, and SEQ ID NO: 69.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 41, SEQ ID NO. 57 and SEQ ID NO. 71.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO:43, SEQ ID NO:59, and SEQ ID NO: 73.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 45, SEQ ID NO. 49 and SEQ ID NO. 61.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 33, SEQ ID NO. 49 and SEQ ID NO. 61.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 17, SEQ ID NO. 49 and SEQ ID NO. 61.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 19, SEQ ID NO. 51 and SEQ ID NO. 63.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 19, SEQ ID NO. 51 and SEQ ID NO. 65.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 19, SEQ ID NO. 53 and SEQ ID NO. 67.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 21, SEQ ID NO. 55 and SEQ ID NO. 69.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 23, SEQ ID NO. 57 and SEQ ID NO. 71.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 25, SEQ ID NO. 59 and SEQ ID NO. 73.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 27, SEQ ID NO. 49 and SEQ ID NO. 61.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 17,SEQ ID NO:33,SEQ ID NO:49 and SEQ ID NO. 61.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 19,SEQ ID NO:35,SEQ ID NO:51 and SEQ ID NO. 63.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 19,SEQ ID NO:35,SEQ ID NO:51 and SEQ ID NO. 65.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 19,SEQ ID NO:37,SEQ ID NO:53 and SEQ ID NO. 67.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 21,SEQ ID NO:39,SEQ ID NO:55 and SEQ ID NO. 69.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 23,SEQ ID NO:41,SEQ ID NO:57 and SEQ ID NO. 71.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 25,SEQ ID NO:43,SEQ ID NO:59 and SEQ ID NO. 73.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 27,SEQ ID NO:45,SEQ ID NO:49 and SEQ ID NO. 61.

In some embodiments, the reporter gene may comprise the amino acid sequences of SEQ ID NO. 27,SEQ ID NO:33,SEQ ID NO:49 and SEQ ID NO. 61.

In one embodiment, the reporter gene comprises the amino acid sequence of SEQ ID NO. 01 or a variant thereof comprising a sequence having at least about 80% -100% sequence identity to the amino acid sequence of SEQ ID NO. 01, including any percent identity in the range of 80% -100%, e.g., 81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98, or 99% sequence identity.

In one embodiment, the reporter gene comprises the amino acid sequence of SEQ ID NO. 03 or a variant thereof comprising a sequence having at least about 80% -100% sequence identity to the amino acid sequence of SEQ ID NO. 03, including any percent identity in the range of 80% -100%, e.g., 81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98, or 99% sequence identity.

In one embodiment, the reporter gene comprises the amino acid sequence of SEQ ID NO. 05 or a variant thereof comprising a sequence having at least about 80% -100% sequence identity to the amino acid sequence of SEQ ID NO. 05, including any percent identity in the range of 80% -100%, e.g., 81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98, or 99% sequence identity.

In one embodiment, the reporter gene comprises the amino acid sequence of SEQ ID NO:07 or a variant thereof comprising a sequence having at least about 80% -100% sequence identity to the amino acid sequence of SEQ ID NO:07, including any percent identity in the range of 80% -100%, e.g., 81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98, or 99% sequence identity.

In one embodiment, the reporter gene comprises the amino acid sequence of SEQ ID NO. 09 or a variant thereof comprising a sequence having at least about 80% -100% sequence identity to the amino acid sequence of SEQ ID NO. 09, including any percent identity in the range of 80% -100%, e.g., 81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98, or 99% sequence identity.

In one embodiment, the reporter gene comprises the amino acid sequence of SEQ ID NO. 11 or a variant thereof comprising a sequence having at least about 80% -100% sequence identity to the amino acid sequence of SEQ ID NO. 11, including any percent identity in the range of 80% -100%, e.g., 81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98, or 99% sequence identity.

In one embodiment, the reporter gene comprises the amino acid sequence of SEQ ID NO. 13 or a variant thereof comprising a sequence having at least about 80% -100% sequence identity to the amino acid sequence of SEQ ID NO. 13, including any percent identity in the range of 80% -100%, e.g., 81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98, or 99% sequence identity.

In one embodiment, the reporter gene comprises the amino acid sequence of SEQ ID NO. 15 or a variant thereof comprising a sequence having at least about 80% -100% sequence identity to the amino acid sequence of SEQ ID NO. 15, including any percent identity in the range of 80% -100%, e.g., 81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98, or 99% sequence identity.

In one embodiment, the reporter gene comprises the amino acid sequence of SEQ ID NO. 01 or a variant thereof comprising a sequence having at least about 80% -100% sequence identity to the amino acid sequence of SEQ ID NO. 01, said sequence identity comprising any percent identity in the range of 80% -100%, e.g., 81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98, or 99% sequence identity, wherein the mitochondrial targeting sequence does not comprise the amino acid sequence of SEQ ID NO. 29.

In another specific embodiment, the reporter gene comprises the amino acid sequence of SEQ ID NO. 15 or a variant thereof comprising a sequence having at least about 80% -100% sequence identity to the amino acid sequence of SEQ ID NO. 15, said sequence identity comprising any percent identity in the range of 80% -100%, e.g., 81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98, or 99% sequence identity, wherein the mitochondrial targeting sequence does not comprise the amino acid sequence of SEQ ID NO. 31.

In some embodiments, the polypeptides or proteins provided herein comprise the amino acid sequence of SEQ ID NO. 01,SEQ ID NO:03,SEQ ID NO:05,SEQ ID NO:07,SEQ ID NO:09,SEQ ID NO:11,SEQ ID NO:13, or SEQ ID NO. 15, with one or more additions, substitutions and/or deletions.

In some embodiments, the peptides or polypeptides and/or proteins of the invention comprise synthetic peptides, peptides containing one or more unnatural amino acids, peptidomimetics, conjugated synthetic peptides (e.g., conjugated to functional groups (e.g., fluorophores, luminescent substrates, etc.)).

It will be appreciated that a reporter gene of the invention may comprise one or more linkers operably binding to sequences. The linker is typically a short peptide sequence of 2 to 30 amino acid residues, typically consisting of glycine and/or serine residues. The linker amino acid sequence is typically very short, e.g., 20 or fewer amino acids (i.e., 20,19,18,17,16,15,14,13,12,11,10,9,8,7,6,5,4,3,2, or 1 amino acid). Examples include short peptide sequences that promote cloning, poly glycine linkers (Glyn, where n=2, 3,4,5,6,7,8,9,10 or more), histidine tags (Hisn, where n=3, 4,5,6,7,8,9,10 or more), linkers consisting of glycine and serine residues ([ Gly-Ser ] n, [ Gly-Ser-Gly ] n, [ Ser-Ala-Gly ] n, and [ Gly-Ser ] n, where n= 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15 or more), GSAT, SEG, and Z-EGFR linkers. The linker may include restriction sites that facilitate cloning and manipulation. Other suitable linker amino acid sequences will be apparent to those skilled in the art. (see, e.g., argos P.J. mol. Biol. (1990) 211 (4): 943-958;Crasto et al.Protein Eng. (2000) 13:309-312;George et al.Protein Eng. (2002) 15:871-879;Arai et al.Protein Eng. (2001) 14:529-532; and the Registry of Standard Biological Parts (partsregistration. Org/protein_domains/Linker).

In some embodiments, a marker sequence may be added to a reporter gene of the application. In some embodiments, the marker sequence is located at the N-terminus or the C-terminus of the reporter gene. In other embodiments, the marker sequence may be inserted anywhere within the reporter gene. Exemplary labels that may be used in the practice of the present application may include His-labels, strep-labels, TAP-labels, S-labels, SBP-labels, arg-labels, calmodulin-binding peptide labels, cellulose binding domain labels, dsbA-labels, c-myc labels, glutathione S-transferase labels, FLAG-labels, HAT-labels, maltose-binding protein labels, nusA-labels, and thioredoxin labels.

The reporter gene can also be fused to other fluorescent proteins or bioluminescent proteins, or to biologically active domains or polypeptide fragments of other fluorescent proteins or bioluminescent proteins, or to variants of other fluorescent proteins or bioluminescent proteins having fluorescent or bioluminescent properties (e.g., green Fluorescent Protein (GFP) or luciferase).

Any luciferase may be used to construct the reporter gene. Luciferase sequences from a variety of substances are well known in the art, such as, but not limited to, deep sea shrimp Oplophorus luciferase, firefly luciferase, click beetle luciferase, renilla luciferase, gaussia luciferase, metridia luciferase, vargula luciferase, bacterial luciferases (e.g., vibrio fermi, vibrio haweyi and Vibrio hawaii) and dinoflagellate luciferase), any of which may be incorporated into the bioluminescence fusion protein. Representative luciferase sequences are shown in the National Center for Biotechnology Information (NCBI) database, see, e.g., NCBI accession numbers: JQ437370, AFJ15586, AHH41349, AHH41346, HV216898, HV216897, Q9GV45, AB644228, M63501, AY015988, EF535511, AY015993, EU239244, AB371097, AB371096, EU025117, AB519703, AB674506, U89490, M25666, xm_003190150, xm_003602031, yp_004273613, yp_004216833, yp_003275551, kep44836, yp_004749, efr93032, yp_206879, yp_206878, abg 26173, wp_005438583, wp_005384122, p07740, ef492542, af085332, af4060, af4059, eu025117, ay3664, U03687, and all of which are incorporated herein by reference for all days. Any of these sequences, or variants thereof comprising sequences having at least about 80-100% sequence identity to these sequences, can be used to construct the bioluminescent fusion proteins described herein, or nucleic acids encoding bioluminescent fusion proteins, wherein sequence identity comprises any percent identity in the range of 80-100%, e.g., 81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98, or 99% sequence identity.

In some embodiments, the reporter gene comprises a luciferase derived from Oplophorus gracihrostris. Such bioluminescent fusion proteins can produce light from a chemiluminescent substrate (which includes coelenterazine and coelenterazine analogs, see, e.g., U.S. patent application US20120117667, which is incorporated herein by reference in its entirety).

In one embodiment, the bioluminescent fusion protein comprises a NanoLuc luciferase, which is an engineered Oplophorus gracihrostris luciferase obtained from Promega Corporation (Madison, wis.). The molecular weight of the NanoLuc luciferase is 19.1kDa, which is an ATP-independent luciferase that uses the coelenterazine analog furimazine as a chemiluminescent substrate to produce high intensity luminescence. A representative amino acid sequence of the nanoLuc luciferase is shown in SEQ ID NO. 51. In one embodiment, a polypeptide comprising the sequence of SEQ ID NO. 51 or a variant thereof is used to construct a reporter gene encoding a protein, wherein the luciferase is capable of catalyzing a light-generating reaction of a chemiluminescent substrate useful for measuring OMA1 protease activity, and wherein the variant comprises a sequence having at least about 80-100% sequence identity with the amino acid sequence of SEQ ID NO. 51, including any percent identity in the range of 80-100%, e.g., 81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98, or 99% sequence identity.

Any fluorescent protein can be used to construct the reporter gene. Examples of fluorescent proteins include, but are not limited to: green Fluorescent Protein (GFP), cyan Fluorescent Protein (CFP), blue Fluorescent Protein (BFP) and Yellow Fluorescent Protein (YFP), wherein the color of the fluorescence depends on the wavelength of the emitted light, green fluorescent protein emits light of 520-565nm, cyan fluorescent protein emits light of 500-520nm, blue fluorescent protein emits light of 450-500nm, yellow fluorescent protein emits light of 565-590nm, and red fluorescent protein described further below emits light of 625-740 nm. Furthermore, fluorescent proteins as used herein include, for example, those proteins genetically engineered to improve properties such as, but not limited to: improving protein expression, changing excitation or emission wavelength, improving brightness, pH tolerance, stability or speed of fluorophore formation or fluorophore dissociation, photoactivity or reduced oligomerization or photobleaching.

Fluorescent proteins useful in the present invention include those that emit a variety of different spectra including violet, blue, cyan, green, yellow, orange and red. As further described below, fluorescent proteins as used herein also include, but are not limited to: blue Fluorescent Protein (BFP) and Cyan Fluorescent Protein (CFP) generated by random mutation of GFP and Yellow Fluorescent Protein (YFP) of rational design. BFP has a Tyr66His substitution relative to GFP and its peak in absorption spectrum shifts to 384nm at an emission wavelength of 448nm (Heim et al, proc. Natl. Acad. Sci. (1994) 91:12501). CFP is brighter than BFP and has higher photostability due to the substitution of Tyr66Trp, which has an absorption/emission spectral range at a median level between BFP and EGFP (Heim et al, proc. Natl. Acad. Sci. (1994) 91:12501;Heim and Tsien,Curr.Biol. (1996) 6:178-182;and Ellenberg et al, biotechniques (1998) 25:838); the Thr203Tyr CFP variant is referred to as "CGFP" and has excitation and emission wavelengths at a median level between CFP and EGFP. The rationally designed YFP has absorption and emission spectra that are red shifted relative to the green fluorescent protein (Ormo et al, science 273:1392 (1996); heim and Tsien, supra, 1996). Various variants of YFP exhibit improved properties, including but not limited to: YFP variants "Citrine" (YFP-Val 68Leu/Gln69Met; griesbeck et al, J.biol. Chem. (2001) 276:29188-29194) and "Venus" (YFP-Phe 46Leu/Phe64Leu/Met153Thr/Val63Ala/Ser175 Gly), variants "Venus" are a very bright and rapidly maturing YFP (Nagai et al, nature Biotech. (2002) 20:87-90). Those skilled in the art will appreciate that these fluorescent proteins, as well as various other fluorescent proteins derived from GFP, for example, or other naturally occurring fluorescent proteins, may also be used in the present invention.

The fluorescent protein used in the present invention may also be a long wavelength fluorescent protein, for example, a red or far red fluorescent protein, which may be used to reduce or eliminate background fluorescence of samples derived from eukaryotic cells or tissues. These red fluorescent proteins include naturally occurring and genetically modified versions of sea anemonin (Discosoma striata proteins), which include but are not limited to: dsRed (DsRed 1 or drFP583; matz et al, nat. Biotech. (1999) 17:969-973); dsRed2 (Tersikh et al, J.biol.chem. (2002) 277:7633-7636); t1 (dsRed-Express; clontech; palo Alto, calif.; bevis and Glick, nature Biotech. (2002) 20:83-87); dsRed variant mRFP1 (Campbell et al, proc. Natl. Acad. Sci. USA (2002) 99:7877-7882). These red fluorescent proteins also include naturally occurring and genetically modified forms of the protein of Karspanflower sea anemone (Heteractis crispa), such as HcRed (Gurskaya et al, FEBS Lett (2001) 507:16).

Fluorescent proteins useful in the reporter gene can be derived from any of a number of different species, including marine species, such as a.victoria and other coelenterazine marine organisms. Useful fluorescent proteins include, but are not limited to: renilla diffracted fluorescent proteins, such as dimeric Renilla GFP, with narrower excitation peaks (498 nm) and emission peaks (509 nm) (Peele et al, J.prot.chem. (2001) 507-519); sea anemone (Anemonia sulcata) fluorescent proteins, such as DsRed proteins, e.g., asFP595 (Lukyanov et al, J.biol. Chem. (2000) 275:25879-25882); coral (Discosoma) fluorescent proteins, e.g., discosoma striata red fluorescent protein, e.g., dsFP593 (frapkov et al, FEBS lett (2000) 479:127-130); fluorescent proteins of sea anemone (Heteractis crispa), such as HcRed and HcRed-2A (Gurskaya et al, FEBS Lett. (2001) 507:16); and Entacmeae quadricolor fluorescent proteins, including red fluorescent proteins such as eqFP611 (Wiedenmann et al, proc. Natl. Acad. Sci. USA (2002) 99:11646-11651), the sequences of which are incorporated herein by reference. Those skilled in the art will appreciate that these fluorescent proteins, as well as many other fluorescent proteins (including species homologs of the naturally occurring proteins described above, as well as engineered fluorescent proteins) may be used to design the reporter genes of the present invention.

In some embodiments, the fluorescent protein according to the invention is a polypeptide having fluorescent properties in the presence of bilirubin. Such fluorescent proteins are a group of polypeptides having a commonality that emit fluorescence having a prescribed wavelength by irradiation of excitation light in the presence of bilirubin or the like, but do not emit fluorescence by irradiation of the same excitation light in the absence of bilirubin or the like. Examples of fluorescent polypeptides having such properties are polypeptides derived from eel, more specifically from jel in japan, which are known as UnaG (SEQ ID NO: 75) and variants thereof, all of which sequences are incorporated herein by reference. Although UnaG was originally isolated from jel, the source of the fluorescent polypeptide is not limited thereto.

In one embodiment, the reporter gene comprises a sequence or subsequence of the amino acid sequence of SEQ ID NO. 75 or a variant thereof comprising a sequence having at least about 80-100% sequence identity to SEQ ID NO. 75, said sequence identity comprising any percent identity in the range of 80-100%, e.g., 81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98, or 99% sequence identity, wherein the subsequence is operably bound to a fluorescent protein. Such fluorescent protein may be any polypeptide composed of amino acids linked by peptide bonds, but is not limited thereto. For example, a fluorescent polypeptide may comprise a different structure than the polypeptide. Non-limiting examples of structures other than polypeptides include sugar chains and isoprenoid groups.

The invention also provides a bioluminescent fusion protein comprising at least one fluorescent protein and at least one luciferase, wherein the fluorescent protein is operably linked to the luciferase to allow Bioluminescence Resonance Energy Transfer (BRET) to occur between the fluorescent protein as a fluorescent BRET acceptor and the luciferase reaction product as a bioluminescent BRET donor.

In some embodiments, the bioluminescent fusion protein comprises a fluorescent protein comprising the amino acid sequence of SEQ ID NO. 63 or a variant thereof comprising a sequence having at least about 80-100% sequence identity to SEQ ID NO. 63, said sequence identity comprising any percent identity in the range of 80-100%, e.g., 81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98, or 99% sequence identity, wherein the fluorescent protein emits orange-red light in response to absorption of cyan excitation light.

In some embodiments, the invention includes a Bioluminescence Resonance Energy Transfer (BRET) system comprising a bioluminescence fusion protein described herein and a chemiluminescent substrate (e.g., coelenterazine analog (e.g., furimazine), or other luciferase substrate). The BRET system may also include a light detector or imaging device for detecting light emitted from the bioluminescent fusion protein, such as, but not limited to, an optical microscope, a digital microscope, a photometer, a photo-sensitive coupled (CCD) image sensor, a Complementary Metal Oxide Semiconductor (CMOS) image sensor, or a digital camera.

