WO2012051306A2

WO2012051306A2 - Compositions and methods for modulating mitochondrial proteases

Info

Publication number: WO2012051306A2
Application number: PCT/US2011/055974
Authority: WO
Inventors: Shaharyar Khan
Original assignee: Gencia Corporation
Priority date: 2010-10-12
Filing date: 2011-10-12
Publication date: 2012-04-19
Also published as: WO2012051306A3

Abstract

Compositions and methods for modulating oxidative phosphorylation in a subject are provided. The compositions include an effective amount of mitochondrial protease inhibitor conjugated to a mitochondrial targeting moiety to inhibit or reduce proteolysis of TFAM. In a preferred embodiment, the protease inhibitor inhibits Lon protease activity on TFAM resulting in increased TFAM levels and/or increased TFAM half-life. Increases in TFAM concentration or increased TFAM half-life stimulate oxidative metabolism causing an increase in oxidative phosphorylation. A preferred mitochondrial targeting moiety is a liphophilic cation such as triphenyl phosphonium.

Description

COMPOSITIONS AND METHODS FOR MODULATING MITOCHONDRIAL PROTEASES

CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit of and priority to U.S. patent provisional application number 61/392,380 filed on October 12, 2010.

FIELD OF THE INVENTION

The invention is generally directed to field of protease inhibitors, in particular to mitochondrially targeted protease inhibitors.

BACKGROUND OF THE INVENTION

Most proteins possess a half-life and are degraded. In Escherichia coli, many proteins are generally degraded by ATP-dependent intracellular proteases, two families of which have been well characterized: the Lon and Clp serine proteases. Lon is a member of the super family of ATPases associated with diverse cellular activities (AAA ATPases), and forms a homooligomeric, ring-shaped structure.

Ubiquitin, 20s and 26s protease complexes are the best studied ATP- dependent systems which contribute to the control of protein turnover in eukaryotic cells. Besides these, other proteases appear to be involved in ATP-consuming proteolysis. Homo logs of the bacterial Clp protease were found in various eukaryotic cells. An extensively characterized E. coli Lon protease which is involved in the rapid degradation of short-lived regulatory and abnormal proteins also appears to have homologs in eukaryotes. ATP- dependent enzymes similar to Lon protease exist in mitochondria, where they actively degrade mitochondrial proteins.

Lon is the major protease in the mitochondrial matrix and is well conserved among species. Lon contributes to protein quality control surveillance in mitochondria by degrading preferentially oxidatively modified or misfolded proteins before they aggregate. In bacteria, in addition to proteolysis of damaged proteins, Lon also plays a key role in turnover of specific unstable proteins involved in a variety of biological processes. Similarly, the steroidogenic acute regulatory protein StAR, several subunits of cytochrome c oxidase, and oxidized mitochondrial aconitase are known to be Lon substrates in animal mitochondria. In addition to its proteolytic function, mitochondrial Lon has the ability to bind DNA in vitro, and has been shown to interact with mtDNA in human cultured cells.

Mitochondrial Lon is also the protease responsible for degrading proteins involved in mitochondrial replication, transcription and translation. Activity of mitochondrial Lon on these protein substrates reduces their availability for promoting mtDNA maintenance and activity.

It is an object of the invention to provide methods and compositions for modulating mitochondrial protease activity in a subject.

It is another object of the invention to provide methods and compositions for modulating oxidative phosphorylation in a subject.

It is still another object of the invention to provide methods for identifying modulators of mitochondrial protease activity.

SUMMARY OF THE INVENTION

Compositions and methods for modulating oxidative phosphorylation in a subject are provided. The compositions include an effective amount of mitochondrial protease inhibitor to inhibit or reduce proteolysis of transcription factor A - mitochondrial (TFAM). In a preferred embodiment, the protease inhibitor inhibits Lon protease activity on TFAM resulting in increased TFAM levels and/or increased TFAM half-life. Increases in TFAM concentration or increased TFAM half-life stimulate oxidative metabolism causing an increase in oxidative phosphorylation.

Targeting protease inhibitors to mitochondria to inhibit mitochondrial Lon protease provides a means of increasing TFAM levels. The composition can contain one or more protease inhibitors targeted to selectively localize within the mitochondria of a cell. The protease inhibitors can be targeted to the mitochondria by attaching the protease inhibitor to one or more mitochondrial targeting agents.

In certain embodiments, the mitochondrially targeted protease inhibitor is represented by the general structure shown below

A-B-(C)_n

wherein,

A is a Lon protease inhibitor B is absent, or is a linker;

n is an integer between one a eight; and

C is a mitochondrial targeting agent.

In certain embodiments, the Lon protease inhibitor is MG132, MG262, Dansyl 89-98 Abu boronate, 3,4-dichloroisocoumarin (DCI) or coumarinic derivatives or isomers thereof, clasto-lactacystin-P-lactone or a derivative thereof. In preferred embodiments, the mitochondrial targeting agent is a cationic phosphonium group, such as an alkyltriarylphosphonium group or a tetraalkylphosphonium group.

In some embodiments, the mitochondrially targeted protease inhibitor is defined by Formu

Formula I

wherein,

E is an electrophilic moiety;

L is absent, or is a linking group;

Z is a mitochondrial targeting agent; and

R is an alkyl group, an aryl group, or a heterocyclic group

In some embodiments of Formula I, L is absent, or is C2-C12 alkyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylarylalkyl, or aryl group optionally substituted with between one and five substituents individually selected from alkyl, cyclopropyl, cyclobutyl ether, amine, halogen, hydroxyl, ether, nitrile, CF₃, ester, amide, urea, carbamate, thioether, and aryl.

In some embodiments of Formula I, E is an aldehyde, vinyl sulfone, epoxyketone, β-lactone, or boronate moiety. In certain embodiments, E is an aldehyde or boronate moiety.