In some embodiments, the polynucleotide sequence encoding the reporter polypeptide is a codon optimized for expression in a bacterial host cell (e.g., e.coli). In other embodiments, the polynucleotide sequence encoding the reporter polypeptide is a codon optimized for expression in a eukaryotic host cell or organism (e.g., fungus, yeast, worm, mouse, rat, hamster, guinea pig, monkey, or human). In other embodiments, the polynucleotide sequence encoding the reporter polypeptide is a codon optimized for expression in a mammalian host cell or organism (e.g., mouse, rat, hamster, guinea pig, monkey). In some embodiments, the polynucleotide sequences encoding the reporter polypeptides provided herein include the sequence of SEQ ID NO. 02,SEQ ID NO:04,SEQ ID NO:06,SEQ ID NO:08,SEQ ID NO:10,SEQ ID NO:12,SEQ ID NO:14, or SEQ ID NO. 16, or variants thereof.

In some embodiments, the invention provides expression vectors comprising an expression control sequence operably linked to a nucleic acid molecule encoding a reporter polypeptide. In other embodiments, the invention provides viruses comprising nucleic acid molecules encoding reporter polypeptides. In other embodiments, the invention provides recombinant host cells comprising a nucleic acid molecule encoding a reporter polypeptide.

Another aspect of the invention relates to methods comprising the reporter genes disclosed herein. Those skilled in the art will appreciate from the disclosure and examples herein that modifications, alterations, or design of the invention, and the like, are within the scope of the invention. Reporter genes are widely used in research in various aspects of various fields, such as the fields of biology, chemistry or pharmacy. Thus, in light of the disclosure herein, the present invention also pertains to methods of finding applications in various fields. The invention may include, but is not limited to, monitoring of clinical conditions, diagnosis, monitoring of therapeutic drugs, biological research, drugs, compound detection and monitoring, and the like. Other applications include drug development, for example, high throughput molecular screening or safety and toxicity studies.

The methods of use according to the present invention include a reporter gene, which may include a targeting signal, a complementing element, and a recognition domain, wherein the recognition domain separates the complementing element, whereby each element is functional. According to the invention, the recognition portion may form a complex, whereby the recognition portion is hydrolyzed and the functional element is separated. This typically causes the element function to be eliminated, which can be measured and correlated with the formation of a complex or other related event.

In some embodiments, the reporter gene is expressed, such as in a cell or whole organism. The recombinant host is then exposed to at least one experimental condition and a signal of a certain intensity is generated based on said condition. Alternatively, the reporter gene may be isolated from the host and used in the detection method. Methods for protein isolation are known to the person skilled in the art and are described, for example, in Protein Purification Applications: A Practical Approach, (Simon Roe, ed., 2001). The signal may be detected in the detection in any suitable way, which may be direct or indirect and may especially comprise a light detector or an imaging device, such as, but not limited to, an optical microscope, a digital microscope, a photometer, a photo-coupled (CCD) image sensor, a Complementary Metal Oxide Semiconductor (CMOS) image sensor, a photomultiplier tube or a digital camera. The signal may be further processed, integrated or compared with signals obtained under other conditions (e.g., control conditions), and correlated with experimental conditions.

In some embodiments, such a signal is detected by measuring a change in a detectable label (i.e., a detectable group) that is part of the reporter gene. In some embodiments, the reporter molecule comprises a detectable group that provides an indication of a cleavage event. In some embodiments, cleavage can be detected by a change in the length of the polypeptide (e.g., gel electrophoresis, size exclusion chromatography, immunofluorescence, etc.) or by other biochemical and physical changes occurring on the reporter molecule. In some embodiments, the reporter molecule comprises a label that facilitates cleavage detection. In some embodiments, the reporter molecule comprises a cleavable enzyme (e.g., a bioluminescent enzyme), wherein the cleavage event alters the function of the enzyme. In some embodiments, the reporter molecule comprises a cleavable detectable group (e.g., a fluorescent protein), wherein the cleavage event alters the group and the alteration is detectable. In some embodiments, cleavage is detected using a FRET pair or a BRET pair, wherein a change in fluorescence is indicative of a cleavage event. Methods for detecting and monitoring protein cleavage are well known in the art and any such method may be used to detect cleavage of the reporter molecule of the invention. In some embodiments, the reporter gene of the invention is conjugated to at least one other reporter gene for dual reading, or in some embodiments, for multi-mode reading.

In some embodiments, the signals from these assays may be associated with the formation of complexes, which may comprise any number of small molecules, compounds, molecules, peptides, polypeptides, or proteins or other compositions. In some embodiments, the signal is associated with a complex formed by the compound and the polypeptide or protein (e.g., a drug-target interaction). In some embodiments, the signal is associated with a complex formed by the molecule and a protease (e.g., OMA1 protease). Such methods are particularly useful for identifying protease inhibitors (e.g., OMA1 inhibitors). In some embodiments, the invention provides methods of screening for an OMA1 inhibitor, wherein OMA1 hydrolyzes the recognition peptide, thereby eliminating the signal from the reporter gene. Thus, this screening method identified potential OMA1 inhibitors as compounds or molecules that are capable of retaining reporter gene signals, and this screening was useful for reducing the likelihood of identifying false positives (e.g., compounds that block signals produced by functional elements other than OMA 1).

In some embodiments, the invention provides methods of detecting the presence of one or more protease activities in a sample comprising: a) Combining the sample with a reporter molecule comprising a targeting signal, a complementing element of a detectable group and a recognition element, wherein the recognition element complementing element is separated such that the detectable group is functional; and b) detecting cleavage of the recognition element by separation of the complementing element and a change in the function of the detectable group. In some embodiments, a protease activity can be detected using the method. In some embodiments, 2,3,4,5,6,7,8,9, or 10 protease activities can be detected using the methods. In some embodiments, one or more protease activities may be detected using the methods. In some embodiments, OMA1 protease activity may be detected using the methods. In other embodiments, YME1L1 protease activity can be detected using the methods. In yet another embodiment, AFG3L2 protease activity can be detected using the method. In some embodiments, i-AAA protease activity may be detected using the methods. In other embodiments, m-AAA protease activity may be detected using the methods. In other embodiments, the PARL protease activity may be detected using the methods.

The present invention provides a method of identifying a protease inhibitor, the method comprising the steps of: a) Binding a molecule to a reporter protein comprising a complementing element of a detectable group and a recognition element, and b) detecting a change in the function of the detectable group, wherein the molecule is recognized as a protease inhibitor when no significant change in the function of the detectable group has occurred. Optionally, the method further comprises a step of allowing protease activity to occur after step a) (e.g., a step of activating the protease). Optionally, the reporter protein may further comprise a targeting signal.

The present invention provides a method of identifying a cell permeable protease inhibitor, the method comprising the steps of: a) Binding the molecule to a recombinant host expressing a reporter protein comprising a complementing element of a detectable group and a recognition element, wherein the recognition element separates the complementing element such that the detectable group is functional; and b) detecting cleavage of the recognition element by separation of the complementing element and a change in the function of the detectable group, wherein the molecule is recognized as a protease inhibitor when no significant change in the function of the detecting group occurs. Optionally, the method may further comprise a step of allowing protease activity to occur after step a) (e.g., a step of activating the protease). Optionally, the reporter protein may further comprise a targeting signal.

The present invention provides a method of screening for a cell permeable protease inhibitor, the method comprising the steps of: a) obtaining a library of compounds, b) exposing a recombinant host expressing a reporter protein comprising complementary elements and recognition elements of a detectable group to the library of compounds, and c) measuring a change in the function of the detectable group, wherein the compound is said to be a protease inhibitor when no significant change in the function of the detectable group has occurred. Optionally, the method may further comprise a step of allowing protease activity to occur after step a) (e.g., a step of activating the protease). Optionally, the reporter protein may further comprise a targeting signal.

In other embodiments, the invention provides methods of measuring mitochondrial toxicity of a compound, wherein a recognition peptide is hydrolyzed and reporter gene signal disappears after exposure to the compound. These methods are particularly useful in determining toxic concentrations in cellular assays by correlating (e.g., dose-response relationship) the concentration of a compound with the intensity of a signal from a reporter gene. In some embodiments, the present invention provides methods of detecting toxicity (or potential for toxicity) that can be used to select compounds suitable for in vivo administration that do not produce adverse toxicity, or compounds intended for in vivo administration that do not produce adverse toxicity, or compounds that are potentially useful for in vivo administration that do not produce adverse toxicity, such as, but not limited to mitochondrial toxicity.

The present invention provides a method of predicting in vivo toxicity (e.g., mitochondrial toxicity) of a molecule, the method comprising the steps of: a) Binding the molecule to a recombinant host expressing a reporter protein comprising a complementing element of a detectable group and a recognition element, wherein the recognition element separates the complementing element such that the detectable group is functional, and b) detecting cleavage of the recognition element and a change in the function of the detectable group resulting from the separation of the complementing element, wherein an increase in cleavage is detected indicative of an increase in molecular toxicity. Optionally, the reporter protein may further comprise a targeting signal. Optionally, the method may further comprise a step of generating protease activity (e.g., a step of activating a protease) after step a).

The present invention provides a method of selecting a compound having reduced in vivo toxicity (e.g., mitochondrial toxicity), the method comprising the steps of: a) Binding the compound to a recombinant host expressing a reporter protein comprising a complementing element of a detectable group and a recognition element, wherein the recognition element separates the complementing element such that the detectable group is functional, b) detecting cleavage of the recognition element by separation of the complementing element and a change in the function of the detectable group; and c) selecting one or more compounds for which no or reduced cleavage is detected. Optionally, the method may further comprise step d): administering the selected compound to a mammalian, e.g., human, subject after step c). Optionally, the reporter protein may further comprise a targeting signal. Optionally, the method may further comprise a step of allowing protease activity to occur after step a) (e.g., a step of activating the protease).

Provided herein are several forms of using a reporter gene in assays. In some embodiments, these forms of use for the reporter gene are performed in vitro and in other embodiments, these forms of use for the reporter gene are performed in vivo, in other embodiments, from in vitro to in vivo. In some embodiments, the reporter gene is transiently expressed in the host cell, and in other embodiments, the stably transfected cells express the reporter gene. The recombinant genes, recombinant proteins and recombinant cells or recombinant organisms may be provided separately, may be combined or as a kit, as separate diagnostic kit components and/or research kit components, or as separate reagents that may be customized for a single assay.

These kits may contain the reporter gene along with appropriate instructions and other reagents necessary for making or using the above-described components. The kit may comprise the reporter polypeptide or recombinant construct for producing the reporter polypeptide, and/or the cell (either transfected or alone) in a separate container. In addition, instructions (e.g., written, tape, VCR, CD-ROM, DVD, flash memory, SD card, etc.) for using the reporter gene of the invention (e.g., as a reporter gene for determining mitochondrial toxicity) can be included in the kit. The kit may also include other packaged reagents and materials (e.g., transfection reagents, buffers, culture media, etc.). As discussed above, the reporter gene may be used, inter alia, for fluorescence detection or bioluminescence detection. Thus, the kit may also comprise reagents for performing these assays or medical imaging. In some embodiments, the kit further comprises a chemiluminescent substrate, BRET system, or reporter construct using a fluorescent protein or bioluminescent fusion protein as described herein.

The reporter genes of the present invention can be prepared in any of a variety of ways, all of which are known in the art. The person skilled in the art may further introduce mutations by any method to enhance at least one property of the polypeptides, enzymes and/or proteins encoded by the reporter genes of the invention, such as signal-to-noise ratio, signal stability, signal specificity and signal intensity.

In one embodiment, the reporter genes provided herein are produced using recombinant techniques. Those skilled in the art will be able to determine the nucleotide sequence encoding the desired polypeptide by standard techniques and teachings herein. Basic literature disclosing common methods of recombinant technology includes: sambrook et al Molecular Cloning, A Laboratory Manual (2 nd ed.1989); kriegler, gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al, eds., 1994)). Recombinant techniques can be used to clone sequences encoding polypeptides useful in the present invention, which can be mutated in vitro by appropriate base pair substitution to create codons for the desired amino acids. Such changes may include changes of only one base pair, affecting a single amino acid, or may include changes of several base pairs. Alternatively, mutations can be effected using mismatched primers that hybridize to the parent nucleotide sequence (typically the cDNA corresponding to the RNA sequence) at a lower melting temperature than the mismatched duplex. The primers can be made specific by keeping the primer length and base composition within relatively narrow limits and keeping the mutant base centered. See, for example, innis et al, (1990) PCR applications: protocols for Functional Genomics; zoller and Smith, methods enzymes (1983) 100:468. The use of DNA polymerase affects primer extension and clones containing mutant DNA obtained by aggregation of the primer extended strand and the cloned products are selected. This selection was accomplished using mutant primers as hybridization markers. This technique can also be applied to create multiple point mutations. See, e.g., dalbie-McFarland et al Proc. Natl. Acad. Sci USA (1982) 79:6409. The sequences encoding the polypeptides may also be prepared synthetically, e.g., based on known sequences. The polynucleotide sequence may be designed using appropriate codons for the particular amino acid sequence desired. Complete sequences are typically assembled by overlapping oligonucleotides prepared by standard methods and assembled into complete coding sequences. See, e.g., edge Nature (1981) 292:756; nambair et al science (1984) 223:1299; jay et al J.biol.chem. (1984) 259:6311; stemmer et al Gene (1995) 164:49-53.

Once the coding sequences are isolated and/or synthesized, they can be cloned into any suitable vector or replicon for expression. As clearly shown in the teachings herein, a variety of different vectors encoding the polypeptides of the invention can be prepared for expression in prokaryotic or eukaryotic cells. A non-limiting example of such a carrier is shown in fig. 1A. Those skilled in the art will be able to select or design suitable vectors useful in the present invention. A variety of cloning vectors are known in the art and selection of an appropriate cloning vector is a choice that can be made by those skilled in the art.

The gene may be placed under the control of a promoter, a ribosome binding site (for bacterial expression) and optionally an operator (collectively referred to herein as a "control" element), such that the DNA sequence encoding the desired polypeptide is transcribed into RNA of a host cell transformed with a vector comprising such an expression construct.

Other regulatory sequences that allow for the regulation of the expression of the protein sequence relative to the growth of the host cell are also desirable. Such regulatory sequences are known to those skilled in the art and examples include those that cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. For example, expression of a protein from a eukaryotic cell vector may be regulated using an inducible promoter. The level of expression is correlated with the concentration of the inducing agent (e.g., tetracycline or ecdysone) by incorporating the responsive element of the inducing agent into the promoter under the influence of the inducible promoter. In general, high levels of expression are obtained from inducible promoters in the presence of only an inducing agent, with minimal basal expression levels. Inducible expression vectors are typically selected if expression of the protein of interest is detrimental to eukaryotic cells. Other types of adjusting elements may also be present in the carrier.

Typically, expression vectors are used to transform suitable host cells. A variety of mammalian cell lines are known in the art, including immortalized cell lines obtained from the American Type Culture Collection (ATCC), such as, but not limited to: chinese Hamster Ovary (CHO) cells, heLa cells, baby mouse kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., hep G2), hek293 cells, and the like. Similarly, bacteria such as E.coli, B.subtilis and Streptococcus will find use in the expression constructs of the invention. Yeast hosts useful in the present invention include, inter alia, saccharomyces cerevisiae, candida albicans, candida maltosa, hansenula polymorpha, kluyveromyces fragilis (Kluyveromyces fragilis), kluyveromyces lactis, pichia pastoris (Pichia guillerimondii), pichia pastoris, schizosaccharomyces pombe, and yarrowia lipolytica. Insect cells for baculovirus expression vectors include Egyptian mosquito, spodoptera frugiperda (Autographa californica), silkworm, drosophila melanogaster, spodoptera frugiperda (Spodoptera frupperda), and Spodoptera frugiperda.

Based on the chosen expression system and host, the reporter gene of the present invention is prepared by growing a host cell transformed with the expression vector at elevated temperature under a variety of conditions, thereby expressing the protein encoded by the target reporter gene. The selection of suitable growth conditions is within the ability of those skilled in the art.

In some embodiments, the reporter gene of the invention is disposed within an expression cassette for expression within a eukaryotic cell. Expression cassettes typically include a control element operably linked to a coding sequence that allows for expression of the gene in a host species. Typical promoters for mammalian cell expression include, for example, the SV40 early promoter, the CMV promoter (e.g., CMV immediate early promoter), the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP) and the herpes simplex virus promoter, among others. Other non-viral promoters (e.g., promoters derived from murine metallothionein genes) have also been found useful for mammalian expression. Typically, there are also transcription termination and polyadenylation sequences located 3' of the translation termination codon. Preferably, there is also a sequence for optimizing the initiation of translation, which is located 5' to the coding sequence. Examples of transcription terminator/polyadenylation signals include those derived from SV40, which are described in the Sambrook et al literature, supra, and bovine growth hormone terminator sequences.

Enhancer sequences may also be used in the present invention to increase the expression level of mammalian constructs. Examples include the SV40 early gene enhancer (described in Dijkema et al, EMPO J. (1985) 4:761), the enhancer/promoter derived from the Long Terminal Repeat (LTR) of the Rous sarcoma virus (described in Gorman et al, proc. Natl. Acad. Sci. USA (1982) 79:6777), and elements derived from human CMV (described in Boshare et al, cell (1985) 41:521), e.g., elements included in the CMV intron A sequence.

Alternative targeting sequences may also be used to direct the location of the polypeptide or protein encoded by the reporter gene to a particular tissue, cell type (e.g., muscle cell, heart cell or nerve cell), cell compartment (e.g., mitochondria or other organelles, serosa) or protein. For example, the construct may comprise a polynucleotide sequence encoding a secreted protein signal sequence, a membrane protein signal sequence, a nuclear localization signal sequence, an endoplasmic reticulum localization sequence, a peroxisome localization sequence, a mitochondrial localization sequence or a protein-protein interaction motif sequence. See, e.g., protein Targeting, transport, and Translocation (r.dalbey and Gunnar von Heijne eds., academic Press, 2002); protein Targeting Protocols (Methods in Molecular Biology, R.A. clegg ed., humana Press, 1998); protein Engineering and Design (s.j.park j.r.cochran eds., CRC Press, 2009); protein-Protein Interactions: methods and Applications (Methods in Molecular Biology, H.Fu ed., humana Press, 2004); emantelsson et al Biochim Biophys Acta (2001) 1541 (1-2): 114-119; hurley et al, annu, rev, biophys, biomol, structure (2000) 29:49-79; jans et al Bioessays (2000) 22 (6): 532-544; christophe et al cell Signal (2000) 12 (5): 337-341; stanley mol. Membr. Biol. (1996) 13 (1): 19-27; cosson et al Cold Spring Harb.Symp.Quant.biol. (1995) 60:113-117; emmott et al EMBO Rep. (2009)) 10 (3) 231-238; gurkan et al adv.exp.Med.biol. (2007) 607:73-83; romanelli et al j. Neurochem. (2008) 105 (6): 2055-2068; terlecky et al adv. Drug Deliv. Rev. (2007) 59 (8): 739-747; arnoys et al acta histochem. (2007) 109 (2): 89-110; brown et al Kidney int (2000) 57 (3): 816-824; jadwin et al febs letters (2012) 586 (17): 2586-2596; liu et al febs letters (2012) 586 (17): 2597-2605; romero et al adv. Pharmacol. (2011) 62:279-314; obenauer et al methods mol. Biol. (2004) 261:445-468, the entire contents of which are incorporated herein by reference.

Transformation of host cells with recombinant DNA may be performed by those conventional techniques well known to those skilled in the art. Where the host is a prokaryote (e.g., E.coli), competent cells capable of uptake of DNA are prepared by harvesting cells after the log phase of growth and subsequently treating the cells by CaCl2 methods by procedures well known in the art. Alternatively, mgCl2 or RbCl may be used. Transformation may also be performed after formation of protoplasts of the host cell or by electroporation.

When the host is eukaryotic, these methods of transfecting DNA may be used, for example, calcium phosphate co-precipitation, conventional mechanical process steps (e.g., microinjection, electroporation, insertion of packaged plasmid or viral vector into liposomes). Eukaryotic cells can also be co-transfected with a DNA sequence encoding a reporter molecule of the invention, and another exogenous DNA molecule encoding an alternative phenotype (e.g., a herpes simplex thymidine kinase gene).

A variety of virus-based systems have been developed for transferring genes into mammalian cells. These systems include adenoviruses, retroviruses (gamma-retroviruses and lentiviruses), poxviruses, adeno-associated viruses, baculoviruses and herpes simplex viruses (see, e.g., warnock et al methods mol. Biol. (2011) 737:1-25;Walther et al.Drugs (2000) 60 (2): 249-271;and Lundstrom Trends Biotechnol. (2003) 21 (3): 117-122, which is incorporated herein by reference). In one embodiment of the invention, reporter gene #01 is delivered to High-Five insect cells via baculovirus.

In some embodiments, the reporter gene can be produced by integrating (i.e., knocking in) the transgene into the chromosome of the eukaryotic cell to produce a recombinant host cell. Such knockins may be random or position specific and are preferentially directed to mammalian cells. Many methods for knocking transgenes into a host are known in the art. Typical site-specific integration methods involve the following steps: 1) Introducing a targeting vector containing a gene of interest into eukaryotic cells and 2) screening and selecting transfected cells that integrate the gene of interest at a specific gene location.

In some embodiments, the cells are transiently transfected or stably transformed or transfected with one or more vectors encoding a reporter gene (e.g., comprising a targeting signal, two complementing elements, and a recognition element). In some embodiments, the transgenic organism produces a protein encoding a reporter gene necessary for performing the assays described herein. In other embodiments, the reporter gene is expressed in a cell (e.g., lymphoblastic, dermal fibroblast, or myoblast) from the subject.

Cell-free reconstitution systems may also be used to express the reporter genes provided herein. Typically, these systems may include cell lysates derived from simultaneous translation or combined transcriptional translation of recombinant genetic material encoding experimental and control reporter enzymes or proteins.

Another aspect of the invention relates to methods of treatment of pathological conditions or diseases amenable to OMA1 modulators.

In some embodiments, the drug with OMA1 modulating properties is tiranavir, pazopanib hydrochloride, sorafenib, sunitinib, ibrutinib, regorafenib, celecoxib, raloxifene, actinomycin, exendine, cabotinib, tamoxifen citrate, pexidanib, daunorubicin hydrochloride, dabrafenib mesylate, lazotinib, pentarubicin, trimetinib, emtrictinib, bosutinib hydrochloride, idarubicin hydrochloride, flecaitinib, celecoxib Li Nisuo, rebaudinib, ceritinib, imatinib, doxorubicin hydrochloride, valettac, geretene, mitotane, or octtinib.

In one aspect, pazopanib hydrochloride or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises pazopanib hydrochloride.

In one aspect, pazopanib hydrochloride is used for the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses pazopanib hydrochloride as a component thereof.

In one aspect, sorafenib, or a solvate, prodrug, analog, or pharmaceutically acceptable salt thereof, is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises sorafenib.

In one aspect, sorafenib is used to prepare a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses sorafenib as a component thereof.

In one aspect, kavain or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises kavain.

In one aspect, kavain is used in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses kavain as a component thereof.

In one aspect, sunitinib, or a solvate, prodrug, analog, or pharmaceutically acceptable salt thereof, is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises sunitinib.

In one aspect, sunitinib is used for the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses sunitinib as a component thereof.

In one aspect, ibrutinib, or a solvate, prodrug, analog, or pharmaceutically acceptable salt thereof, is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises ibrutinib.

In one aspect, ibrutinib is used for the manufacture of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses ibrutinib as a component thereof.

In one aspect, regorafenib, or a solvate, prodrug, analog, or pharmaceutically acceptable salt thereof, is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises regorafenib.

In one aspect, regorafenib is used for the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses regorafenib as a component thereof.

In one aspect, celecoxib or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises celecoxib.

In one aspect, celecoxib is used for the manufacture of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses celecoxib as a component thereof.

In one aspect, raloxifene or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises raloxifene.

In one aspect, raloxifene is used for the manufacture of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses raloxifene as a component thereof.

In one aspect, actinomycin or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises an actinomycin.

In one aspect, actinomycin is used in the manufacture of a personalized medicine for the treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses actinomycin as a component thereof.

In one aspect, the exendin or solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises azepine.

In one aspect, exendin is used for the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses exendin as a component thereof.

In one aspect, cabozatinib, or a solvate, prodrug, analog, or pharmaceutically acceptable salt thereof, is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises cabotinib.

In one aspect, cabozantinib is used for the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses cabotinib as a component thereof.

In one aspect, tamoxifen citrate or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises tamoxifen citrate.

In one aspect, tamoxifen citrate is used in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses tamoxifen citrate as a component thereof.

In one aspect, pexidanib or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises pexidanib.

In one aspect, pexidanib is used for the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, the process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses pexidantinib as a component thereof.

In one aspect, daunorubicin hydrochloride, or a solvate, prodrug, analog, or pharmaceutically acceptable salt thereof, is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises daunorubicin hydrochloride.

In one aspect, daunorubicin hydrochloride is used in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses daunorubicin hydrochloride as a component thereof.

In one aspect, dabrafenib mesylate, or a solvate, prodrug, analog, or pharmaceutically acceptable salt thereof, is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises dabrafenib mesylate.

In one aspect, dabrafenib mesylate is used in the manufacture of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses dabrafenib mesylate as a component thereof.

In one aspect, the laratinib or solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises loratidine.

In one aspect, loratidine is used in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses laratinib as a component thereof.

In one aspect, valrubicin or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises valrubicin.

In one aspect, valrubicin is used for the manufacture of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses valrubicin as a component thereof.

In one aspect, trametetinib, or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof, is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises trametinib.

In one aspect, trametinib is used for the manufacture of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses trametinib as a component thereof.

In one aspect, emtrictinib, or a solvate, prodrug, analog, or pharmaceutically acceptable salt thereof, is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises emtrictinib.

In one aspect, emtrictinib is used for the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses emtrictinib as a component thereof.

In one aspect, bosutinib or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises bosutinib.

In one aspect, bosutinib is used for the manufacture of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses bosutinib as a component thereof.

In one aspect, idarubicin hydrochloride or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises idarubicin hydrochloride.

In one aspect, idarubicin hydrochloride is used for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses idarubicin hydrochloride as a component thereof.

In one aspect, the criatinib, or a solvate, prodrug, analog, or pharmaceutically acceptable salt thereof, is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises lixisenatide.

In one aspect, the criatinib is used to prepare a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses tutoritinib as a component thereof.

In one aspect, plug Li Nisuo or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises plug Li Nisuo.

In one aspect, plug Li Nisuo is used to prepare a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses plug Li Nisuo as a component thereof.

In one aspect, the rebaudiana or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises rebaudinib.

In one aspect, rebaudinib is used to prepare a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses rebaudinib as an ingredient thereof.

In one aspect, ceritinib or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises ceritinib.

In one aspect, ceritinib is used for the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses ceritinib as a component thereof.

In one aspect, imatinib or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises imatinib.

In one aspect, imatinib is used for the preparation of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses imatinib as a component thereof.

In one aspect, doxorubicin hydrochloride or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises doxorubicin hydrochloride.

In one aspect, doxorubicin hydrochloride is used for the preparation of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses doxorubicin hydrochloride as its ingredient.

In one aspect, the valnemulin, or a solvate, prodrug, analog, or pharmaceutically acceptable salt thereof, is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises valnemulin.

In one aspect, the valnemulin is used to prepare a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses valnemulin as a component thereof.

In one aspect, the gefitinib or solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises gefitinib.

In one aspect, gefitinib is used in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses gefitinib as a component thereof.

In one aspect, mitotane or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises mitotane.

In one aspect, mitotane is used for the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses mitotane as a component thereof.

In one aspect, the octreotide or solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises octenib.

In one aspect, the use of octreotide for the manufacture of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses octenib as a component thereof.

In one aspect, telanavir or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprises telanavir.

In one aspect, telanavir is used to prepare a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

In one aspect, a process for preparing a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity uses telanavir as a component thereof.

It is to be understood that the various aspects and embodiments provided herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Examples

The following are examples of specific embodiments for practicing the invention. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way. In these examples, the accuracy of the numbers used (e.g. concentrations) has been ensured as much as possible, but some experimental errors and deviations are certainly allowed.

Example 1 provides a non-limiting example of the production and use of the reporter gene of the invention and illustrates the function of the reporter gene. For this reason, the preparation and use of reporter gene #01 (SEQ ID NO: 01) and reporter gene #15 (SEQ ID NO: 15) are described and the functions of both are compared. The general design of the reporter gene of the present invention is illustrated in fig. 1A and its principle of operation is illustrated in fig. 1B. In general, the reporter genes (specifically, reporter gene #01 and reporter gene # 15) can be prepared as follows. The DNA sequence of the reporter gene may be codon optimized on silica gel or by any other means for expression in a suitable host. The DNA sequences of reporter genes #01 and #15 are codon optimized for expression in humans (SEQ ID NO:02 and SEQ ID NO:16, respectively). The (optimized) sequences were synthesized under the control of the CMV promoter and cloned into pcdna3.1 expression vectors (see fig. 1 for an illustration of such vectors). Of course, for best results, the expression system and host should be matched. In this example, the host is a Hek293T cell maintained under standard culture conditions with 10% fetal bovine serum and 1% Pen/Strep DMEM. In the experiments Hek293T cells were seeded at 80% confluence in clear white tissue culture treated 96-well plates and transfected with vectors carrying reporter #01 and reporter #15, after 24 hours incubated in optmem or optmem with 10 μm CCCP for 30 min at 37 ℃. After 30 minutes, the OptiMEM cell culture medium was replaced with a 1:100 dilution of luciferase substrate (Promega) in OptiMEM and bioluminescence was measured by Fluoroskan Ascent FL (Thermo Scientific) using a 517nm single pass filter and integration time of 200 milliseconds. The results are shown in fig. 3 as bar graphs with standard deviation. This example, which is for illustration only, shows: (A) The bioluminescence signals from reporter genes #01 and #15 were significantly reduced after CCCP treatment. CCCP treatment activates OMA1 and peptide hydrolyzes the reporter genes encoded by reporter genes #01 and # 15. This cleavage event results in loss of reporter activity, thereby eliminating the bioluminescent signal; and (B) reporter #15 produces a much stronger signal than reporter #01, almost an order of magnitude stronger. (note that the scales on the two y-axes in fig. 3 are different). This finding is very surprising and is quite contrary to what is expected. Reporter gene #01 contains a larger portion of the OPA1 amino terminus as a mitochondrial targeting sequence that is cleaved by mitochondrial processing peptidases following mitochondrial introduction (see example 2). Reporter #15 was originally expected as a control for reporter # 01. Thus, reporter #15 lacks the first 66 amino acids of reporter #01, which 66 amino acids are believed to be necessary for mitochondrial induction. Those of ordinary skill in the art, even those of ordinary skill in the art like me, would expect that reporter #15 would remain in the cytoplasm and would not be transferred into mitochondria because reporter #15 does not have a mitochondrial import sequence. Surprisingly and contrary to what is expected, reporter gene #15 is transferred into mitochondria (see also example 7). Moreover, it is incredible that the performance of reporter #15 is much better than that of reporter # 01.

Example 2 relates to fig. 4 and illustrates hydrolysis of reporter gene #01 and reporter gene # 15. In this example, transfected cells were selected by puromycin. Reporter cells were exposed to 10 μmcccp for 30 min, followed by harvesting the cells in RIPA buffer and processing them on 12% SDS-PAGE followed by Western blot analysis. As shown in FIG. 4A, reporter #01 and reporter #15 migrate in the same size, just below the 34kDa standard. Full-length reporter #01 had the expected size of 39.6kDa, but was not detected in Western blot analysis. This suggests that the mitochondrial import sequence of reporter #01 is efficiently cleaved, most likely by mitochondrial processing peptidases. Meanwhile, reporter #15 was not processed after introduction, but migrated to the expected size of 31.9kDa (example 7 established mitochondrial introduction by showing co-migration of reporter #15 and OPA1 and OMA1 in the mitochondrial-rich fraction). Reporter #15 appears to be more abundant and more stable, which may explain the reason for its better performance. CCCP treatment resulted in processing of both reporter #01 and reporter # 15. This provides additional evidence that reporter #15 is positively located in mitochondria, where reporter #15 is recognized and hydrolyzed by OMA 1. It is well known in the art that the chelator phenanthroline inhibits OMA1 protease in vitro (see, e.g., ehses et al J Cell Biol (2009) 187 (7): 1023-36;and Head et al.J Cell Biol (2009) 187 (7): 959-66). Thus, phenanthroline is used as a positive control in the detection of a reporter gene. If reporter #15 is indeed cleaved by OMA1, then phenanthroline should prevent such cleavage, at least to some extent by inhibiting OMA1 and thereby preserving the signal. Hek293T cells were transfected with reporter gene #15 as described in the examples above. After 24 hours, the transfected cells were incubated with 500. Mu.M phenanthroline in OptiMEM. After 1 hour of pretreatment, the CCCP in optmem/o-diazofilm was added to a final concentration of 10 μm for CCCP and the cells were incubated for an additional 30 minutes at 37 ℃. After 30 minutes, the medium was replaced with furimazine (1:100 in OptiMEM) and bioluminescence was measured as described in example 1. The results of this illustrated example are provided in fig. 4B, which shows that the bioluminescence signal generated by reporter #15 was eliminated when cells were treated with 10 μm CCCP, but the bioluminescence signal was largely retained when cells were pretreated with 500 μm phenanthroline. Thus, this example demonstrates that (1) reporter #15 is indeed cleaved by OMA1, and that (2) the described method is very useful for identifying OMA1 inhibitors.

Example 3 relates to fig. 5 and shows a time-course experiment to illustrate the detection window and the detection effectiveness. Hek293T cells in 96-well plates were transfected with reporter genes #01 and #15, cells were incubated with 10 μΜ CCCP for 30 minutes after 24 hours, after which time the medium was replaced with furimazine and bioluminescence was measured at the indicated time points as described in the examples above. As shown in FIG. 5A, reporter gene #01 and reporter gene #15 show comparable kinetics, reaching maximum bioluminescence signal at 2 to 5 minutes, after which the signal decays slowly over time. The effectiveness of the test can be assessed and evaluated by its Z-prime score (Z'), which takes into account the mean signal (mean) and the standard deviation (s.d.) with respect to the positive and negative controls of the test. Z 'is calculated by subtracting the sum of 3 times the standard deviation divided by the difference between the mean and 1, Z' >0.5 is considered to be sufficiently effective for high throughput screening. Figure 5C provides a Z' score for this specific non-limiting example, which demonstrates the effectiveness of the reporter gene #15 assay for high throughput drug screening.

Example 4 relates to fig. 6 and establishes that the targeting sequence itself is sufficient for the OMA1 protease to recognize the reporter gene. In this example, the S1-cleavage site of reporter #15 was replaced with a TEV cleavage site (see also example 6). Neural 2A cells were transfected with reporter #15-S1 and reporter #15-TEV and selected by puromycin. FIG. 6 shows the valinomycin dose response relationship for reporter #15-S1 and reporter #15-TEV, measured essentially as described in the previous examples. Valinomycin activates OMA1 proteases (see, e.g., ehses et al J Cell Biol (2009) 187 (7): 1023-36; and Head et al J Cell Biol (2009) 187 (7): 959-66), which can be monitored by a decrease in the bioluminescence of the reporter gene. Both reporter #15-S1 and reporter #15-TEV showed similar responses to valinomycin with half maximal effective concentration (EC 50) values in the lower nanomolar range. This result is surprising and departs from all teachings of the art, which asserts (without exaggeration) that OMA1 recognizes the S1-cleavage site (see, e.g., US10739331B 2). FIG. 7 provides an overview of the arrangement of elements of different reporter genes, which include, inter alia, the amino acid sequences of SEQ ID NO:01 (Rep#01), SEQ ID NO:03 (Rep#04), SEQ ID NO:09 (Rep#08), SEQ ID NO:13 (Rep#10), and SEQ ID NO:15 (Rep#15). FIG. 8 shows Western blot analysis of Hek293T cells expressing Rep#01,Rep#04,Rep#08,Rep#10,and Rep#15 (incubated with or without CCCP). Hydrolysis of reporter gene #01 (FIG. 8A, asterisk) and reporter gene #15 (FIG. 8E, asterisk) was confirmed in CCCP treated cells, although hydrolysis of other reporter genes did not proceed very well. For example, reporter gene #08 (FIG. 8C) lacks part of the targeting sequence of Rep #15' and has only a short recognition peptide of 4 amino acids (SEQ ID NO: 39), which appears to be unrecognized by OMA 1.