In some embodiments of Formula I, Z is a polypeptide containing positively charged amino acid residues, a cationic ammonium group, or a cationic phosphonium group. In certain embodiments, the mitochondrial targeting agent is an alkyltriarylphosphonium group or a

tetraalkylphosphonium group. In preferred embodiments, the mitochondrial targeting agent is an alkyltriphenylphosphonium group.

In certain embodiments, the mitochondrially targeted protease inhibitor is defined by Formula la

Formula la

wherein,

E is an electrophilic moiety;

L is absent, or is a linking group;

Z is a mitochondrial targeting agent; and

R is an alkyl group, an aryl group, or a heterocyclic group

In some embodiments of Formula la, L is absent, or is C2-C12 alkyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylarylalkyl, or aryl group optionally substituted with between one and five substituents individually selected from alkyl, cyclopropyl, cyclobutyl ether, amine, halogen, hydroxyl, ether, nitrile, CF₃, ester, amide, urea, carbamate, thioether, and aryl.

In some embodiments of Formula la, E is an aldehyde, vinyl sulfone, epoxyketone, β-lactone, or boronate moiety. In certain embodiments, E is an aldehyde or boronate moiety.

In some embodiments of Formula la, Z is a polypeptide containing positively charged amino acid residues, a cationic ammonium group, or a cationic phosphonium group. In certain embodiments, the mitochondrial targeting agent is an alkyltriarylphosphonium group or a

One or more Lon protease inhibitors can also be targeted to the mitochondria using a mitochondrial delivery vehicle, such as a lipid raft, mitochondrially targeted nanoparticle, or mitochondriotropic liposome. The protease inhibitors can be encapsulated in, dispersed in, or associated with a lipid raft, mitochondrially targeted nanoparticle, or mitochondriotropic liposome. In some of these embodiments, the Lon protease inhibitor is MG132, MG262, Dansyl 89-98 Abu boronate, 3,4-dichloroisocoumarin (DCI) or coumarinic derivatives or isomers thereof, clasto-lactacystin-β- lactone or a derivative thereof, or combinations thereof.

Modified TFAM is also provided in which the amino acid sequence of TFAM is altered to inhibit or reduce proteolysis by mitochondrial proteases, in particular Lon protease.

Methods for identifying mitochondrial protease inhibitors are also provided.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

In describing and claiming the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below.

The term "polypeptides" includes proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R),

Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).

"Variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

Modifications and changes can be made in the structure of the polypeptides of in disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties.

In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8);

tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly, where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamnine (+0.2); glycine (0); proline (-0.5 ± 1); threonine (-0.4); alanine (-0.5); histidine (- 0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);

isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn,

Gin), (lie: Leu, Val), (Leu: lie, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: He, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%>, 80%>, 90%>, and 95% sequence identity to the polypeptide of interest. As used herein, the term "treating" includes alleviating one or more symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms.

"Operably linked" refers to a juxtaposition wherein the components are configured so as to perform their usual function. For example, control sequences or promoters operably linked to a coding sequence are capable of effecting the expression of the coding sequence, and an organelle localization sequence operably linked to protein will direct the linked protein to be localized at the specific organelle.

"Localization Signal or Sequence or Domain" or "Targeting Signal or Sequence or Domain" are used interchangeably and refer to a signal that directs a molecule to a specific cell, tissue, organelle, or intracellular region. The signal can be polynucleotide, polypeptide, or carbohydrate moiety or can be an organic or inorganic compound sufficient to direct an attached molecule to a desired location. Exemplary organelle localization signals include nuclear localization signals known in the art and other organelle localization signals known in the art such as those provided in Table 1 and described in Emanuelson et al., Predicting Subcellular Localization of Proteins Based on Their N-terminal Amino Acid Sequence. Journal of Molecular Biology. 300(4): 1005-16, 2000 Jul 21, and in Cline and Henry, Import and Routing of Nucleus-encoded Chloroplast Proteins. Annual Review of Cell &

Developmental Biology. 12:1-26, 1996, the disclosures of which are incorporated herein by reference in their entirety. It will be appreciated that the entire sequence listed in Table 1 need not be included, and modifications including truncations of these sequences are within the scope of the disclosure provided the sequences operate to direct a linked molecule to a specific organelle. Organelle localization signals of the present disclosure can have 80 to 100% homology to the sequences in Table 1.

One class of suitable organelle localization signals include those that do not interact with the targeted organelle in a receptor:ligand mechanism. For example, organelle localization signals include signals having or conferring a net charge, for example a positive charge. Positively charged signals can be used to target negatively charged organelles such as the mitochondria. Negatively charged signals can be used to target positively charged organelles.

As used herein, the term "organelle" refers to cellular membrane bound structures such as the chloroplast, mitochondrion, and nucleus. The term "organelle" includes natural and synthetic organelles.

As used herein, the term "non-nuclear organelle" refers to any cellular membrane bound structure present in a cell, except the nucleus.

"Alkyl", as used herein, refers to the radical of saturated or unsaturated aliphatic groups, including straight-chain alkyl, alkenyl, or alkynyl groups, branched-chain alkyl, alkenyl, or alkynyl groups, cycloalkyl, cycloalkenyl, or cycloalkynyl (alicyclic) groups, alkyl substituted cycloalkyl, cycloalkenyl, or cycloalkynyl groups, and cycloalkyl substituted alkyl, alkenyl, or alkynyl groups. Unless otherwise indicated, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., Ci- C₃₀ for straight chain, C₃-C₃₀ for branched chain), more preferably 20 or fewer carbon atoms, more preferably 12 or fewer carbon atoms, and most preferably 8 or fewer carbon atoms. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure. The ranges provided above are inclusive of all values between the minimum value and the maximum value.