Example 5 illustrates how mitochondrial toxicity can be measured using the reporter gene and detection methods of the invention. The OMA1-OPA1 pathway essentially acts as a precursor to mitochondrial degeneration (canary-in-the-coramine). Mitochondria rapidly activate OMA1 upon exposure to toxins, which can be monitored by the reporter gene and detection methods of the invention. Sorafenib and telanavir are two drugs that exhibit mitochondrial toxicity (see, e.g., FDA labeling). Hek293T cells expressing reporter genes #01 and #15 were exposed to CCCP (sorafenib and telanavir) for 30 minutes, then medium was replaced with furimazine (1:100), and bioluminescence was measured. The results of this illustrated example are provided in fig. 9, fig. 9 showing that CCCP (sorafenib and telanavir) significantly reduced bioluminescence of reporter genes #01 and # 15. Fig. 10 shows the dose response curves of telanavir and kavain (CAS registry # 500-64-1), wherein kavain and telanavir share common structural features (see, e.g., WO 9530670). (the dose response curve for sorafenib is provided in fig. 21D). This example shows how the reporter gene of the invention is useful in detecting mitochondrial toxicity.

The following non-limiting examples illustrate in more detail the in vivo assessment of OMA1 protease activity using the artificial luciferase reporter gene of the invention stably expressed in Hek293T cells. The reporter cell line designated 293TR15F6 or Luke-S1 was deposited with the ATCC patent deposit unit under the Budapest treaty at 4.7.2021, accession number PTA-127022. These examples demonstrate that engineered luciferases incorporating a truncated portion of the amino terminus of OPA1 are successfully transferred into mitochondria where they are hydrolysed under experimental conditions, as are OPA 1. Further, these examples show that the Z-prime value for the assay performed in 384-well plates is 0.68. These examples also illustrate two complementary drug screening methods for OMA1 activator and OMA1 inhibitor, respectively, which were successfully performed in pilot scale screening. As already explained, activation of OMA1 results in cleavage of the reporter gene and loss of luciferase activity. Screening for 1,280 compounds of different molecular structures that significantly reduced bioluminescence gave 195 compounds (15.2%). Moreover, 30 (18.1%) of 166 approved cancer drugs are able to activate OMA1, but only 27 (6.9%) of 390 natural products are able to activate OMA1. Given that (i) OMA1 and OPA1 are associated with a variety of diseases (e.g., neurodegenerative and cardiac diseases) and (ii) chemotherapy-induced neuropathy and cardiac toxicity are common side effects in many drugs, it can be clearly seen that the detection methods described herein are very useful in predicting these side effects. Moreover, the detection methods described herein are also very useful in the design of better cancer therapies in that (a) OMA1 activation is avoided, thereby limiting adverse side effects, or (B) OMA1 activation specifically in malignant cells results in apoptosis, thereby inhibiting tumor growth. All such embodiments of use and the methods and applications derived therefrom are within the scope of the invention. Furthermore, the detection methods of the present invention may be used to identify potential OMA1 inhibitors or compounds that counteract OMA1 activation (as exemplified in the examples below).

Example 6 relates to FIG. 11 and incorporates the Luke-S1 reporter cell line, which is based on a modified nanoLuc complementation system (Dixon, et al ACS Chem Biol (2016) 11 (2): 400-8). NanoLuc is an engineered luciferase that converts a cell permeable substrate, imidozopyrazinone, to give off light (Hall, et al ACS Chem Biol (2012) 7 (11): 1848-57). As illustrated in FIG. 11A, the Luke-S1 reporter gene (having the protein sequence SEQ ID NO:15 and the DNA sequence SEQ ID NO: 16) has the last 11 n-terminal amino acids of nanoLuc (referred to as SmBiT), the c-terminus is linked to the remaining 156 amino acids of the luciferase (referred to as LgBiT) via a 24 amino acid linker, which encodes an OPA 1S 1 cleavage site having the protein sequence SEQ ID NO:45 and the DNA sequence SEQ ID NO:46.Luke-S1 is targeted to the mitochondrial inner membrane by 86 amino acid portions of the c-terminus of OPA1, which has the protein sequence SEQ ID NO 27 and the DNA sequence SEQ ID NO 28. A reporter gene with the S1 site replaced by a TEV cleavage site (called Luke-TEV) and a native nanoLuc enzyme (called Luke) were used as controls. As shown in FIG. 11, both Luke-S1 and Luke-TEV assembled into functional luciferase with 37.9. Mu.M.+ -. 5.1 standard deviation (SE) and 30.9. Mu.M.+ -. 4.8SE Michaelis-Menten substrate affinity (K) _M ). The K is _M The values are within the range of the native nanoLuc enzyme (K _M [Luke]31.3. Mu.M.+ -. 3.7 SE). However, vmax is significantly reduced by about an order of magnitude (V _max [Luke-S1]:120μM±6SE；V _max [Luke-TEV]:53μM±3SE；V _max [Luke]:537μM±23SE)。

Example 7 relates to FIG. 12 and establishes that both the Luke-S1 reporter gene and the Luke-TEV reporter gene are hydrolyzed under conditions that activate OMA 1. In general, OMA1 shows very little activity under physiological conditions, but OMA1 cleaves OPA1 in cells treated with proton carrier CCCP or ionophore valinomycin (see also Ehses et al J Cell Biol (2009) 187 (7): 1023-36; and Head et al J Cell Biol (2009) 187 (7): 959-66). OPA1 hydrolysis was monitored by western blot analysis. FIG. 12A shows complete disappearance of the L-OPA1 isoform in Hek293T cells, e.g., after 30 minutes of treatment with 3. Mu.M CCCP. FIG. 12B shows that valinomycin is more potent, 100nM valinomycin being sufficient for cleavage of OPA 1. FIG. 12C shows that 3. Mu.M CCCP and 100nM valinomycin also induced Luke-S1 reporter cleavage in Western immunoblot analysis. The LgBiT antibody recognizes proteins that migrate in untreated Luke-S1 cells below the 34kDa standard. The predicted size of full-length Luke-S1 is 31.8kDa. In cells treated with CCCP and valomycin, the 31.8kDa band tended to be horizontal and bands migrating above the 15kDa standard became much more prominent, about 19kDa according to the approximate size of the band, which corresponds to the reporter gene hydrolysed at the S1 site. Surprisingly, luke-TEV also cleaved after CCCP or valinomycin treatment, but showed a different cleavage pattern with only a slight size reduction (fig. 12C). Notably, intima anchoring is sufficient to be recognized by OMA 1. The fact that Luke-TEV also cleaves indicates that OMA1 is accidental in its substrate recognition and that the OPA 1S 1 cleavage site is not necessary for the design of OMA1 detection methods. This is contrary to the teachings of the prior art (see for example US10739331B 2). FIG. 12D shows cell debris with Luke-S1 and Luke-TEV obtained by different centrifugation treatments in mitochondrial enriched debris. Full-length Luke-S1 reporter gene, OPA1 and OMA1 were detected in mitochondrial-rich fragments (fig. 12D). Luke-TEV also migrates with OPA1 and OMA1 according to different centrifugation treatments. Neither the lysate S-OPA1 nor LgBiT in valomycin-treated cells was actively released from mitochondria, but it was still mainly present in mitochondrial-rich fragments. This concept also confirms that mitochondrial integrity is not affected by the cell fragmentation step and that the mitochondrial outer membrane is still intact.

Example 8 relates to fig. 13 and shows the response of in vivo protease detection to CCCP and valinomycin. Luke-S1 reporter cells were incubated for 30 min at half maximum Effective Concentration (EC) with increased concentration of CCCP ₅₀ ) 398.4nM (95% confidence interval: 291.3 to 545.0nM, FIG. 13A). Dose response relationship of valinomycin shows EC ₅₀ CCCP at 17.6nM (95% confidence interval: 12.2 to 25.4nM, FIG. 13B) also acts on Luke-TEV, EC of Luke-TEV ₅₀ 566.4nM (95% confidence interval: 352.2 to 910.7nM, FIG. 13C), whereas valinomycin only produces a very small signal decrease (FIG. 13D). This suggests that Luke-TEV is likely to cleave near its amino terminus, since valinomycin does not lose most of the luciferase activity of Luke-TEV, further emphasizing the difficulties and challenges of designing a practically functional OMA1 detection method. Interestingly, CCCP completely abrogated Luke bioluminescence (its EC ₅₀ 967.9nM (95% confidence interval: 793.5 to 1,898nM; FIG. 13E)). At the same time, valinomycin also did not play a role (fig. 13F). The interference of the NanoLuc reporter system on CCCP detection has been previously noted and this interference phenomenon is most likely explained by the phenomenon observed above (Pereira et al J Mol Biol (2019) 431 (8): 1689-1699).

Example 9 relates to fig. 14 and provides support data establishing the specificity of Luke-S1 while showing its temporal resolution. As illustrated by the above examples, valinomycin resulted in a significant decrease in Luke-S1 signal due to cleavage of the reporter gene. This also occurs in Luke-S1 cells transfected with control siRNA and treated with 100nM valomycin for 30 min. In contrast, luke-S1 cells transfected with OMA1siRNA showed no significant signal reduction when treated with valinomycin (fig. 14A, one-way ANOVA analysis for multiple group comparison: p=0.003). In these experiments, siRNA mediated OMA1 knockdown reduced protein levels by about 70% (fig. 14B). This data further demonstrates that the Luke-S1 reporter gene is recognized and cleaved by OMA1 protease. Exposing the reporter cells to both valinomycin and luciferase substrates and immediately recording real-time bioluminescence can help to better understand the kinetics of OMA1 protease and the transient resolution of the reporter. Luke-S1 showed a significant decrease in signal from the beginning of the measurement relative to cells without valinomycin (fig. 14C). At the first 15 minutes, the signal drops more rapidly in the presence of valinomycin, and finally the signal stabilizes at a significantly lower intensity level (fig. 14C). On the other hand, luke-TEV showed that the change from the beginning of the measurement and after about 15 minutes was much smaller, there was no difference between cells treated with and without valinomycin (fig. 14D). At the beginning of the measurement, the Luke signal intensity fills the photomultiplier of the measuring instrument. Nevertheless, after 10 minutes, there was no significant difference in bioluminescence levels or signal decay under the two treatment conditions (fig. 14E). (the rapid and continuous signal decay observed in Luke cells is most likely due to substrate depletion). Taken together, these findings demonstrate that (i) OMA1 is rapidly activated by valinomycin and (ii) the Luke-S1 reporter gene has a higher dynamic range and responds rapidly to OMA1 activation.

Example 10 provides an exemplary example illustrating drug screening of 1,280 chemically distinct compounds to screen for OMA1 activators. For this drug screening, luke-S1 cells were incubated with 10 μm test compound in 384 well plates for 60 min, followed by addition of luciferase substrate. Valomycin treated cells in columns #2 and #23 of each plate served as a positive control against which all measurements were normalized (see figure 15A, which is a snapshot of 384 well plates for the drug screen). The mean bioluminescence of untreated cells when normalized to valomycin-treated cells (100% + -4.9 SD; FIG. 15B) in this assay was 372.1% + -24.4 Standard Deviation (SD). The calculated Z-prime value is 0.68. The average standard deviation of 128 controls in all plates was 100% ± 14.5SD (fig. 15C). The higher variability of the actual screening is due in part to the plate drift exceeding 5 minutes, which requires measurement for each plate (see also fig. 15D). This screening method screens molecules that activate OMA1 in a manner comparable to valinomycin. For this reason, hit threshold was defined to be within 3 standard deviations of the control with bioluminescence reduced to valomycin treatment ((< 143.5%; fig. 15c & d, dashed line). 195 (15.2%) of 1,280 test molecules reduced the signal below 143.5% threshold (fig. 15c & d.) the detection method was also able to pick out 26 of any of the compounds screened that interfered with luciferase itself inhibited bioluminescence, even 56.5% below 3×sd threshold. 26 molecules (2.0%) were likely to inhibit luciferase, whereas instead of binding to oma1, independent screening found that 2.7% of 42,000 test compounds inhibited NanoLuc enzyme by at least 30% (Ho et al.acs Biol (2013) 8 (5): 1009-17).

Example 11 provides an exemplary embodiment illustrating drug screening of 3,520 compounds of different chemical structures to screen for OMA1 inhibitors. For this screening, luke-S1 cells were pre-incubated with 10 μm test compound for 1 to 2 hours, followed by the addition of valinomycin (final concentration 100 nM) for an additional 30 minutes. The purpose of this screening is to identify compounds that are capable of counteracting the induction of OMA1 activation by valinomycin. For this reason, untreated cells acted as a control, and all measurements were normalized to the control (see fig. 16A, which is a snapshot of the 384 well plates of the screen). The average standard deviation of 352 controls in 11 plates was 100% ± 10.4SD. Test molecules capable of maintaining bioluminescence levels in valomycin-treated Luke-S1 cells within 3 x SD of untreated cells were considered as molecules in the screen. Thus, the hit threshold is set to >68.8% (fig. 16b & c, dotted line). The average signal of 3,520 test molecules after valinomycin addition was 30.7% ± 7.0SD.26 molecules (0.7%) quenched bioluminescence to 9.8% below the 3×sd threshold, most likely due to interference with NanoLuc. One test compound had a 71.4% signal, which was within three standard deviations of untreated cells. However, this molecule was not confirmed to be selected in the retest of six different concentrations (fig. 16D).

Example 12 relates to fig. 17-21 and illustrates broad spectrum activation of OMA1 by different kinds of cancer drugs. It is known in the art that cancer drugs such as cisplatin and sorafenib can promote OPA1 cleavage (see, e.g., zhao et al lab Invest (2013) 93 (1): 8-19;Kong et al.JBiol Chem (2014) 289 (39): 27134-45). Survival data for individuals with cancer are also significantly correlated with the gene expression levels of OMA1 (see US10906931B 2) and, quite surprisingly, most FDA approved cancer drugs (30 out of 166 different classes of cancer drugs (18.1%) trigger OMA1 activation in Luke-S1 assays, whereas only 27 out of 390 natural products (6.9%) activate OMA1. Luke-S1 reporter cells were incubated with 10 μm drug for 60 min, followed by the addition of luciferase substrate and measurement of bioluminescence. The signal intensity of this study was normalized to untreated cells (100% ± 12.5SD; valomycin-treated control: 18.5% ± 5.6 SD) and the significance level was defined as a decrease in signal by at least three standard deviations (> 37.5% decrease) from untreated signal. As shown in fig. 17, 30 of the 166 approved cancer drugs (18.1%) reduced bioluminescence by 37.5% to 85.6%. 18 of these drugs were kinase inhibitors (60.0%), which account for only 39.2% of all drugs collected (65 out of 166). This too high proportion is statistically significant (Fisher accurate detection: p=0.013). Kinase inhibitors are well known clinically for cardiotoxicity, and different approaches have been developed for preclinical evaluation and cardiotoxicity prediction (see, e.g., shalma et al Sci Transl Med (2017) 9 (377): eaaf 2584). OPA1 mutations and conditional YME1L1 knockouts are known in the art to impair cardiac function in mice (see, e.g., chen et al j Am Heart Assoc (2012) 1 (5): e00301; piquereau et al cardioview Res (2012) 94 (3): 408-17;Wai et al.Science (2015) 350 (6265): aad0116; le Page et al plos One (2016) 11 (10): e 0164066). On the other hand, OMA1 elimination protects cardiomyocytes in three different mouse models of heart failure (see Acin-Perez et al Sci Transl Med (2018) 10 (434): ean 4935). Also, missense variants in human OMA1 increased the risk of mortality from heart failure in two sets of experiments with 599 individuals and 999 individuals (see Hu et al cardiova Drugs Ther (2020) 34 (3): 345-356). Previously, cardiotoxicity has prompted research into the area of cross-reactivity of multi-targeted tyrosine kinase inhibitors with mitochondria (Will et al, toxicol Sci (2008) 106 (1): 153-61). Combining all of these different data with the discovery that kinase inhibitors activate a large number of OMA1, it is clear that kinase inhibitors do react with OMA1, a mechanism that results in cardiotoxicity in individuals taking these drugs. Within the framework of this concept, the Luke-S1 assay gives a straightforward and economical method for large-scale detection of unwanted cytotoxicity. Moreover, each of the drugs and molecules described herein, as well as any of the drugs and molecules described herein, may be used to modulate OMA1 activity in a subject suffering from a disease that may be amenable to OMA1 modulation therapy, such diseases being known in the art and may include, inter alia, the diseases disclosed in US10906931B 2.

Without further elaboration, it is to be understood that one skilled in the art can utilize the foregoing description and examples to their fullest extent. Accordingly, the preferred embodiments described above are merely illustrative of the present invention and are not intended to limit the disclosure in any way. Although the foregoing invention has been described in some detail by way of illustration and example, it is to be understood that the invention is not limited to the specific details disclosed herein, but is capable of modification and variation within the scope of the following claims. In addition, each reference provided herein is incorporated by reference in its entirety as if each reference was individually incorporated by reference. In the event of a conflict between the present disclosure and the references provided herein, the present disclosure will control.