The alkyl groups may also be substituted with one or more groups including, but not limited to, halogen, hydroxy, amino, thio, ether, ester, carboxy, oxo, and aldehyde groups. The alkyl groups may also contain one or more heteroatoms within the carbon backbone. Preferably the

heteroatoms incorporated into the carbon backbone are oxygen, nitrogen, sulfur, and combinations thereof. In certain embodiments, the alkyl group contains between one and four heteroatoms.

"Alkenyl" and "Alkynyl", as used herein, refer to unsaturated aliphatic groups containing one or more double or triple bonds analogous in length (e.g., C₂-C₃₀) and possible substitution to the alkyl groups described above.

"Aryl", as used herein, refers to 5-, 6- and 7-membered aromatic ring. The ring may be a carbocyclic, heterocyclic, fused carbocyclic, fused heterocyclic, bicarbocyclic, or biheterocyclic ring system, optionally substituted by halogens, alkyl-, alkenyl-, and alkynyl-groups. Broadly defined, "Ar", as used herein, includes 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "heteroaryl", "aryl heterocycles", or "heteroaromatics". The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, --CF , --CN, or the like. The term "Ar" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocycles. Examples of heterocyclic ring include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,

decahydroquinolinyl, 2H,6H-l,5,2-dithiazinyl, dihydrofuro[2,3

b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, lH-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H- indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3- oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl,

phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-l ,2,5-thiadiazinyl, 1 ,2,3-thiadiazolyl, 1 ,2,4-thiadiazolyl, 1 ,2,5- thiadiazolyl, 1 ,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl,

thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl.

"Alkylaryl", as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or hetero aromatic group).

"Heterocycle" or "heterocyclic", as used herein, refers to a cyclic radical attached via a ring carbon or nitrogen of a monocyclic or bicyclic ring containing 3-10 ring atoms, and preferably from 5-6 ring atoms, consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H, O, (Ci_₄) alkyl, phenyl or benzyl, and optionally containing one or more double or triple bonds, and optionally substituted with one or more substituents. The term "heterocycle" also encompasses substituted and unsubstituted heteroaryl rings. Examples of heterocyclic ring include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H- 1 ,5 ,2-dithiazinyl,

dihydrofuro[2,3-¾]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, lH-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,

methylenedioxyphenyl, morpholinyl, naphthyridinyl,

octahydroisoquinolinyl, oxadiazolyl, 1 ,2,3-oxadiazolyl, 1 ,2,4-oxadiazolyl, 1 ,2,5-oxadiazolyl, 1 ,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,

pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H- quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl,

tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1 ,2,5- thiadiazinyl, 1 ,2,3-thiadiazolyl, 1 ,2,4-thiadiazolyl, 1 ,2,5-thiadiazolyl, 1 ,3,4- thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl.

"Heteroaryl", as used herein, refers to a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and 1 , 2, 3, or 4 heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(Y) where Y is absent or is H, O, (Ci-Cg) alkyl, phenyl or benzyl. Non-limiting examples of heteroaryl groups include furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide), quinolyl (or its N-oxide) and the like. The term "heteroaryl" can include radicals of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto. Examples of heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyraxolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl (or its N-oxide), thientyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide), quinolyl (or its N-oxide), and the like.

The term "substituted" as used herein, refers to all permissible substituents of the compounds described herein. In the broadest sense, the permissible substituents include acyclic and cyclic, branched and

unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats.

Representative substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic, substituted C3-C20 cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, and polypeptide groups.

Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is understood that "substitution" or "substituted" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e. a compound that does not spontaneously undergo transf

"Linker" or "Linking Group", as used herein, refer to a group or moiety which is at minimum bivalent, and connects a protease inhibitor to a mitochondrial targeting agent. The linker can be composed of any assembly of atoms, including oligomeric and polymeric chains; however, the total number of atoms in the spacer group is preferably between 3 and 200 atoms, more preferably between 3 and 150 atoms, more preferably between 3 and 100 atoms, most preferably between 3 and 50 atoms. In some embodiments, the linker is hydrophilic. In some embodiments, the linker is an alkyl group, an alkylaryl group, an oligo- or polyethylene glycol chain, or an oligo- or poly(amino acid) chain. In some embodiments, the linker may also include one or more cleavable subunits, such as a disulfide group, one or more hydrolysable functional groups, such as an ester or amide, one or more metal complexes, such as a polyhistidine-nickel chelate complex, one or more hydrogen bond donor-acceptor pairs, one or more biomolecule/bioconjugate pairs (such as biotin-avidin or biotin-streptavidin pair), as well as combinations thereof.

The terms "Analog" and "Derivative" are used herein

interchangeably, and refer to a compound having a structure similar to that a parent compound, but varying from the parent compound by a difference in one or more certain components. The analog or derivative can differ from the parent compound in one or more atoms, functional groups, or substructures, which are replaced with other atoms, groups, or substructures. An analog or derivative can be imagined to be formed, at least theoretically, from the parent compound via some chemical or physical process.

As generally used herein "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications

commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers and/or excipients include those include compounds or materials generally recognized as safe (GRAS) by the U.S. Food and Drug Administration.

II. Mitochondrially Targeted Lon Inhibitors

Mitochondrially targeted protease inhibitors are provided. The protease inhibitors can be targeted to the mitochondria by conjugating, associating, linking, encapsulating, or dispersing the protease inhibitor with or in a mitochondrial targeting agent.

In some embodiments, the mitochondrially targeted protease inhibitor is a protease inhibitor attached to one or more mitochondrial targeting agents.

The protease inhibitor and the mitochondrial targeting agent may be linked in any arrangement using suitable covalent or non-covalent means. In some cases, the protease inhibitor is directly attached to the mitochondrial targeting agent. In other embodiments, the mitochondrial targeting agent is attached to the protease inhibitor through a linker. In certain embodiments, the mitochondrially targeted protease inhibitor is represented by the general structure shown below

A-B-(C)_n

wherein,

A is a Lon protease inhibitor

B is absent, or is a linker;

n is an integer between one a eight; and

C is a mitochondrial targeting agent.

In certain embodiments, the Lon protease inhibitor is MG132, MG262, Dansyl 89-98 Abu boronate, 3,4-dichloroisocoumarin (DCI) or coumarinic derivatives or isomers thereof, clasto-lactacystin-P-lactone or a derivative thereof. The mitochondrially targeted protease inhibitor can include any suitable mitochondrial targeting agent. In preferred

embodiments, the mitochondrial targeting agent is a cationic phosphonium group, such as an alkyltriarylphosphonium group or a tetraalkylphosphonium group.

In some instances, the protease inhibitor retains its biological activity when linked to the mitochondrial targeting agent. In these instances, the protease inhibitor may be linked via a non-cleavable linking group or bond.

In some cases, the protease inhibitor may be linked to one or more mitochondrial targeting agents such that, upon entering the mitochondria, the protease inhibitor can be cleaved from the mitochondrial targeting agent. The protease inhibitor and the mitochondrial targeting agent can be linked to permit cleavage by a variety of mechanisms, including simple hydrolysis and/or enzymatic cleavage.

In one embodiment, the protease inhibitor is bound directly to the mitochondrial targeting agent and the protease inhibitor is released hydrolytically and/or enzymatically. In another embodiment, the creatine moiety is bound to the targeting agent via a linker and the linker is cleaved hydrolytically and/or enzymatically. In some embodiments, the linker is a non-peptide linker which is cleaved within the mitochondria. In other embodiments, the linker is a peptide linker which is cleaved within the mitochondria. In still other embodiments, the creatine moiety is not cleaved from the targeting agent provided the creatine moiety retains the desired biological activity.

In some instances, the Lon protease inhibitor contains a peptidyl segment and an electrophilic moiety. In certain embodiments, the Lon protease inhibitor contains a peptidyl segment of between two to six amino acid residues selected from alanine, valine, leucine, isoleucine, glycine, phenylalanine, tyrosine, and threonine. In some embodiments, the Lon protease inhibitor contains an electrophilic moiety selected from aldehyde, vinyl sulfone, epoxyketone, β-lactone, or boronate moiety. Suitable inhibitors of this type are known in the art, and include MG132, MG262, and Dansyl 89-98 Abu boronate, as well as analogs and derivatives thereof. The Lon protease inhibitor may also be 3,4-dichloroisocoumarin (DO) or coumarinic derivatives or isomers thereof, and clasto-lactacystin-P-lactone, or derivatives thereof.

In preferred embodiments, the mitochondrially targeted protease inhibitor contains a Lon protease inhibitor covalently attached to a mitochondrial targeting agent. In some embodiments, the Lon protease inhibitor is a derivative or analog of MG132, MG262, or Dansyl 89-98 Abu boronate.

Formula I

wherein,

E is an electrophilic moiety;

L is absent, or is a linking group;

Z is a mitochondrial targeting agent; and

R is an alkyl group, an aryl group, or a heterocyclic group In some embodiments of Formula I, L is absent, or is C₂-C₁₂ alkyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylarylalkyl, or aryl group optionally substituted with between one and five substituents individually selected from alkyl, cyclopropyl, cyclobutyl ether, amine, halogen, hydroxyl, ether, nitrile, CF₃, ester, amide, urea, carbamate, thioether, and aryl.

Formula la

wherein,

E is an electrophilic moiety;

L is absent, or is a linking group;

Z is a mitochondrial targeting agent; and

R is an alkyl group, an aryl group, or a heterocyclic group

In some embodiments of Formula la, L is absent, or is C₂-C₁₂ alkyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylarylalkyl, or aryl group optionally substituted with between one and five substituents individually selected from alkyl, cyclopropyl, cyclobutyl ether, amine, halogen, hydroxyl, ether, nitrile, CF₃, ester, amide, urea, carbamate, thioether, and aryl. In some embodiments of Formula la, E is an aldehyde, vinyl sulfone, epoxyketone, β-lactone, or boronate moiety. In certain embodiments, E is an aldehyde or boronate moiety.

A. Lon Inhibitors

Mitochondrial dysfunction is involved in a wide range of diseases from ischaemia-reperfusion to neurodegenerative diseases. In part, this is due to oxidative damage from free radicals generated during oxidative phosphorylation. Accumulation of oxidatively modified proteins is avoided through the action of ATP-dependent proteases within the mitochondria, such as Lon. Increased expression of Lon in rats is associated with enhanced mitochondrial biogenesis.

Although Lon is a serine protease, it is relatively unreactive towards most serine protease inhibitors. Both phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate (DIFP) required concentrations in the low millimolar range to achieve even 50% inhibition of the proteolytic activity. Interestingly, the enzyme is also susceptible to deactivation by certain cysteine protease inhibitors, such as Nethylmaleimide (NEM),

iodoacetamide, and dansyl fluoride, although these are equally as poor at inhibiting proteolysis.

The compositions can contain any suitable Lon protease inhibitor. In certain cases, the protease inhibitor contains a peptidyl segment and an electrophilic moiety. The peptidyl segment may contain any number of amino acid residues. In some cases, the peptidyl segment of the inhibitor contains between two and eight amino acid residues, more preferably between two and six amino acid residues, most preferably between two and five amino acid residues. In some embodiments, the peptidyl segment contains one or more amino acid residues with hydrophobic side chains. In some embodiments, the peptidyl segment contains one or more amino acid residues with uncharged, polar sidechains. In certain embodiments, the peptidyl segment contains one or more amino acids selected from alanine, valine, leucine, isoleucine, glycine, phenylalanine, tyrosine, and threonine.