Sequence listing

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Phe Ile Glu Gly Thr Leu Pro Met Gly Val Val Arg Pro Leu Thr Glu

450 455 460

Val Glu Met Asp His Tyr Arg Glu Pro Phe Leu Asn Pro Val Asp Arg

465 470 475 480

Glu Pro Leu Trp Arg Phe Pro Asn Glu Leu Pro Ile Ala Gly Glu Pro

485 490 495

Ala Asn Ile Val Ala Leu Val Glu Glu Tyr Met Asp Trp Leu His Gln

500 505 510

Ser Pro Val Pro Lys Leu Leu Phe Trp Gly Thr Pro Gly Val Leu Ile

515 520 525

Pro Pro Ala Glu Ala Ala Arg Leu Ala Lys Ser Leu Pro Asn Cys Lys

530 535 540

Ala Val Asp Ile Gly Pro Gly Leu Asn Leu Leu Gln Glu Asp Asn Pro

545 550 555 560

Asp Leu Ile Gly Ser Glu Ile Ala Arg Trp Leu Ser Thr Leu Glu Ile

565 570 575

Ser Gly

<210> 6

<211> 1737

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 6

atgtggggaa taaaaggaag tttaccacta caaaaactac atctggtttc acgaagcatt 60

tatcattcac atcatcctac cttaaagctt caacgacccc aattaaggac atcctttcag 120

cagttctctt ctctgacaaa ccttccttta cgtaaactga aattctctcc aattaaatat 180

ggctaccagc ctcgcaggaa tttttggcca gcaagattag ctacgagact cttaaaactt 240

cgctatctca tactaggatc ggctgttggg ggtggctaca cagccaaaaa gacttttgtc 300

ttcacactcg aagatttcgt tggggactgg cgacagacag ccggctacaa cctggaccaa 360

gtccttgaac agggaggtgt gtccagtttg tttcagaatc tcggggtgtc cgtaactccg 420

atccaaagga ttgtcctgag cggtgaaaat gggctgaaga tcgacatcca tgtcatcatc 480

ccgtatgaag gtctgagcgg cgaccaaatg ggccagatcg aaaaaatttt taaggtggtg 540

taccctgtgg atgatcatca ctttaaggtg atcctgcact atggcacact ggtaatcgac 600

ggggttacgc cgaacatgat cgactatttc ggacggccgt atgaaggcat cgccgtgttc 660

gacggcaaaa agatcactgt aacagggacc ctgtggaacg gcaacaaaat tatcgacgag 720

cgcctgatca accccgacgg ctccctgctg ttccgagtaa ccatcaacgg agtgaccggc 780

tggcggctgt gcgaacgcat tctggcgccg gaagaaacgg cgtttagagc aacagatcgt 840

ggaggatccg aaatcggtac tggctttcca ttcgaccccc attatgtgga agtcctgggc 900

gagcgcatgc actacgtcga tgttggtccg cgcgatggca cccctgtgct gttcctgcac 960

ggtaacccga cctcctccta cgtgtggcgc aacatcatcc cgcatgttgc accgacccat 1020

cgctgcattg ctccagacct gatcggtatg ggcaaatccg acaaaccaga cctgggttat 1080

ttcttcgacg accacgtccg cttcatggat gccttcatcg aagccctggg tctggaagag 1140

gtcgtcctgg tcattcacga ctggggctcc gctctgggtt tccactgggc caagcgcaat 1200

ccagagcgcg tcaaaggtat tgcatttatg gagttcatcc gccctatccc gacctgggac 1260

gaatggccag aatttgcccg cgagaccttc caggccttcc gcaccaccga cgtcggccgc 1320

aagctgatca tcgatcagaa cgtttttatc gagggtacgc tgccgatggg tgtcgtccgc 1380

ccgctgactg aagtcgagat ggaccattac cgcgagccgt tcctgaatcc tgttgaccgc 1440

gagccactgt ggcgcttccc aaacgagctg ccaatcgccg gtgagccagc gaacatcgtc 1500

gcgctggtcg aagaatacat ggactggctg caccagtccc ctgtcccgaa gctgctgttc 1560

tggggcaccc caggcgttct gatcccaccg gccgaagccg ctcgcctggc caaaagcctg 1620

cctaactgca aggctgtgga catcggcccg ggtctgaatc tgctgcaaga agacaacccg 1680

gacctgatcg gcagcgagat cgcgcgctgg ctgtctactc tggagatttc cggttaa 1737

<210> 7

<211> 658

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 7

Met Trp Gly Ile Lys Gly Ser Leu Pro Leu Gln Lys Leu His Leu Val

1 5 10 15

Ser Arg Ser Ile Tyr His Ser His His Pro Thr Leu Lys Leu Gln Arg

20 25 30

Pro Gln Leu Arg Thr Ser Phe Gln Gln Phe Ser Ser Leu Thr Asn Leu

35 40 45

Pro Leu Arg Lys Leu Lys Phe Ser Pro Ile Lys Tyr Gly Tyr Gln Pro

50 55 60

Arg Arg Asn Phe Trp Pro Ala Arg Leu Ala Thr Arg Leu Leu Lys Leu

65 70 75 80

Arg Tyr Leu Ile Leu Gly Ser Ala Val Gly Gly Gly Tyr Thr Ala Lys

85 90 95

Lys Thr Phe Asp Thr Ala Ile Leu Ser Val Val Pro Phe His His Gly

100 105 110

Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu Ile Cys Gly Phe Arg Val

115 120 125

Val Leu Met Tyr Arg Phe Glu Glu Glu Leu Phe Leu Arg Ser Leu Gln

130 135 140

Asp Tyr Lys Ile Gln Ser Ala Leu Leu Val Pro Thr Leu Phe Ser Phe

145 150 155 160

Phe Ala Lys Ser Thr Leu Ile Asp Lys Tyr Asp Leu Ser Asn Leu His

165 170 175

Glu Ile Ala Ser Gly Gly Ala Pro Leu Ser Lys Glu Val Gly Glu Ala

180 185 190

Val Ala Lys Arg Phe His Leu Pro Gly Ile Arg Gln Gly Tyr Gly Leu

195 200 205

Thr Glu Thr Thr Ser Ala Ile Leu Ile Thr Pro Glu Gly Asp Asp Lys

210 215 220

Pro Gly Ala Val Gly Lys Val Val Pro Phe Phe Glu Ala Lys Val Val

225 230 235 240

Asp Leu Asp Thr Gly Lys Thr Leu Gly Val Asn Gln Arg Gly Glu Leu

245 250 255

Cys Val Arg Gly Pro Met Ile Met Ser Gly Tyr Val Asn Asn Pro Glu

260 265 270

Ala Thr Asn Ala Leu Ile Asp Lys Asp Gly Trp Leu His Ser Gly Asp

275 280 285

Ile Ala Tyr Trp Asp Glu Asp Glu His Phe Phe Ile Val Asp Arg Leu

290 295 300

Lys Ser Leu Ile Lys Tyr Lys Gly Tyr Gln Val Ala Pro Ala Glu Leu

305 310 315 320

Glu Ser Ile Leu Leu Gln His Pro Asn Ile Phe Asp Ala Gly Val Ala

325 330 335

Gly Leu Pro Asp Asp Asp Ala Gly Glu Leu Pro Ala Ala Val Val Val

340 345 350

Leu Glu His Gly Lys Thr Met Thr Glu Lys Glu Ile Val Asp Tyr Val

355 360 365

Ala Ser Gln Val Thr Thr Ala Lys Lys Leu Arg Gly Gly Val Val Phe

370 375 380

Val Asp Glu Val Pro Lys Gly Leu Thr Gly Lys Leu Asp Ala Arg Lys

385 390 395 400

Ile Arg Glu Ile Leu Ile Lys Ala Lys Lys Ala Ser Pro Glu Glu Thr

405 410 415

Ala Phe Arg Ala Thr Asp Arg Gly Ser Glu Ser Ala Lys Asn Ile Lys

420 425 430

Lys Gly Pro Ala Pro Phe Tyr Pro Leu Glu Asp Gly Thr Ala Gly Glu

435 440 445

Gln Leu His Lys Ala Met Lys Arg Tyr Ala Leu Val Pro Gly Thr Ile

450 455 460

Ala Phe Thr Asp Ala His Ile Glu Val Asp Ile Thr Tyr Ala Glu Tyr

465 470 475 480

Phe Glu Met Ser Val Arg Leu Ala Glu Ala Met Lys Arg Tyr Gly Leu

485 490 495

Asn Thr Asn His Arg Ile Val Val Cys Ser Glu Asn Ser Leu Gln Phe

500 505 510

Phe Met Pro Val Leu Gly Ala Leu Phe Ile Gly Val Ala Val Ala Pro

515 520 525

Ala Asn Asp Ile Tyr Asn Glu Arg Glu Leu Leu Asn Ser Met Gly Ile

530 535 540

Ser Gln Pro Thr Val Val Phe Val Ser Lys Lys Gly Leu Gln Lys Ile

545 550 555 560

Leu Asn Val Gln Lys Lys Leu Pro Ile Ile Gln Lys Ile Ile Ile Met

565 570 575

Asp Ser Lys Thr Asp Tyr Gln Gly Phe Gln Ser Met Tyr Thr Phe Val

580 585 590

Thr Ser His Leu Pro Pro Gly Phe Asn Glu Tyr Asp Phe Val Pro Glu

595 600 605

Ser Phe Asp Arg Asp Lys Thr Ile Ala Leu Ile Met Asn Ser Ser Gly

610 615 620

Ser Thr Gly Leu Pro Lys Gly Val Ala Leu Pro His Arg Thr Ala Cys

625 630 635 640

Val Arg Phe Ser His Ala Arg Asp Pro Ile Phe Gly Asn Gln Ile Ile

645 650 655

Pro Val

<210> 8

<211> 1977

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 8

atgtggggaa taaaaggaag tttaccacta caaaaactac atctggtttc acgaagcatt 60

tatcattcac atcatcctac cttaaagctt caacgacccc aattaaggac atcctttcag 120

cagttctctt ctctgacaaa ccttccttta cgtaaactga aattctctcc aattaaatat 180

ggctaccagc ctcgcaggaa tttttggcca gcaagattag ctacgagact cttaaaactt 240

cgctatctca tactaggatc ggctgttggg ggtggctaca cagccaaaaa gacttttgac 300

accgctatcc tcagcgtggt gccatttcac cacggcttcg gcatgttcac cacgctgggc 360

tacttgatct gcggctttcg ggtcgtgctc atgtaccgct tcgaggagga gctattcttg 420

cgcagcttgc aagactataa gattcaatct gccctgctgg tgcccacact atttagcttc 480

ttcgctaaga gcactctcat cgacaagtac gacctaagca acttgcacga gatcgccagc 540

ggcggggcgc cgctcagcaa ggaggtaggt gaggccgtgg ccaaacgctt ccacctacca 600

ggcatccgcc agggctacgg cctgacagaa acaaccagcg ccattctgat cacccccgaa 660

ggggacgaca agcctggcgc agtaggcaag gtggtgccct tcttcgaggc taaggtggtg 720

gacttggaca ctggtaagac actgggtgtg aaccagcgcg gcgagctgtg cgtccgtggc 780

cccatgatca tgagcggcta cgttaacaac cccgaggcta caaacgctct catcgacaag 840

gacggctggc tgcacagcgg cgacatcgcc tactgggacg aggacgagca cttcttcatc 900

gtggaccggc tgaagagcct gatcaaatac aagggctacc aggtagcccc agccgaactg 960

gagagcatcc tgctgcaaca ccccaacatc ttcgacgccg gggtcgccgg cctgcccgac 1020

gacgatgccg gcgagctgcc cgccgcagtc gtcgtgctgg aacacggtaa aaccatgacc 1080

gagaaggaga tcgtggacta tgtggccagc caggttacaa ccgccaagaa gctgcgcggt 1140

ggtgttgtgt tcgtggacga ggtgcctaaa ggactgaccg gcaagttgga cgcccgcaag 1200

atccgcgaga ttctcattaa ggccaagaag gctagcccgg aagaaacggc gtttagagca 1260

acagatcgtg gatctgaatc tgccaaaaac attaagaagg gcccagcgcc attctaccca 1320

ctcgaagacg ggaccgccgg cgagcagctg cacaaagcca tgaagcgcta cgccctggtg 1380

cccggcacca tcgcctttac cgacgcacat atcgaggtgg acattaccta cgccgagtac 1440

ttcgagatga gcgttcggct ggcagaagct atgaagcgct atgggctgaa tacaaaccat 1500

cggatcgtgg tgtgcagcga gaatagcttg cagttcttca tgcccgtgtt gggtgccctg 1560

ttcatcggtg tggctgtggc cccagctaac gacatctaca acgagcgcga gctgctgaac 1620

agcatgggca tcagccagcc caccgtcgta ttcgtgagca agaaagggct gcaaaagatc 1680

ctcaacgtgc aaaagaagct accgatcata caaaagatca tcatcatgga tagcaagacc 1740

gactaccagg gcttccaaag catgtacacc ttcgtgactt cccatttgcc acccggcttc 1800

aacgagtacg acttcgtgcc cgagagcttc gaccgggaca aaaccatcgc cctgatcatg 1860

aacagtagtg gcagtaccgg attgcccaag ggcgtagccc taccgcaccg caccgcttgt 1920

gtccgattca gtcatgcccg cgaccccatc ttcggcaacc agatcatccc cgtttaa 1977

<210> 9

<211> 237

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 9

Met Trp Gly Ile Lys Gly Ser Leu Pro Leu Gln Lys Leu His Leu Val

1 5 10 15

Ser Arg Ser Ile Tyr His Ser His His Pro Thr Leu Lys Leu Gln Arg

20 25 30

Pro Gln Leu Arg Thr Ser Phe Gln Gln Phe Ser Ser Leu Thr Asn Leu

35 40 45

Pro Leu Arg Lys Leu Lys Phe Ser Pro Ile Lys Tyr Gly Tyr Gln Pro

50 55 60

Arg Arg Asn Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln

65 70 75 80

Thr Ala Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser

85 90 95

Ser Leu Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile

100 105 110

Val Leu Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile

115 120 125

Pro Tyr Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile

130 135 140

Phe Lys Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu

145 150 155 160

His Tyr Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp

165 170 175

Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys

180 185 190

Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu

195 200 205

Arg Leu Ile Asn Pro Asp Gly Ser Leu Ala Phe Arg Ala Thr Ile Asn

210 215 220

Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala

225 230 235

<210> 10

<211> 714

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 10

atgtggggaa taaaaggaag tttaccacta caaaaactac atctggtttc acgaagcatt 60

tatcattcac atcatcctac cttaaagctt caacgacccc aattaaggac atcctttcag 120

cagttctctt ctctgacaaa ccttccttta cgtaaactga aattctctcc aattaaatat 180

ggctaccagc ctcgcaggaa tgtcttcaca ctcgaagatt tcgttgggga ctggcgacag 240

acagccggct acaacctgga ccaagtcctt gaacagggag gtgtgtccag tttgtttcag 300

aatctcgggg tgtccgtaac tccgatccaa aggattgtcc tgagcggtga aaatgggctg 360

aagatcgaca tccatgtcat catcccgtat gaaggtctga gcggcgacca aatgggccag 420

atcgaaaaaa tttttaaggt ggtgtaccct gtggatgatc atcactttaa ggtgatcctg 480

cactatggca cactggtaat cgacggggtt acgccgaaca tgatcgacta tttcggacgg 540

ccgtatgaag gcatcgccgt gttcgacggc aaaaagatca ctgtaacagg gaccctgtgg 600

aacggcaaca aaattatcga cgagcgcctg atcaaccccg acggctccct ggcgttccga 660

gcaaccatca acggagtgac cggctggcgg ctgtgcgaac gcattctggc gtaa 714

<210> 11

<211> 284

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 11

Met Trp Gly Ile Lys Gly Ser Leu Pro Leu Gln Lys Leu His Leu Val

1 5 10 15

Ser Arg Ser Ile Tyr His Ser His His Pro Thr Leu Lys Leu Gln Arg

20 25 30

Pro Gln Leu Arg Thr Ser Phe Gln Gln Phe Ser Ser Leu Thr Asn Leu

35 40 45

Pro Leu Arg Lys Leu Lys Phe Ser Pro Ile Lys Tyr Gly Tyr Gln Pro

50 55 60

Arg Arg Asn Phe Trp Pro Ala Arg Leu Ala Thr Arg Leu Leu Lys Leu

65 70 75 80

Arg Tyr Leu Ile Leu Gly Ser Ala Val Gly Gly Gly Tyr Thr Ala Lys

85 90 95

Lys Thr Phe Asp Gln Trp Lys Asp Met Val Glu Lys Phe Val Gly Thr

100 105 110

Trp Lys Ile Ala Asp Ser His Asn Phe Gly Glu Tyr Leu Lys Ala Ile

115 120 125

Gly Ala Pro Lys Glu Leu Ser Asp Gly Gly Asp Ala Thr Thr Pro Thr

130 135 140

Leu Tyr Ile Ser Gln Lys Asp Gly Asp Lys Met Thr Val Lys Ile Glu

145 150 155 160

Asn Gly Pro Pro Thr Phe Leu Asp Thr Gln Val Lys Phe Lys Leu Gly

165 170 175

Glu Glu Phe Asp Glu Phe Pro Ser Asp Arg Arg Lys Glu Tyr Ile Asp

180 185 190

Phe Gly Ser Pro Glu Glu Thr Ala Phe Arg Ala Thr Asp Arg Gly Ser

195 200 205

Glu Ser Asp Lys His Phe Arg Lys Gly Leu Leu Gly Glu Leu Ile Leu

210 215 220

Leu Gln Gln Gln Lys Gly Val Lys Ser Val Val Asn Leu Val Gly Glu

225 230 235 240

Lys Leu Val Tyr Val Gln Lys Trp Asp Gly Lys Glu Thr Thr Tyr Val

245 250 255

Arg Glu Ile Lys Asp Gly Lys Leu Val Val Thr Leu Thr Met Gly Asp

260 265 270

Val Val Ala Val Arg Ser Tyr Arg Arg Ala Thr Glu

275 280

<210> 12

<211> 852

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 12

atgtggggaa taaaaggaag tttaccacta caaaaactac atctggtttc acgaagcatt 60

tatcattcac atcatcctac cttaaagctt caacgacccc aattaaggac atcctttcag 120

cagttctctt ctctgacaaa ccttccttta cgtaaactga aattctctcc aattaaatat 180

ggctaccagc ctcgcaggaa tttttggcca gcaagattag ctacgagact cttaaaactt 240

cgctatctca tactaggatc ggctgttggg ggtggctaca cagccaaaaa gacttttgat 300

cagtggaaag atatggttga gaagtttgtt ggcacttgga agatagctga tagtcataac 360

tttggagaat acctgaaggc cattggtgca ccgaaagaac tttctgatgg cggagacgca 420

acaactccca cactctatat cagccaaaag gacggggaca aaatgacagt aaagatcgaa 480

aacggaccac ccactttcct ggatacgcag gtgaagttta agctcggaga ggagttcgat 540

gaatttccca gcgaccgccg gaaagagtac atcgacttcg gcagccccga ggagaccgcc 600

ttcagggcca ccgacagggg cagcgagagc gacaagcact tcaggaaggg cctgctgggc 660

gagctgatcc tgctgcagca gcagaaaggc gtgaagagcg tagtgaactt agttggagaa 720

aaactggtct atgttcagaa atgggacggt aaggagacca cttatgtgcg agaaattaag 780

gacggaaagc ttgtggtgac gctgactatg ggagacgtcg tagcagtgcg gagttataga 840

cgcgcgaccg aa 852

<210> 13

<211> 286

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 13

Met Trp Gly Ile Lys Gly Ser Leu Pro Leu Gln Lys Leu His Leu Val

1 5 10 15

Ser Arg Ser Ile Tyr His Ser His His Pro Thr Leu Lys Leu Gln Arg

20 25 30

Pro Gln Leu Arg Thr Ser Phe Gln Gln Phe Ser Ser Leu Thr Asn Leu

35 40 45

Pro Leu Arg Lys Leu Lys Phe Ser Pro Ile Lys Tyr Gly Tyr Gln Pro

50 55 60

Arg Arg Asn Phe Trp Pro Ala Arg Leu Ala Thr Arg Leu Leu Lys Leu

65 70 75 80

Arg Tyr Leu Ile Leu Gly Ser Ala Val Gly Gly Gly Tyr Thr Ala Lys

85 90 95

Lys Thr Phe Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln

100 105 110

Thr Ala Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser

115 120 125

Ser Leu Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile

130 135 140

Val Leu Ser Gly Ser Pro Glu Glu Thr Ala Phe Arg Ala Thr Asp Arg

145 150 155 160

Gly Ser Glu Ser Asp Lys His Phe Arg Lys Ile Asp Ile His Val Ile

165 170 175

Ile Pro Tyr Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys

180 185 190

Ile Phe Lys Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile

195 200 205

Leu His Tyr Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile

210 215 220

Asp Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys

225 230 235 240

Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp

245 250 255

Glu Arg Leu Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile

260 265 270

Asn Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala

275 280 285

<210> 14

<211> 861

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 14

atgtggggaa taaaaggaag tttaccacta caaaaactac atctggtttc acgaagcatt 60

tatcattcac atcatcctac cttaaagctt caacgacccc aattaaggac atcctttcag 120

cagttctctt ctctgacaaa ccttccttta cgtaaactga aattctctcc aattaaatat 180

ggctaccagc ctcgcaggaa tttttggcca gcaagattag ctacgagact cttaaaactt 240

cgctatctca tactaggatc ggctgttggg ggtggctaca cagccaaaaa gacttttgtc 300

ttcacactcg aagatttcgt tggggactgg cgacagacag ccggctacaa cctggaccaa 360

gtccttgaac agggaggtgt gtccagtttg tttcagaatc tcggggtgtc cgtaactccg 420

atccaaagga ttgtcctgag cggttctccg gaagaaacgg cgtttagagc aacagatcgt 480

ggatctgaaa gtgacaagca ttttagaaag atcgacatcc atgtcatcat cccgtatgaa 540

ggtctgagcg gcgaccaaat gggccagatc gaaaaaattt ttaaggtggt gtaccctgtg 600

gatgatcatc actttaaggt gatcctgcac tatggcacac tggtaatcga cggggttacg 660

ccgaacatga tcgactattt cggacggccg tatgaaggca tcgccgtgtt cgacggcaaa 720

aagatcactg taacagggac cctgtggaac ggcaacaaaa ttatcgacga gcgcctgatc 780

aaccccgacg gctccctgct gttccgagta accatcaacg gagtgaccgg ctggcggctg 840

tgcgaacgca ttctggcgta a 861

<210> 15

<211> 280

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 15

Met Phe Trp Pro Ala Arg Leu Ala Thr Arg Leu Leu Lys Leu Arg Tyr

1 5 10 15

Leu Ile Leu Gly Ser Ala Val Gly Gly Gly Tyr Thr Ala Lys Lys Thr

20 25 30

Phe Asp Gln Trp Lys Asp Met Ile Pro Asp Leu Ser Glu Tyr Lys Trp

35 40 45