The electrophilic moiety can be any electrophilic moiety suitable for reaction with a nucleophilic amino acid residue present in the Lon enzyme. In certain embodiments, the electrophilic moiety is an aldehyde, vinyl sulfone, epoxyketone, β-lactone, or boronate moiety. In preferred embodiments, the electrophilic moiety is a boronate.

Lon inhibitors are known in the art, can be identified using screening methods, such as an ATP-dependent FRETN 89-98 peptidase assay. See, for example Frase, H. et al. Biochemistry, 45(27); 8264-74 (2006).

Representative inhibitors include, but are not limited to MG132, MG262, Dansyl 89-98 Abu boronate, 3,4-dichloroisocoumarin (DCI) or coumarinic derivatives or isomers thereof, and clasto-lactacystin-P-lactone, the structures of which are shown below. See Lee, I. and Suzuki, C. K. Biochim. Biophys.

-735 (2008).

Dansyl 89-98 Abu boronate

In some embodiments, the Lon inhibitor is a derivative of MG132, MG262, Dansyl 89-98 Abu boronate, 3,4-dichloroisocoumarin (DCI), or clasto-lactacystin-P-lactone.

B. Mitochondrial Targeting Agents

Mitochondrially targeted protease inhibitors contain a protease inhibitor connected to or associated with one or more mitochondrial targeting agents. In some instances, the protease inhibitors are functionalized with a single mitochondrial targeting agent. Alternatively, protease inhibitors can be functionalized with more than one mitochondrial targeting agent. For example, protease inhibitor can bound to a linker, optionally containing one or more branch points, to which multiple mitochondrial targeting agents are attached.

In the case of mitochondrially targeted protease inhibitors containing a plurality of mitochondrial targeting agents, the agents may be the same or different. In some embodiments, a mitochondrially targeted protease inhibitor is functionalized with multiple copies of the same agent. In alternative embodiments, a mitochondrially targeted protease inhibitors is functionalized with two or more different mitochondrial targeting agents.

Mitochondrial targeting agents are known in the art, and include lipophilic cations that convey a positive charge to the compound under physiological conditions, such as cationic phosphonium and ammonium groups, peptide targeting moieties, and mitochondrial delivery vehicles.

Preferably the mitochondrial targeting agent does not permanently damage the mitochondrion, for example the mitochondrial membrane, or otherwise impair mitochondrial function. 1. Lipophilic Cations

The mitochondrial protease inhibitors can be connected to one or more lipophilic cations that convey a positive charge to the compound under physiological conditions, such as cationic phosphonium and ammonium groups.

In the case of cationic phosphonium and ammonium groups, the selection of carbon substituents on the cationic atom will affect the target activity, the ability of the therapeutic drug to localize within the

mitochondria, and the pharmacokinetic properties (ADME) of the drug. Generally, the substituents on the cation are chosen to distribute the localization of the positive charge and to provide a lipophilic environment in the vicinity of the positive charge to shield the cation from direct interaction with lipophilic biological barriers. Additional pharmacokinetic properties, including oral bioavailability, volume of distribution, and clearance are also dependent on the balance between lipophilic and hydrophilic attributes.

Representative lipophilic cations include, but are not limited to, phosphonium groups represented by the general formula (PR₄)⁺X^~, wherein X is an anion and R is, independently for each occurrence, an alkyl, alkylaryl, alkylcycloalkyl, heterocyclo, alkylheterocyclo, and aryl group, optionally substituted with between one and five substituents selected from alkyl, aklylaryl, cycloalkyl, aryl, hydroxy, alkyl ether, aryl ether, nitrile, fluorine, chlorine, bromine, CF₃, thioether, amide, urea, ester, and carbamate.

Preferably, between two and three of the R groups are aryl groups. In cases where alkylaryl and/or aryl substituents are attached to the

phosphonium ion, the aryl component is preferably a phenyl or a 5-6 membered heteroaryl ring, optionally substituted with between one and two substituents such as halogen, alkyl, alkoxy, CF₃, and nitrile.

In a preferred embodiment, the mitrochondrial targeting agent is an alkyltriphenylphosphonium, tetraphenylphosphonium, or

tetraalkylphosphonium group. Suitable alkyltriphenylphosphonium moieties include, but are not limited to, those alkyltriphenylphosphonium moieties containing a Ci-C₆ straight chain alkylene group having from 1 to 6 carbons, such as a methylene, ethylene, propylene, or butylene group. Suitable tetraalkylphosphonium groups include, but are not limited to those alkyltriphenylphosphonium moieties containing one Ci-C₆ straight chain alkylene group having from 1 to 6 carbons, such as a methylene, ethylene, propylene, or butylene group, and 3 Ci-Ci₈ linear, branched, or cyclic alkyl groups.

Other lipophilic cations include quaternary ammonium groups represented by the general formula (ΝΡ )⁺Χ^~, wherein X is an anion and R is, independently for each occurrence, an alkyl, alkylaryl, alkylcycloalkyl, heterocyclo, alkylheterocyclo, and aryl group, optionally substituted with between one and five substituents selected from alkyl, aklylaryl, cycloalkyl, aryl, hydroxy, alkyl ether, aryl ether, nitrile, fluorine, chlorine, bromine, CF₃, thioether, amide, urea, ester, and carbamate, including tetraalkylammonium groups, tetraphenylammonium groups, and alkyltriphenylammonium groups. The lipophilic cation can also be tetraphenylarsonium, Rhodamine G and derivatives thereof, oligo- or polyarginine, oligo- or polylysine, as well as delocalized lipophilic cations containing one to three carbimino, sulfimino, or phosphinimino units as described in Kolomeitsev et al., Tet. Let., Vol. 44, No. 33, 5795-5798 (2003). Particularly preferred mitochondrial targeting agents contain a cationic triphenylphosphonium group.

Liphophilic cations are preferred mitochondrial targeting agents because they can pass directly through phospholipid bilayers without requiring a specific uptake mechanism, and they accumulate substantially within mitochondria due to the large membrane potential. The large hydrophobic radius of the triphenylphosphine (TPP) cation enables it to pass easily through the phospholipid bilayer relative to other cations. In one embodiment the disclosed compounds include TPP derivatives modified to increase hydrophobicity. For example, the hydrophobicity of the targeting agent can be increased by increasing the length of the carbon chain linker as described in Asin-Cayuela et al, FEBS Lett. , 30:571 (1-3), 9-16 (2004).

Without wishing to be bound to one theory, it is believed that lipophilic cations are taken up from a positively charged cellular

compartment into a negatively charged compartment until a sufficiently large concentration gradient is built up to equalize the electrochemical potential of the molecules in the two compartments. For every 60 mV increase in membrane potential, there will be approximately tenfold accumulation of the lipophilic cation within mitochondria. Because the plasma membrane has a negative 30-60 mV potential on the inside, lipophilic cations will accumulate 5 to 10 fold in the cytosol. Lipophilic cations within the cytosol will accumulate in mitochondria because the mitochondrial membrane potential is typically about 140 to 180 mV.

Methods for conjugating lipophilic cations to other molecules are known in the art. See for example U.S. Patent No. 7,232,809 which is incorporated by reference in its entirety.

2. Polypeptide Targeting Moieties

The mitochondrial targeting agent can also be a polypeptide including positively charged amino acids. Thus, some embodiments include PTDs that are cationic or amphipathic. Protein transduction domains (PTD), also known as a cell penetrating peptides (CPP), are polypeptides including positively charged amino acids. Therefore, the mitochondrial targeting agent can be a PTD or a CPP. "Protein Transduction Domain" refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compounds that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to the compounds disclosed herein facilitates the molecule traversing membranes, for example going from extracellular space to intracellular space, or cytosol to within an organelle such as the mitochondria. PTDs are known in the art, and include but are not limited to small regions of proteins that are able to cross a cell membrane in a receptor-independent mechanism (Kabouridis, P., Trends in Biotechnology (11):498-503 (2003)). Although several of PTDs have been documented, the two most commonly employed PTDs are derived from TAT (Frankel and Pabo, Cell, 55(6): 1189-93(1988)) protein of HIV and Antennapedia transcription factor from Drosophila, whose PTD is known as Penetratin (Derossi et al, J Biol Chem., 269(14): 10444-50 (1994)).

The Antennapedia homeodomain is 68 amino acid residues long and contains four alpha helices. Penetratin is an active domain of this protein which consists of a 16 amino acid sequence derived from the third helix of Antennapedia. TAT protein consists of 86 amino acids and is involved in the replication of HIV- 1. The TAT PTD consists of an 11 amino acid sequence domain (residues 47 to 57; YGRKKRRQRRR (SEQ ID NO: l)) of the parent protein that appears to be critical for uptake. Additionally, the basic domain Tat(49-57) or RKKRRQRRR (SEQ ID NO:2) has been shown to be a PTD. In the current literature TAT has been favored for fusion to proteins of interest for cellular import. Several modifications to TAT, including substitutions of Glutatmine to Alanine, i.e., Q- A, have demonstrated an increase in cellular uptake anywhere from 90% (Wender et al, Proc Natl Acad Sci USA., 97(24): 13003-8 (2000)) to up to 33 fold in mammalian cells. (Ho et al, Cancer Res., 61(2):474-7 (2001)) The most efficient uptake of modified proteins was revealed by mutagenesis experiments of TAT-PTD, showing that an 11 arginine stretch was several orders of magnitude more efficient as an intercellular delivery vehicle.

PTDs can include a sequence of multiple arginine residues, referred to herein as poly-arginine or poly-ARG. In some embodiments the sequence of arginine residues is consecutive. In some embodiments the sequence of arginine residues is non-consecutive. A poly-ARG can include at least 7 arginine residues, more preferably at least 8 arginine residues. In some embodiments, the poly-ARG includes between 7 and 15 arginine residues, more preferably between 8 and 15 arginine residues. In some embodiments the poly-ARG includes between 7 and 15, more preferably between 8 and 15 consecutive arginine residues. An example of a poly-ARG is RRRRRRR (SEQ ID NO:3). Additional exemplary PTDs include but are not limited to; PTD-5 - RRQRRTSKLMKR (SEQ. ID. NO:4); Transportan

GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:5); KALA - WEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:6); and RQIKIWFQNRRMKWKK (SEQ ID NO:7).

Mitochondrial targeting agents can include short peptide sequences (Yousif, et al, Chembiochem., 10(13):2131 (2009), for example

mitochondrial transporters-synthetic cell-permeable peptides, also known as mitochondria-penetrating peptides (MPPs), that are able to enter

mitochondria. MPPs are typically cationic, but also lipophilic; this combination of characteristics facilitates permeation of the hydrophobic mitochondrial membrane. For example, MPPs can include alternating cationic and hydrophobic residues (Horton, et al, Chem Biol, 15(4):375-82 (2008)). Some MPPs include delocalized lipophilic cations (DLCs) in the peptide sequence instead of, or in addition to natural cationic amino acids (Kelley, et al, Pharm. Res., 2011 Aug 11 [Epub ahead of print]). Other variants can be based on an oligomeric carbohydrate scaffold, for example attaching guanidinium moieties due to their delocalized cationic form (Yousif, et al, Chembiochem., 10(13):2131 (2009).

Mitochondrial targeting agents also include mitochondrial localization signals or mitochondrial targeting signals. Many mitochondrial proteins are synthesized as cytosolic precursor proteins containing a leader sequence, also known as a presequence, or peptide signal sequence.