Ile Val Pro Asp Ile Val Trp Glu Ile Asp Glu Tyr Ile Asp Phe Glu

50 55 60

Lys Ile Arg Lys Ala Leu Pro Ser Ser Glu Asp Leu Val Lys Leu Ala

65 70 75 80

Pro Asp Phe Asp Lys Ile Val Val Thr Gly Tyr Arg Leu Phe Glu Glu

85 90 95

Ile Leu Ser Gly Ser Pro Glu Glu Thr Ala Phe Arg Ala Thr Asp Arg

100 105 110

Gly Ser Glu Ser Asp Lys His Phe Arg Lys Val Phe Thr Leu Glu Asp

115 120 125

Phe Val Gly Asp Trp Glu Gln Thr Ala Ala Tyr Asn Leu Asp Gln Val

130 135 140

Leu Glu Gln Gly Gly Val Ser Ser Leu Leu Gln Asn Leu Ala Val Ser

145 150 155 160

Val Thr Pro Ile Gln Arg Ile Val Arg Ser Gly Glu Asn Ala Leu Lys

165 170 175

Ile Asp Ile His Val Ile Ile Pro Tyr Glu Gly Leu Ser Ala Asp Gln

180 185 190

Met Ala Gln Ile Glu Glu Val Phe Lys Val Val Tyr Pro Val Asp Asp

195 200 205

His His Phe Lys Val Ile Leu Pro Tyr Gly Thr Leu Val Ile Asp Gly

210 215 220

Val Thr Pro Asn Met Leu Asn Tyr Phe Gly Arg Pro Tyr Glu Gly Ile

225 230 235 240

Ala Val Phe Asp Gly Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn

245 250 255

Gly Asn Lys Ile Ile Asp Glu Arg Leu Ile Thr Pro Asp Gly Ser Met

260 265 270

Leu Phe Arg Val Thr Ile Asn Ser

275 280

<210> 16

<211> 843

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 16

atgttttggc cagcaagatt agctacgaga ctcttaaaac ttcgctatct catactagga 60

tcggctgttg ggggtggcta cacagccaaa aagacttttg atcagtggaa agatatgata 120

ccggacctta gtgaatataa atggattgtg cctgacattg tgtgggaaat tgatgagtat 180

atcgattttg agaaaattag aaaagccctt cctagttcag aagaccttgt aaagttagca 240

ccagactttg acaagattgt tgtgaccggc taccggctgt tcgaggagat tctgtcaggt 300

tctccggaag aaacggcgtt tagagcaaca gatcgtggat ctgaaagtga caagcatttt 360

agaaaggtct tcacactcga agatttcgtt ggggactggg aacagacagc cgcctacaac 420

ctggaccaag tccttgaaca gggaggtgtg tccagtttgc tgcagaatct cgccgtgtcc 480

gtaactccga tccaaaggat tgtccggagc ggtgaaaatg ccctgaagat cgacatccat 540

gtcatcatcc cgtatgaagg tctgagcgcc gaccaaatgg cccagatcga agaggtgttt 600

aaggtggtgt accctgtgga tgatcatcac tttaaggtga tcctgcccta tggcacactg 660

gtaatcgacg gggttacgcc gaacatgctg aactatttcg gacggccgta tgaaggcatc 720

gccgtgttcg acggcaaaaa gatcactgta acagggaccc tgtggaacgg caacaaaatt 780

atcgacgagc gcctgatcac ccccgacggc tccatgctgt tccgagtaac catcaacagc 840

taa 843

<210> 17

<211> 152

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 17

Trp Gly Ile Lys Gly Ser Leu Pro Leu Gln Lys Leu His Leu Val Ser

1 5 10 15

Arg Ser Ile Tyr His Ser His His Pro Thr Leu Lys Leu Gln Arg Pro

20 25 30

Gln Leu Arg Thr Ser Phe Gln Gln Phe Ser Ser Leu Thr Asn Leu Pro

35 40 45

Leu Arg Lys Leu Lys Phe Ser Pro Ile Lys Tyr Gly Tyr Gln Pro Arg

50 55 60

Arg Asn Phe Trp Pro Ala Arg Leu Ala Thr Arg Leu Leu Lys Leu Arg

65 70 75 80

Tyr Leu Ile Leu Gly Ser Ala Val Gly Gly Gly Tyr Thr Ala Lys Lys

85 90 95

Thr Phe Asp Gln Trp Lys Asp Met Ile Pro Asp Leu Ser Glu Tyr Lys

100 105 110

Trp Ile Val Pro Asp Ile Val Trp Glu Ile Asp Glu Tyr Ile Asp Phe

115 120 125

Glu Lys Ile Arg Lys Ala Leu Pro Ser Ser Glu Asp Leu Val Lys Leu

130 135 140

Ala Pro Asp Phe Asp Lys Ile Val

145 150

<210> 18

<211> 456

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 18

tggggaataa aaggaagttt accactacaa aaactacatc tggtttcacg aagcatttat 60

cattcacatc atcctacctt aaagcttcaa cgaccccaat taaggacatc ctttcagcag 120

ttctcttctc tgacaaacct tcctttacgt aaactgaaat tctctccaat taaatatggc 180

taccagcctc gcaggaattt ttggccagca agattagcta cgagactctt aaaacttcgc 240

tatctcatac taggatcggc tgttgggggt ggctacacag ccaaaaagac ttttgatcag 300

tggaaagata tgataccgga ccttagtgaa tataaatgga ttgtgcctga cattgtgtgg 360

gaaattgatg agtatatcga ttttgagaaa attagaaaag cccttcctag ttcagaagac 420

cttgtaaagt tagcaccaga ctttgacaag attgtt 456

<210> 19

<211> 98

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 19

Trp Gly Ile Lys Gly Ser Leu Pro Leu Gln Lys Leu His Leu Val Ser

1 5 10 15

Arg Ser Ile Tyr His Ser His His Pro Thr Leu Lys Leu Gln Arg Pro

20 25 30

Gln Leu Arg Thr Ser Phe Gln Gln Phe Ser Ser Leu Thr Asn Leu Pro

35 40 45

Leu Arg Lys Leu Lys Phe Ser Pro Ile Lys Tyr Gly Tyr Gln Pro Arg

50 55 60

Arg Asn Phe Trp Pro Ala Arg Leu Ala Thr Arg Leu Leu Lys Leu Arg

65 70 75 80

Tyr Leu Ile Leu Gly Ser Ala Val Gly Gly Gly Tyr Thr Ala Lys Lys

85 90 95

Thr Phe

<210> 20

<211> 294

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 20

tggggaataa aaggaagttt accactacaa aaactacatc tggtttcacg aagcatttat 60

cattcacatc atcctacctt aaagcttcaa cgaccccaat taaggacatc ctttcagcag 120

ttctcttctc tgacaaacct tcctttacgt aaactgaaat tctctccaat taaatatggc 180

taccagcctc gcaggaattt ttggccagca agattagcta cgagactctt aaaacttcgc 240

tatctcatac taggatcggc tgttgggggt ggctacacag ccaaaaagac tttt 294

<210> 21

<211> 66

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 21

Trp Gly Ile Lys Gly Ser Leu Pro Leu Gln Lys Leu His Leu Val Ser

1 5 10 15

Arg Ser Ile Tyr His Ser His His Pro Thr Leu Lys Leu Gln Arg Pro

20 25 30

Gln Leu Arg Thr Ser Phe Gln Gln Phe Ser Ser Leu Thr Asn Leu Pro

35 40 45

Leu Arg Lys Leu Lys Phe Ser Pro Ile Lys Tyr Gly Tyr Gln Pro Arg

50 55 60

Arg Asn

65

<210> 22

<211> 198

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 22

tggggaataa aaggaagttt accactacaa aaactacatc tggtttcacg aagcatttat 60

cattcacatc atcctacctt aaagcttcaa cgaccccaat taaggacatc ctttcagcag 120

ttctcttctc tgacaaacct tcctttacgt aaactgaaat tctctccaat taaatatggc 180

taccagcctc gcaggaat 198

<210> 23

<211> 103

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 23

Trp Gly Ile Lys Gly Ser Leu Pro Leu Gln Lys Leu His Leu Val Ser

1 5 10 15

Arg Ser Ile Tyr His Ser His His Pro Thr Leu Lys Leu Gln Arg Pro

20 25 30

Gln Leu Arg Thr Ser Phe Gln Gln Phe Ser Ser Leu Thr Asn Leu Pro

35 40 45

Leu Arg Lys Leu Lys Phe Ser Pro Ile Lys Tyr Gly Tyr Gln Pro Arg

50 55 60

Arg Asn Phe Trp Pro Ala Arg Leu Ala Thr Arg Leu Leu Lys Leu Arg

65 70 75 80

Tyr Leu Ile Leu Gly Ser Ala Val Gly Gly Gly Tyr Thr Ala Lys Lys

85 90 95

Thr Phe Asp Gln Trp Lys Asp

100

<210> 24

<211> 309

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 24

tggggaataa aaggaagttt accactacaa aaactacatc tggtttcacg aagcatttat 60

cattcacatc atcctacctt aaagcttcaa cgaccccaat taaggacatc ctttcagcag 120

ttctcttctc tgacaaacct tcctttacgt aaactgaaat tctctccaat taaatatggc 180

taccagcctc gcaggaattt ttggccagca agattagcta cgagactctt aaaacttcgc 240

tatctcatac taggatcggc tgttgggggt ggctacacag ccaaaaagac ttttgatcag 300

tggaaagat 309

<210> 25

<211> 32

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 25

Phe Trp Pro Ala Arg Leu Ala Thr Arg Leu Leu Lys Leu Arg Tyr Leu

1 5 10 15

Ile Leu Gly Ser Ala Val Gly Gly Gly Tyr Thr Ala Lys Lys Thr Phe

20 25 30

<210> 26

<211> 96

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 26

ttttggccag caagattagc tacgagactc ttaaaacttc gctatctcat actaggatcg 60

gctgttgggg gtggctacac agccaaaaag actttt 96

<210> 27

<211> 86

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 27

Phe Trp Pro Ala Arg Leu Ala Thr Arg Leu Leu Lys Leu Arg Tyr Leu

1 5 10 15

Ile Leu Gly Ser Ala Val Gly Gly Gly Tyr Thr Ala Lys Lys Thr Phe

20 25 30

Asp Gln Trp Lys Asp Met Ile Pro Asp Leu Ser Glu Tyr Lys Trp Ile

35 40 45

Val Pro Asp Ile Val Trp Glu Ile Asp Glu Tyr Ile Asp Phe Glu Lys

50 55 60

Ile Arg Lys Ala Leu Pro Ser Ser Glu Asp Leu Val Lys Leu Ala Pro

65 70 75 80

Asp Phe Asp Lys Ile Val

85

<210> 28

<211> 258

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 28

ttttggccag caagattagc tacgagactc ttaaaacttc gctatctcat actaggatcg 60

gctgttgggg gtggctacac agccaaaaag acttttgatc agtggaaaga tatgataccg 120

gaccttagtg aatataaatg gattgtgcct gacattgtgt gggaaattga tgagtatatc 180

gattttgaga aaattagaaa agcccttcct agttcagaag accttgtaaa gttagcacca 240

gactttgaca agattgtt 258

<210> 29

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 29

Arg Leu Arg Arg Ala Ala Val Ala Cys Glu Val Cys Gln Ser Leu Val

1 5 10 15

Lys His Ser Ser

20

<210> 30

<211> 60

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 30

cgactacgtc gggccgctgt ggcctgtgag gtctgccagt ctttagtgaa acacagctct 60

<210> 31

<211> 86

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 31

Trp Arg Leu Arg Arg Ala Ala Val Ala Cys Glu Val Cys Gln Ser Leu

1 5 10 15

Val Lys His Ser Ser Gly Ile Lys Gly Ser Leu Pro Leu Gln Lys Leu

20 25 30

His Leu Val Ser Arg Ser Ile Tyr His Ser His His Pro Thr Leu Lys

35 40 45

Leu Gln Arg Pro Gln Leu Arg Thr Ser Phe Gln Gln Phe Ser Ser Leu

50 55 60

Thr Asn Leu Pro Leu Arg Lys Leu Lys Phe Ser Pro Ile Lys Tyr Gly

65 70 75 80

Tyr Gln Pro Arg Arg Asn

85

<210> 32

<211> 258

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 32

tggcgactac gtcgggccgc tgtggcctgt gaggtctgcc agtctttagt gaaacacagc 60

tctggaataa aaggaagttt accactacaa aaactacatc tggtttcacg aagcatttat 120

cattcacatc atcctacctt aaagcttcaa cgaccccaat taaggacatc ctttcagcag 180

ttctcttctc tgacaaacct tcctttacgt aaactgaaat tctctccaat taaatatggc 240

taccagcctc gcaggaat 258

<210> 33

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 33

Gly Ser Pro Glu Glu Thr Ala Phe Arg Ala Thr Asp Arg Gly Ser Glu

1 5 10 15

Ser Asp Lys His Phe Arg Lys

20

<210> 34

<211> 69

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 34

ggttctccgg aagaaacggc gtttagagca acagatcgtg gatctgaaag tgacaagcat 60

tttagaaag 69

<210> 35

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 35

Pro Glu Glu Thr Ala Phe Arg Ala Thr Asp Arg Gly

1 5 10

<210> 36

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 36

ccggaagaaa cggcgtttag agcaacagat cgtgga 36

<210> 37

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 37

Pro Glu Glu Thr Ala Phe Arg Ala Thr Asp Arg Gly Ser Glu

1 5 10

<210> 38

<211> 42

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 38

ccggaagaaa cggcgtttag agcaacagat cgtggatctg aa 42

<210> 39

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 39

Ala Phe Arg Ala

1

<210> 40

<211> 12

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 40

gcgtttagag ca 12

<210> 41

<211> 40

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 41

Glu Tyr Ile Asp Phe Gly Ser Pro Glu Glu Thr Ala Phe Arg Ala Thr

1 5 10 15

Asp Arg Gly Ser Glu Ser Asp Lys His Phe Arg Lys Gly Leu Leu Gly

20 25 30

Glu Leu Ile Leu Leu Gln Gln Gln

35 40

<210> 42

<211> 120

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 42

gagtacatcg acttcggcag ccccgaggag accgccttca gggccaccga caggggcagc 60

gagagcgaca agcacttcag gaagggcctg ctgggcgagc tgatcctgct gcagcagcag 120

<210> 43

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 43

Ser Pro Glu Glu Thr Ala Phe Arg Ala Thr Asp Arg Gly Ser Glu Ser

1 5 10 15

Asp Lys His Phe Arg

20

<210> 44

<211> 63

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 44

tctccggaag aaacggcgtt tagagcaaca gatcgtggat ctgaaagtga caagcatttt 60

aga 63

<210> 45

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 45

Ser Gly Ser Pro Glu Glu Thr Ala Phe Arg Ala Thr Asp Arg Gly Ser

1 5 10 15

Glu Ser Asp Lys His Phe Arg Lys

20

<210> 46

<211> 72

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 46

tcaggttctc cggaagaaac ggcgtttaga gcaacagatc gtggatctga aagtgacaag 60

cattttagaa ag 72

<210> 47

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 47

Ser Gly Ser Thr Thr Glu Asn Leu Tyr Phe Gln Ser Asp Asn Gly Ser

1 5 10 15

Glu Ser Asp Lys His Phe Arg Lys

20

<210> 48

<211> 72

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 48

tcaggttcta caaccgagaa cctgtacttc cagagcgaca acggatctga aagtgacaag 60

cattttagaa ag 72

<210> 49

<211> 158

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 49

Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Glu Gln Thr Ala Ala

1 5 10 15

Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu Leu

20 25 30

Gln Asn Leu Ala Val Ser Val Thr Pro Ile Gln Arg Ile Val Arg Ser

35 40 45

Gly Glu Asn Ala Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr Glu

50 55 60

Gly Leu Ser Ala Asp Gln Met Ala Gln Ile Glu Glu Val Phe Lys Val

65 70 75 80

Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu Pro Tyr Gly

85 90 95

Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Leu Asn Tyr Phe Gly

100 105 110

Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr Val

115 120 125

Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu Ile

130 135 140

Thr Pro Asp Gly Ser Met Leu Phe Arg Val Thr Ile Asn Ser

145 150 155

<210> 50

<211> 474

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 50

gtcttcacac tcgaagattt cgttggggac tgggaacaga cagccgccta caacctggac 60

caagtccttg aacagggagg tgtgtccagt ttgctgcaga atctcgccgt gtccgtaact 120

ccgatccaaa ggattgtccg gagcggtgaa aatgccctga agatcgacat ccatgtcatc 180

atcccgtatg aaggtctgag cgccgaccaa atggcccaga tcgaagaggt gtttaaggtg 240

gtgtaccctg tggatgatca tcactttaag gtgatcctgc cctatggcac actggtaatc 300

gacggggtta cgccgaacat gctgaactat ttcggacggc cgtatgaagg catcgccgtg 360

ttcgacggca aaaagatcac tgtaacaggg accctgtgga acggcaacaa aattatcgac 420

gagcgcctga tcacccccga cggctccatg ctgttccgag taaccatcaa cagc 474

<210> 51

<211> 170

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 51

Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala Gly

1 5 10 15

Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu Phe

20 25 30

Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu Ser

35 40 45

Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr Glu

50 55 60

Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys Val

65 70 75 80

Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr Gly

85 90 95

Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe Gly

100 105 110

Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr Val

115 120 125

Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu Ile

130 135 140

Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val Thr

145 150 155 160

Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala

165 170

<210> 52

<211> 510

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 52

gtcttcacac tcgaagattt cgttggggac tggcgacaga cagccggcta caacctggac 60

caagtccttg aacagggagg tgtgtccagt ttgtttcaga atctcggggt gtccgtaact 120

ccgatccaaa ggattgtcct gagcggtgaa aatgggctga agatcgacat ccatgtcatc 180

atcccgtatg aaggtctgag cggcgaccaa atgggccaga tcgaaaaaat ttttaaggtg 240

gtgtaccctg tggatgatca tcactttaag gtgatcctgc actatggcac actggtaatc 300

gacggggtta cgccgaacat gatcgactat ttcggacggc cgtatgaagg catcgccgtg 360

ttcgacggca aaaagatcac tgtaacaggg accctgtgga acggcaacaa aattatcgac 420

gagcgcctga tcaaccccga cggctccctg ctgttccgag taaccatcaa cggagtgacc 480

ggctggcggc tgtgcgaacg cattctggcg 510

<210> 53

<211> 232

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 53

Ser Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro Leu Glu

1 5 10 15

Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys Arg Tyr Ala

20 25 30

Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His Ile Glu Val Asp

35 40 45

Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala Glu Ala

50 55 60

Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val Val Cys Ser

65 70 75 80

Glu Asn Ser Leu Gln Phe Phe Met Pro Val Leu Gly Ala Leu Phe Ile

85 90 95

Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg Glu Leu

100 105 110

Leu Asn Ser Met Gly Ile Ser Gln Pro Thr Val Val Phe Val Ser Lys

115 120 125

Lys Gly Leu Gln Lys Ile Leu Asn Val Gln Lys Lys Leu Pro Ile Ile

130 135 140

Gln Lys Ile Ile Ile Met Asp Ser Lys Thr Asp Tyr Gln Gly Phe Gln

145 150 155 160

Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro Gly Phe Asn Glu

165 170 175

Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys Thr Ile Ala Leu

180 185 190

Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys Gly Val Ala Leu

195 200 205

Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala Arg Asp Pro Ile

210 215 220

Phe Gly Asn Gln Ile Ile Pro Val

225 230

<210> 54

<211> 699

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 54

tctgccaaaa acattaagaa gggcccagcg ccattctacc cactcgaaga cgggaccgcc 60

ggcgagcagc tgcacaaagc catgaagcgc tacgccctgg tgcccggcac catcgccttt 120

accgacgcac atatcgaggt ggacattacc tacgccgagt acttcgagat gagcgttcgg 180

ctggcagaag ctatgaagcg ctatgggctg aatacaaacc atcggatcgt ggtgtgcagc 240

gagaatagct tgcagttctt catgcccgtg ttgggtgccc tgttcatcgg tgtggctgtg 300

gccccagcta acgacatcta caacgagcgc gagctgctga acagcatggg catcagccag 360