Typically, cytosolic chaperones deliver the precursor protein to

mitochondrial receptors and the General Import Pore (GIP) (Receptors and GIP are collectively known as Translocase of Outer Membrane or TOM) at the outer membrane. Typically, the precursor protein is translocated through TOM, and the intermembrane space by small TIMs to the TIM23 or 22 (Translocase of Inner Membrane) at the inner membrane. Within the mitochondrial matrix the targeting sequence is cleaved off by mtHsp70.

Mitochondrial localization/targeting signals generally have of a leader sequence of highly positively charged amino acids. This allows the protein to be targeted to the highly negatively charged mitochondria. Unlike receptor: ligand approaches that rely upon stochastic Brownian motion for the ligand to approach the receptor, the mitochondrial localization signal of some embodiments is drawn to mitochondria because of charge.

As discussed above, in order to enter the mitochondria, a protein generally must interact with the mitochondrial import machinery, consisting of the Tim and Tom complexes (Translocase of the Inner/Outer

Mitochondrial Membrane). With regard to the mitochondrial targeting signal, the positive charge draws the linked protein to the complexes and continues to draw the protein into the mitochondria. The Tim and Tom complexes allow the proteins to cross the membranes. Accordingly, one embodiment of the present disclosure delivers compositions of the present disclosure to the inner mitochondrial space utilizing a positively charged targeting signal and the mitochondrial import machinery. In another embodiment, PTD-linked compounds containing a mitochondrial localization signal do not seem to utilize the TOM/TIM complex for entry into the mitochondrial matrix, see Del Gaizo et al. Mol Genet Metab. 80(1- 2): 170-80 (2003). Mitochondrial localization signals are known in the art, see for example, U.S. Published Application No. 2005/0147993.

For mitochondria, several amino -terminal targeting signals have been deduced and are included, in part, in Table 1. Table 1 provides a list of mitochondrial proteins that are translated in the cytosol and transported into the mitochondria. The N-terminal region of the proteins can be used to target molecules to the mitochondrion. The sequences are known in the art. The identification of the specific sequences necessary for translocation of a linked compound into a mitochondria can be determined using predictive software known to those skilled in the art, including the tools located at http://ihg.gsf.de/ihg/mitoprot.html.

Table 1

Localization Signals for Targeting to the Mitochondria.

(verified using Mitochondrial Project MITOP Database—

http://mips.gsf.de/proj/medgen/mitop/)

Using the software referenced above, the predicted sequence that can be used to target the protease inhibitor for Etfa is

MFRAAAPGQLRRAASLLRF (SEQ ID NO: 8). The predicted mitochondrial targeting signal from Did is

MQSWSRVYCSLAKRGHFNRISHGLQGLSAVPLRTY (SEQ ID NO:9).

3. Mitochondrial Delivery Vehicles

The mitochondrial targeting agent can also be a mitochondrial delivery vehicle, such as a lipid raft, mitochondrially targeted nanoparticle, or mitochondriotropic liposome. In such cases, one or more mitochondrial protease inhibitors can be associated with, encapsulated within, dispersed in or on, or covalently attached to the mitochondrial delivery vehicle.

In preferred embodiments, the Lon inhibitors are encapsulated, non- covalently associated with, or dispersed in the mitochondrial delivery vehicle. In these embodiments, a suitable Lon inhibitor can be incorporated without any covalent modification. Representative inhibitors include, but are not limited to MG132, MG262, Dansyl 89-98 Abu boronate, 3,4- dichloroisocoumarin (DCI) or coumarinic derivatives or isomers thereof, and clasto-lactacystin-P-lactone. See Lee, I. and Suzuki, C. K. Biochim. Biophys. Acta. 1784(5):727-735 (2008).

In certain embodiments, Lon protease inhibitors are encapsulated, coupled to, or otherwise associated with mitochondriotropic liposomes. Mitochondriotrophic liposomes are cationic liposomes that can be used to deliver an encapsulated agent to the mitochondria of a cell.

Mitochondriotropic liposomes are known in the art. See, for example, U.S. Patent Application Publication No. US 2008/0095834 to Weissig, et al, which is incorporated herein by reference in its entirety. Mitochondriotropic liposomes are liposomes which contain a hydrophobized amphiphilic delocalized cation, such as a triphenylphosphonium or a quinolinium moiety, incorporated into or conjugate to the lipid membrane of the liposome. As a result, the liposomes can be used to deliver compounds incorporated within them to the mitochondria.

In other embodiments, Lon protease inhibitors are encapsulated within, dispersed in, associated with, or conjugated to a nanoparticle functionalized with one or more mitochondrial targeting agents. For example, the nanoparticle may contain one or be functionalized with one or more lipophilic cations or polypeptide targeting agents.

The nanoparticles may be formed from one or more polymers, copolymers, or polymer blends. In some embodiments, the one or more polymers, copolymers, or polymer blends are biodegradable. Examples of suitable polymers include, but are not limited to, polyhydroxyacids such as poly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolic acids); polycaprolactones; poly(orthoesters); polyanhydrides; poly(phosphazenes); poly(hydroxyalkanoates); poly(lactide-co-caprolactones); polycarbonates such as tyrosine polycarbonates; polyamides (including synthetic and natural polyamides), polypeptides, and poly(amino acids); polyesteramides;

polyesters; poly(dioxanones); poly(alkylene alkylates); hydrophobic polyethers; polyurethanes; polyetheresters; polyacetals ; polycyanoacrylates; polyacrylates; polymethylmethacrylates; polysiloxanes;

poly(oxyethylene)/poly(oxypropylene) copolymers; polyketals;

polyphosphates; polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; poly(maleic acids), poly(alkylene glycols) such as polyethylene glycol (PEG), poly(propylene glycol) (PPG), and copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefmic alcohol), polyvinylpyrrolidone), poly(hydroxy alkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(vinyl alcohol), as well as blends and copolymers thereof. Techniques for preparing suitable polymeric nanoparticles are known in the art, and include solvent evaporation, hot melt particle formation, solvent removal, spray drying, phase inversion, coacervation, and low temperature casting. In some cases, the mitochondrial targeting agents are polypeptides that are covalently linked to the surface of the nanoparticle after particle formulation. In other cases, the mitochondrial targeting agents are lipophilic cations that are covalently bound to the particle surface. In some cases, a cationic polymer is incorporated into the particle to target the particle to the mitochondria.