cccaccgtcg tattcgtgag caagaaaggg ctgcaaaaga tcctcaacgt gcaaaagaag 420

ctaccgatca tacaaaagat catcatcatg gatagcaaga ccgactacca gggcttccaa 480

agcatgtaca ccttcgtgac ttcccatttg ccacccggct tcaacgagta cgacttcgtg 540

cccgagagct tcgaccggga caaaaccatc gccctgatca tgaacagtag tggcagtacc 600

ggattgccca agggcgtagc cctaccgcac cgcaccgctt gtgtccgatt cagtcatgcc 660

cgcgacccca tcttcggcaa ccagatcatc cccgtttaa 699

<210> 55

<211> 150

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 55

Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala Gly

1 5 10 15

Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu Phe

20 25 30

Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu Ser

35 40 45

Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr Glu

50 55 60

Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys Val

65 70 75 80

Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr Gly

85 90 95

Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe Gly

100 105 110

Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr Val

115 120 125

Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu Ile

130 135 140

Asn Pro Asp Gly Ser Leu

145 150

<210> 56

<211> 450

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 56

gtcttcacac tcgaagattt cgttggggac tggcgacaga cagccggcta caacctggac 60

caagtccttg aacagggagg tgtgtccagt ttgtttcaga atctcggggt gtccgtaact 120

ccgatccaaa ggattgtcct gagcggtgaa aatgggctga agatcgacat ccatgtcatc 180

atcccgtatg aaggtctgag cggcgaccaa atgggccaga tcgaaaaaat ttttaaggtg 240

gtgtaccctg tggatgatca tcactttaag gtgatcctgc actatggcac actggtaatc 300

gacggggtta cgccgaacat gatcgactat ttcggacggc cgtatgaagg catcgccgtg 360

ttcgacggca aaaagatcac tgtaacaggg accctgtgga acggcaacaa aattatcgac 420

gagcgcctga tcaaccccga cggctccctg 450

<210> 57

<211> 84

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 57

Met Val Glu Lys Phe Val Gly Thr Trp Lys Ile Ala Asp Ser His Asn

1 5 10 15

Phe Gly Glu Tyr Leu Lys Ala Ile Gly Ala Pro Lys Glu Leu Ser Asp

20 25 30

Gly Gly Asp Ala Thr Thr Pro Thr Leu Tyr Ile Ser Gln Lys Asp Gly

35 40 45

Asp Lys Met Thr Val Lys Ile Glu Asn Gly Pro Pro Thr Phe Leu Asp

50 55 60

Thr Gln Val Lys Phe Lys Leu Gly Glu Glu Phe Asp Glu Phe Pro Ser

65 70 75 80

Asp Arg Arg Lys

<210> 58

<211> 252

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 58

atggttgaga agtttgttgg cacttggaag atagctgata gtcataactt tggagaatac 60

ctgaaggcca ttggtgcacc gaaagaactt tctgatggcg gagacgcaac aactcccaca 120

ctctatatca gccaaaagga cggggacaaa atgacagtaa agatcgaaaa cggaccaccc 180

actttcctgg atacgcaggt gaagtttaag ctcggagagg agttcgatga atttcccagc 240

gaccgccgga aa 252

<210> 59

<211> 49

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 59

Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala Gly

1 5 10 15

Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu Phe

20 25 30

Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu Ser

35 40 45

Gly

<210> 60

<211> 147

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 60

gtcttcacac tcgaagattt cgttggggac tggcgacaga cagccggcta caacctggac 60

caagtccttg aacagggagg tgtgtccagt ttgtttcaga atctcggggt gtccgtaact 120

ccgatccaaa ggattgtcct gagcggt 147

<210> 61

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 61

Val Thr Gly Tyr Arg Leu Phe Glu Glu Ile Leu Ser

1 5 10

<210> 62

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 62

gtgaccggct accggctgtt cgaggagatt ctgtca 36

<210> 63

<211> 233

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 63

Val Ser Lys Gly Glu Glu Leu Ile Lys Glu Asn Met Arg Ser Lys Leu

1 5 10 15

Tyr Leu Glu Gly Ser Val Asn Gly His Gln Phe Lys Cys Thr His Glu

20 25 30

Gly Glu Gly Lys Pro Tyr Glu Gly Lys Gln Thr Asn Arg Ile Lys Val

35 40 45

Val Glu Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr His

50 55 60

Phe Met Tyr Gly Ser Lys Val Phe Ile Lys Tyr Pro Ala Asp Leu Pro

65 70 75 80

Asp Tyr Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Val

85 90 95

Met Val Phe Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp Thr Ser

100 105 110

Leu Gln Asp Gly Glu Leu Ile Tyr Asn Val Lys Val Arg Gly Val Asn

115 120 125

Phe Pro Ala Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly Trp Glu

130 135 140

Pro Ser Thr Glu Thr Met Tyr Pro Ala Asp Gly Gly Leu Glu Gly Arg

145 150 155 160

Cys Asp Lys Ala Leu Lys Leu Val Gly Gly Gly His Leu His Val Asn

165 170 175

Phe Lys Thr Thr Tyr Lys Ser Lys Lys Pro Val Lys Met Pro Gly Val

180 185 190

His Tyr Val Asp Arg Arg Leu Glu Arg Ile Lys Glu Ala Asp Asn Glu

195 200 205

Thr Tyr Val Glu Gln Tyr Glu His Ala Val Ala Arg Tyr Ser Asn Leu

210 215 220

Gly Gly Gly Met Asp Glu Leu Tyr Lys

225 230

<210> 64

<211> 699

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 64

gtgagcaagg gcgaggagct gatcaaggag aacatgagaa gcaagctgta cctggaaggc 60

agcgtgaacg gccaccagtt caagtgcacc cacgaagggg agggcaagcc ctacgagggc 120

aagcagacca acaggatcaa ggtggtggag ggaggccccc tgccgttcgc attcgacatc 180

ctggccaccc actttatgta cgggagcaag gtgttcatca agtaccccgc cgacctcccc 240

gattatttta agcagtcctt ccctgagggc ttcacatggg agagagtcat ggtgttcgaa 300

gacgggggcg tgctgaccgc cacccaggac accagcctcc aggacggcga gctcatctac 360

aacgtcaagg tcagaggggt gaacttccca gccaacggcc ccgtgatgca gaagaaaaca 420

ctgggctggg agcccagcac cgagaccatg taccccgctg acggcggcct ggaaggcaga 480

tgcgacaagg ccctgaagct cgtgggcggg ggccatctgc acgtcaactt caagaccaca 540

tacaagtcca agaaacccgt gaagatgccc ggcgtccact acgtggaccg cagactggaa 600

agaatcaagg aggccgacaa cgagacctac gtcgagcagt acgagcacgc tgtggccaga 660

tactccaacc tgggcggagg catggacgag ctgtacaag 699

<210> 65

<211> 297

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 65

Gly Ser Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro His Tyr Val Glu

1 5 10 15

Val Leu Gly Glu Arg Met His Tyr Val Asp Val Gly Pro Arg Asp Gly

20 25 30

Thr Pro Val Leu Phe Leu His Gly Asn Pro Thr Ser Ser Tyr Val Trp

35 40 45

Arg Asn Ile Ile Pro His Val Ala Pro Thr His Arg Cys Ile Ala Pro

50 55 60

Asp Leu Ile Gly Met Gly Lys Ser Asp Lys Pro Asp Leu Gly Tyr Phe

65 70 75 80

Phe Asp Asp His Val Arg Phe Met Asp Ala Phe Ile Glu Ala Leu Gly

85 90 95

Leu Glu Glu Val Val Leu Val Ile His Asp Trp Gly Ser Ala Leu Gly

100 105 110

Phe His Trp Ala Lys Arg Asn Pro Glu Arg Val Lys Gly Ile Ala Phe

115 120 125

Met Glu Phe Ile Arg Pro Ile Pro Thr Trp Asp Glu Trp Pro Glu Phe

130 135 140

Ala Arg Glu Thr Phe Gln Ala Phe Arg Thr Thr Asp Val Gly Arg Lys

145 150 155 160

Leu Ile Ile Asp Gln Asn Val Phe Ile Glu Gly Thr Leu Pro Met Gly

165 170 175

Val Val Arg Pro Leu Thr Glu Val Glu Met Asp His Tyr Arg Glu Pro

180 185 190

Phe Leu Asn Pro Val Asp Arg Glu Pro Leu Trp Arg Phe Pro Asn Glu

195 200 205

Leu Pro Ile Ala Gly Glu Pro Ala Asn Ile Val Ala Leu Val Glu Glu

210 215 220

Tyr Met Asp Trp Leu His Gln Ser Pro Val Pro Lys Leu Leu Phe Trp

225 230 235 240

Gly Thr Pro Gly Val Leu Ile Pro Pro Ala Glu Ala Ala Arg Leu Ala

245 250 255

Lys Ser Leu Pro Asn Cys Lys Ala Val Asp Ile Gly Pro Gly Leu Asn

260 265 270

Leu Leu Gln Glu Asp Asn Pro Asp Leu Ile Gly Ser Glu Ile Ala Arg

275 280 285

Trp Leu Ser Thr Leu Glu Ile Ser Gly

290 295

<210> 66

<211> 891

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 66

ggatccgaaa tcggtactgg ctttccattc gacccccatt atgtggaagt cctgggcgag 60

cgcatgcact acgtcgatgt tggtccgcgc gatggcaccc ctgtgctgtt cctgcacggt 120

aacccgacct cctcctacgt gtggcgcaac atcatcccgc atgttgcacc gacccatcgc 180

tgcattgctc cagacctgat cggtatgggc aaatccgaca aaccagacct gggttatttc 240

ttcgacgacc acgtccgctt catggatgcc ttcatcgaag ccctgggtct ggaagaggtc 300

gtcctggtca ttcacgactg gggctccgct ctgggtttcc actgggccaa gcgcaatcca 360

gagcgcgtca aaggtattgc atttatggag ttcatccgcc ctatcccgac ctgggacgaa 420

tggccagaat ttgcccgcga gaccttccag gccttccgca ccaccgacgt cggccgcaag 480

ctgatcatcg atcagaacgt ttttatcgag ggtacgctgc cgatgggtgt cgtccgcccg 540

ctgactgaag tcgagatgga ccattaccgc gagccgttcc tgaatcctgt tgaccgcgag 600

ccactgtggc gcttcccaaa cgagctgcca atcgccggtg agccagcgaa catcgtcgcg 660

ctggtcgaag aatacatgga ctggctgcac cagtcccctg tcccgaagct gctgttctgg 720

ggcaccccag gcgttctgat cccaccggcc gaagccgctc gcctggccaa aagcctgcct 780

aactgcaagg ctgtggacat cggcccgggt ctgaatctgc tgcaagaaga caacccggac 840

ctgatcggca gcgagatcgc gcgctggctg tctactctgg agatttccgg t 891

<210> 67

<211> 313

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 67

Asp Thr Ala Ile Leu Ser Val Val Pro Phe His His Gly Phe Gly Met

1 5 10 15

Phe Thr Thr Leu Gly Tyr Leu Ile Cys Gly Phe Arg Val Val Leu Met

20 25 30

Tyr Arg Phe Glu Glu Glu Leu Phe Leu Arg Ser Leu Gln Asp Tyr Lys

35 40 45

Ile Gln Ser Ala Leu Leu Val Pro Thr Leu Phe Ser Phe Phe Ala Lys

50 55 60

Ser Thr Leu Ile Asp Lys Tyr Asp Leu Ser Asn Leu His Glu Ile Ala

65 70 75 80

Ser Gly Gly Ala Pro Leu Ser Lys Glu Val Gly Glu Ala Val Ala Lys

85 90 95

Arg Phe His Leu Pro Gly Ile Arg Gln Gly Tyr Gly Leu Thr Glu Thr

100 105 110

Thr Ser Ala Ile Leu Ile Thr Pro Glu Gly Asp Asp Lys Pro Gly Ala

115 120 125

Val Gly Lys Val Val Pro Phe Phe Glu Ala Lys Val Val Asp Leu Asp

130 135 140

Thr Gly Lys Thr Leu Gly Val Asn Gln Arg Gly Glu Leu Cys Val Arg

145 150 155 160

Gly Pro Met Ile Met Ser Gly Tyr Val Asn Asn Pro Glu Ala Thr Asn

165 170 175

Ala Leu Ile Asp Lys Asp Gly Trp Leu His Ser Gly Asp Ile Ala Tyr

180 185 190

Trp Asp Glu Asp Glu His Phe Phe Ile Val Asp Arg Leu Lys Ser Leu

195 200 205

Ile Lys Tyr Lys Gly Tyr Gln Val Ala Pro Ala Glu Leu Glu Ser Ile

210 215 220

Leu Leu Gln His Pro Asn Ile Phe Asp Ala Gly Val Ala Gly Leu Pro

225 230 235 240

Asp Asp Asp Ala Gly Glu Leu Pro Ala Ala Val Val Val Leu Glu His

245 250 255

Gly Lys Thr Met Thr Glu Lys Glu Ile Val Asp Tyr Val Ala Ser Gln

260 265 270

Val Thr Thr Ala Lys Lys Leu Arg Gly Gly Val Val Phe Val Asp Glu

275 280 285

Val Pro Lys Gly Leu Thr Gly Lys Leu Asp Ala Arg Lys Ile Arg Glu

290 295 300

Ile Leu Ile Lys Ala Lys Lys Ala Ser

305 310

<210> 68

<211> 939

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 68

gacaccgcta tcctcagcgt ggtgccattt caccacggct tcggcatgtt caccacgctg 60

ggctacttga tctgcggctt tcgggtcgtg ctcatgtacc gcttcgagga ggagctattc 120

ttgcgcagct tgcaagacta taagattcaa tctgccctgc tggtgcccac actatttagc 180

ttcttcgcta agagcactct catcgacaag tacgacctaa gcaacttgca cgagatcgcc 240

agcggcgggg cgccgctcag caaggaggta ggtgaggccg tggccaaacg cttccaccta 300

ccaggcatcc gccagggcta cggcctgaca gaaacaacca gcgccattct gatcaccccc 360

gaaggggacg acaagcctgg cgcagtaggc aaggtggtgc ccttcttcga ggctaaggtg 420

gtggacttgg acactggtaa gacactgggt gtgaaccagc gcggcgagct gtgcgtccgt 480

ggccccatga tcatgagcgg ctacgttaac aaccccgagg ctacaaacgc tctcatcgac 540

aaggacggct ggctgcacag cggcgacatc gcctactggg acgaggacga gcacttcttc 600

atcgtggacc ggctgaagag cctgatcaaa tacaagggct accaggtagc cccagccgaa 660

ctggagagca tcctgctgca acaccccaac atcttcgacg ccggggtcgc cggcctgccc 720

gacgacgatg ccggcgagct gcccgccgca gtcgtcgtgc tggaacacgg taaaaccatg 780

accgagaagg agatcgtgga ctatgtggcc agccaggtta caaccgccaa gaagctgcgc 840

ggtggtgttg tgttcgtgga cgaggtgcct aaaggactga ccggcaagtt ggacgcccgc 900

aagatccgcg agattctcat taaggccaag aaggctagc 939

<210> 69

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 69

Thr Ile Asn Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala

1 5 10 15

<210> 70

<211> 48

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 70

accatcaacg gagtgaccgg ctggcggctg tgcgaacgca ttctggcg 48

<210> 71

<211> 56

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 71

Lys Gly Val Lys Ser Val Val Asn Leu Val Gly Glu Lys Leu Val Tyr

1 5 10 15

Val Gln Lys Trp Asp Gly Lys Glu Thr Thr Tyr Val Arg Glu Ile Lys

20 25 30

Asp Gly Lys Leu Val Val Thr Leu Thr Met Gly Asp Val Val Ala Val

35 40 45

Arg Ser Tyr Arg Arg Ala Thr Glu

50 55

<210> 72

<211> 168

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 72

aaaggcgtga agagcgtagt gaacttagtt ggagaaaaac tggtctatgt tcagaaatgg 60

gacggtaagg agaccactta tgtgcgagaa attaaggacg gaaagcttgt ggtgacgctg 120

actatgggag acgtcgtagc agtgcggagt tatagacgcg cgaccgaa 168

<210> 73

<211> 117

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 73

Lys Ile Asp Ile His Val Ile Ile Pro Tyr Glu Gly Leu Ser Gly Asp

1 5 10 15

Gln Met Gly Gln Ile Glu Lys Ile Phe Lys Val Val Tyr Pro Val Asp

20 25 30

Asp His His Phe Lys Val Ile Leu His Tyr Gly Thr Leu Val Ile Asp

35 40 45

Gly Val Thr Pro Asn Met Ile Asp Tyr Phe Gly Arg Pro Tyr Glu Gly

50 55 60

Ile Ala Val Phe Asp Gly Lys Lys Ile Thr Val Thr Gly Thr Leu Trp

65 70 75 80

Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu Ile Asn Pro Asp Gly Ser

85 90 95

Leu Leu Phe Arg Val Thr Ile Asn Gly Val Thr Gly Trp Arg Leu Cys

100 105 110

Glu Arg Ile Leu Ala

115

<210> 74

<211> 351

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 74

aagatcgaca tccatgtcat catcccgtat gaaggtctga gcggcgacca aatgggccag 60

atcgaaaaaa tttttaaggt ggtgtaccct gtggatgatc atcactttaa ggtgatcctg 120

cactatggca cactggtaat cgacggggtt acgccgaaca tgatcgacta tttcggacgg 180

ccgtatgaag gcatcgccgt gttcgacggc aaaaagatca ctgtaacagg gaccctgtgg 240

aacggcaaca aaattatcga cgagcgcctg atcaaccccg acggctccct gctgttccga 300

gtaaccatca acggagtgac cggctggcgg ctgtgcgaac gcattctggc g 351

<210> 75

<211> 139

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 75

Met Val Glu Lys Phe Val Gly Thr Trp Lys Ile Ala Asp Ser His Asn

1 5 10 15

Phe Gly Glu Tyr Leu Lys Ala Ile Gly Ala Pro Lys Glu Leu Ser Asp

20 25 30

Gly Gly Asp Ala Thr Thr Pro Thr Leu Tyr Ile Ser Gln Lys Asp Gly

35 40 45

Asp Lys Met Thr Val Lys Ile Glu Asn Gly Pro Pro Thr Phe Leu Asp

50 55 60

Thr Gln Val Lys Phe Lys Leu Gly Glu Glu Phe Asp Glu Phe Pro Ser

65 70 75 80

Asp Arg Arg Lys Gly Val Lys Ser Val Val Asn Leu Val Gly Glu Lys

85 90 95

Leu Val Tyr Val Gln Lys Trp Asp Gly Lys Glu Thr Thr Tyr Val Arg

100 105 110

Glu Ile Lys Asp Gly Lys Leu Val Val Thr Leu Thr Met Gly Asp Val

115 120 125

Val Ala Val Arg Ser Tyr Arg Arg Ala Thr Glu

130 135

<210> 76

<211> 420

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 76

atggttgaga agtttgttgg cacttggaag atagctgata gtcataactt tggagaatac 60

ctgaaggcca ttggtgcacc gaaagaactt tctgatggcg gagacgcaac aactcccaca 120

ctctatatca gccaaaagga cggggacaaa atgacagtaa agatcgaaaa cggaccaccc 180

actttcctgg atacgcaggt gaagtttaag ctcggagagg agttcgatga atttcccagc 240

gaccgccgga aaggcgtgaa gagcgtagtg aacttagttg gagaaaaact ggtctatgtt 300

cagaaatggg acggtaagga gaccacttat gtgcgagaaa ttaaggacgg aaagcttgtg 360

gtgacgctga ctatgggaga cgtcgtagca gtgcggagtt atagacgcgc gaccgaataa 420

Claims (150)

1. A reporter gene for measuring OMA1 protease activity comprising a targeting sequence and a signal producing domain, wherein the targeting sequence is also a sequence recognized by OMA 1.

2. An OMA1 reporter comprising a mitochondrial targeting sequence that is not cleaved by a mitochondrial processing peptidase.

3. A reporter gene for measuring protease activity, said reporter gene operably binding a functional element to a sequence motif, said functional element selected from the group consisting of: a targeting sequence, an entire enzyme group or protein domain or an "N" fragment of an enzyme group or protein domain, an entire enzyme group or protein domain or a "C" fragment of an enzyme group or protein domain that is complementary to "N" to generate a signal, the sequence motif being recognizable by an OMA1 protease, wherein a decrease in the signal indicates an increase in OMA1 activity.

4. A reporter gene for measuring OMA1 protease activity, wherein the targeting sequence is not a mitochondrial import sequence of OPA 1.

5. The reporter gene for measuring protease activity according to any one of claims 1 to 4, wherein the targeting sequence is 30 amino acids or more, preferably 80 amino acids, but not more than 160 amino acids.

6. The reporter gene for measuring protease activity according to any one of claims 1 to 4, wherein the targeting sequence consists of 50 to 150 amino acids.

7. The reporter gene for measuring protease activity according to any one of claims 1 to 4, wherein the targeting sequence consists of 50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149, or 150 amino acids.

8. The reporter gene for measuring OMA1 protease activity of any one of claims 1 to 4, wherein the targeting sequence has at least 75% identity to SEQ ID NO. 17,SEQ ID NO:19,SEQ ID NO:21,SEQ ID NO:23,SEQ ID NO:25, or SEQ ID NO. 27, or a variant or combination thereof.

9. The reporter gene for measuring OMA1 protease activity according to any of claims 1 to 4, wherein the sequence motif recognizable by OMA1 protease consists of 4 or more amino acids, preferably 23 amino acids, but not more than 50 amino acids.

10. The reporter gene for measuring OMA1 protease activity according to any one of claims 1 to 4, wherein the sequence motif recognized by OMA1 protease consists of 10 to 50 amino acids.

11. The reporter gene for measuring OMA1 protease activity according to any one of claims 1 to 4, wherein the sequence motif recognized by OMA1 protease consists of 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49, or 50 amino acids.

12. The reporter gene for measuring OMA1 protease activity of any one of claims 1 to 4, wherein the sequence motif recognizable by OMA1 protease has at least 75% identity to SEQ ID NO 33,SEQ ID NO:35,SEQ ID NO:37,SEQ ID NO:39,SEQ ID NO:41,SEQ ID NO:43,SEQ ID NO:45 or SEQ ID NO 47 or variants or combinations thereof.

13. The reporter gene for measuring OMA1 protease activity of any one of claims 1 to 4, wherein the entire enzyme group or protein domain or "N" fragment of an enzyme group or protein domain has at least 75% identity to SEQ ID NO 49,SEQ ID NO:51,SEQ ID NO:53,SEQ ID NO:55,SEQ IDNO:57 or SEQ ID NO 59 or variant or combination thereof and the entire enzyme group or protein domain or "C" fragment of an enzyme group or protein domain has at least 75% identity to SEQ ID NO 61,SEQ ID NO:63,SEQ ID NO:65,SEQ ID NO:67,SEQ ID NO:69,SEQ ID NO:71 or SEQ ID NO 73 or variant or combination thereof.

14. A recombinant expression vector comprising the reporter gene of any one of claims 1 to 4.

15. A recombinant host cell comprising the reporter gene of any one of claims 1 to 4.

16. A kit comprising the reporter gene of any one of claims 1 to 4.

17. A method for predicting mitochondrial toxicity comprising the reporter gene of any one of claims 1-4.

18. A method for predicting an adverse event comprising the reporter gene of any one of claims 1 to 4.

19. A method for detecting protease activity comprising the reporter gene of any one of claims 1 to 4.

20. A method for detecting protease activity in a sample, comprising:

a. the sample is bound to a reporter gene,

b. measuring a signal; and

c. the signal value is compared with the signal value of the control,

wherein the signal is inversely related to protease activity.

21. A method for identifying an OMA1 protease inhibitor, comprising:

a. binding a molecule to a reporter gene comprising a functional group separated by an OMA1 cleavage site,

b. the OMA1 protease is activated and the protein,

c. the signal is measured and the signal is measured,

d. molecules that maintain the signal relative to a control that does not contain the molecule are selected.

22. A method for predicting in vivo toxicity of a molecule, the method comprising the steps of:

a. binding the molecule to a recombinant host expressing a reporter gene comprising an X fragment of a signal producing protein separated from a Y fragment of the signal producing protein by a recognition element, the Y fragment being complementary to the X fragment in a signaling manner, and

b. detecting cleavage of the recognition element when the signals emitted by the X-and Y-fragments are altered,

Wherein detection of an increase in cleavage indicates an increase in molecular toxicity.

23. Telanavir or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

24. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising telanavir.

25. Use of telanavir for the manufacture of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

26. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, which uses telanavir as its ingredient.

27. Pazopanib hydrochloride or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

28. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising pazopanib hydrochloride.

29. Use of pazopanib hydrochloride for the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

30. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using pazopanib hydrochloride as its ingredient.

31. Sorafenib, or a solvate, prodrug, analog, or pharmaceutically acceptable salt thereof, for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

32. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising sorafenib.

33. Use of sorafenib in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

34. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using sorafenib as its ingredient.

35. Sunitinib or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

36. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising sunitinib.

37. Use of sunitinib in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

38. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using sunitinib as an ingredient thereof.

39. Ibrutinib or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

40. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising ibrutinib.

41. Use of ibrutinib for the manufacture of a personalized medicine for treating pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

42. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using ibrutinib as an ingredient thereof.

43. Regorafenib or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

44. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising regorafenib.

45. Use of regorafenib for the manufacture of a personalized medicine for treating pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

46. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using regorafenib as its ingredient.

47. Celecoxib or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

48. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising celecoxib.

49. Use of celecoxib in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

50. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using celecoxib as an ingredient thereof.

51. Raloxifene or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

52. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising raloxifene.

53. Use of raloxifene for the manufacture of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

54. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using raloxifene as an ingredient thereof.

55. Actinomycin or a solvate, prodrug, analogue or pharmaceutically acceptable salt thereof, for use in a method of treatment of a pathological condition or disease characterised by pathological OMA1 levels or OMA1 activity.

56. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising an actinomycin.

57. Use of actinomycin in the manufacture of a personalized medicine for the treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

58. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using actinomycin as an ingredient thereof.

59. Encidipine or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof, is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

60. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising azepine.

61. Use of exendin for the manufacture of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

62. A process for the preparation of a personalized medicine for the treatment of pathological symptoms or diseases characterized by pathological OMA1 levels or OMA1 activity, using azepine as its ingredient.

63. Cabatinib, or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof, for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

64. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising cabatinib.

65. Use of cabatinib for the manufacture of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

66. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using cabotinib as an ingredient thereof.

67. Tamoxifen citrate or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

68. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising tamoxifen citrate.

69. Use of tamoxifen citrate in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

70. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using tamoxifen citrate as its ingredient.

71. Pexidanib or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

72. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising pexidanib.

73. Use of pexidanib in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

74. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using pexidanib as its ingredient.

75. Daunorubicin hydrochloride, or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof, for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

76. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising daunorubicin hydrochloride.

77. Use of daunorubicin hydrochloride in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

78. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using daunorubicin hydrochloride as its ingredient.

79. Dabrafenib mesylate, or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof, for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

80. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising dabrafenib mesylate.

81. Use of dabrafenib mesylate in the manufacture of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

82. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using dasafil remafonate as an ingredient thereof.

83. Latification or solvates, prodrugs, analogs or pharmaceutically acceptable salts thereof are useful in methods of treating pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

84. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising loratidine.

85. Use of loratidine for the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

86. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using laratinib as its ingredient.

87. The use of valrubicin or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

88. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising valrubicin.

89. Use of valrubicin in the manufacture of a personalized medicine for treating pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

90. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using valrubicin as its ingredient.

91. Trametinib, or solvates, prodrugs, analogs or pharmaceutically acceptable salts thereof, for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

92. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising trametinib.

93. Use of trametinib in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

94. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using trametinib as an ingredient thereof.

95. Emtrictinib or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

96. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising emtrictinib.

97. Use of emtrictinib for the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

98. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using emtrictinib as a component thereof.

99. Bosutinib or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

100. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising bosutinib.

101. Use of bosutinib in the manufacture of a personalized medicine for treating pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

102. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using bosutinib as an ingredient thereof.

103. Idarubicin hydrochloride or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof, for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

104. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising idarubicin hydrochloride.

105. Use of idarubicin hydrochloride in the manufacture of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

106. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using idarubicin hydrochloride as its ingredient.

107. The use of fig. critinib or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

108. A pharmaceutical composition for treating a disease characterized by pathological OMA1 levels or pathological symptoms or OMA1 activity, comprising critinib.

109. Use of fig. calitinib for the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

110. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using as its ingredient, tutorinib.

111. Plug Li Nisuo or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

112. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising a plug Li Nisuo.

113. Use of plug Li Nisuo in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

114. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using a plug Li Nisuo as a component thereof.

115. Rabociclib or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

116. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising rebaudinib.

117. Use of rebaudinib for the manufacture of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

118. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using rebaudinib as an ingredient thereof.

119. Ceritinib or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

120. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising ceritinib.

121. Use of ceritinib in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

122. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using ceritinib as an ingredient thereof.

123. Imatinib or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

124. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising imatinib.

125. Use of imatinib for the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

126. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using imatinib as an ingredient thereof.

127. Doxorubicin hydrochloride or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

128. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising doxorubicin hydrochloride.

129. Use of doxorubicin hydrochloride in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

130. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using doxorubicin hydrochloride as its ingredient.

131. The use of valnemulin, or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof, in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

132. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising valnemulin.

133. Use of valnemulin in the manufacture of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

134. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using valnemulin as a component thereof.

135. Geranitinib, or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof, for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

136. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising gefitinib.

137. Use of gefitinib for the manufacture of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity.

138. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using gefitinib as its ingredient.

139. Mitotane or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof for use in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

140. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising mitotane.

141. Use of mitotane in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

142. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using mitotane as its ingredient.

143. Ornitanib or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

144. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising octenib.

145. Use of octenib in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

146. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, using octreotide as its ingredient.

147. Kawain or a solvate, prodrug, analog or pharmaceutically acceptable salt thereof is used in a method of treatment of a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

148. A pharmaceutical composition for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity comprising kavain.

149. Use of kavain in the manufacture of a personalized medicine for treating a pathological condition or disease characterized by pathological OMA1 levels or OMA1 activity.

150. A process for the preparation of a personalized medicine for the treatment of pathological conditions or diseases characterized by pathological OMA1 levels or OMA1 activity, which uses kavain as its ingredient.