Lon protease inhibitors can also be targeted to the mitochondria using lipid rafts or other synthetic vesicle compositions. See, for example, U.S. Patent Application Publication No. US 2007/0275924 to Khan, et al. which is incorporated herein by reference in its entirety. The lipid raft

compositions can include cholesterol, and one or more lipids selected from the group consisting of sphingomylein, gangliosides,

phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, phosphatidylinositol, and a mitochondrial targeting agent. In certain embodiments, a polypeptide targeting agent is inserted into the lipid raft to target the raft to the mitochondria. The lipid rafts can be prepared and loaded with one or more protease inhibitors using methods known in the art. See, for example, U.S. Patent No. 6,156,337 to Barenholz, et al.

C. Linkers

Mitochondrially targeted protease inhibitors can optionally contain a linker which connects the protease inhibitor to one or more mitochondrial targeting agents. The linker can be inert, or the linker can have biological activity. The linker must be at minimum bivalent; however, in some embodiments, the linker can be bound to more than one active agent, in which case, the linker is polyvalent.

The linker can be composed of any assembly of atoms, including oligomeric and polymeric chains, which functions to connect one or more of the mitochondrial targeting agents described above to a protease inhibitor. In some cases, the linker is an oligomeric and polymeric chain, such as an oligo- or polyethylene glycol chain, or an oligo- or poly(amino acid) chain. Peptide linkers include peptides that can be cleaved once the compound enters the mitochondria. For example, in some cases, the peptide linker is a mitochondrial localization signal as discussed in detail above. In other cases the linker is a non-polymeric organic functional group, such as an alkyl group or an alkylaryl group. In these embodiments, the total number of atoms in the linker is preferably less than 250 atoms, more preferably between 3 and 200 atoms, more preferably between 3 and 150 atoms, more preferably between 3 and 100 atoms, most preferably between 3 and 50 atoms. In preferred embodiments, the linker is hydrophilic to facilitate passage of the creatine compound across biological membranes.

In many cases, the linker is a linear chain; however, in some embodiments, the linker contains one or more branch points. In the case of branched linker, the terminus of each branch point can be functionalized with a mitochondrial targeting agent. In one such embodiment, a dendritic linker is used, with the protease inhibitor being bound to the focal point of the dendrimer, and multiple mitochondrial targeting agents being bound to the ends of the dendritic branches.

In some embodiments, the linker includes one or more cleavable subunits, such as a disulfide group, a hydrazone group, or a peptide group which can be cleaved by proteolytic enzymes within a cell. In alternative embodiments, the linker contains one or more hydrolysable subunits, such as an ester group. The linker can also contain one or more covalent or non- covalent functional groups to facilitate the assembly and/or separation of the protease inhibitor from the attached mitochondrial targeting agent, including, but not limited to one or more metal complexes, such as polyhistidine-nickel chelate complexes, one or more heteroaromatic rings (such as triazole rings formed by the cycloaddition of an alkyne and an azide), one or more hydrogen bond donor-acceptor pairs, one or more biomolecule/bioconjugate pairs (such as biotin-avidin or biotin-streptavidin pair), as well as

combinations thereof.

One or more ends of the linking group may include a functional group used to facilitate attachment of a protease inhibitor and a mitochondrial targeting agent. The functional group may be an atom or group of atoms that contains at least one atom that is neither carbon nor hydrogen. In some embodiments, the functional group may be a halo functional group, such as a fluoro, chloro, bromo, or iodo group; an oxygen-containing functional group, such as a hydroxyl, carbonyl, aldehyde, acetal, hemiacetal, hemiketal, ester, orthoester, carboxylic acid, carboxylate, or ether group; a nitrogen- containing functional group, such as an amide, amine, imine, azide, cyanate, nitrate, nitrile, nitrite, or pyridyl group; phosphorus containing functional groups, such as a phosphate or phosphono group; or a sulfur-containing functional group, such as a sulfide, sulfonyl, sulfonamido, sulfino, sulfo, sulfmyl, sulfhydryl, carbonothioyl, or disulfide group.

In some embodiments, the functional group is a carboxylic acid, a chemical moiety which can be derived from a carboxylic acid by one or more chemical reactions (such as a condensation reaction to form an ester, amide, or thioester, or a reduction reaction to form an aldehyde or alcohol), an analog of a carboxylic acid in which one or more of the atoms is replaced by a sulfur atom, or an analog of a chemical moiety which could be derived from a carboxylic acid by one or more chemical reactions in which one or more of the atoms is replaced by a sulfur atom. In certain embodiments, the functional group is a secondary amide, tertiary amide, secondary carbamate, tertiary carbamate, urea, carbinol, ether, carboxylic acid, or ester.

D. Synthesis of Mitochondrially Targeted Protease Inhibitors Protease inhibitors functionalized with one or more mitochondrial targeting agents can be synthesized by reacting a suitable protease inhibitor with either a mitochondrial targeting agent or with a linking group. In preferred embodiments, the protease inhibitor and the mitochondrial targeting agent are covalently connected by linker. In such instances, the mitochondrial targeting agent may be prepared by first coupling the mitochondrial targeting agent to the linking group, then reacting the linking group with the protease inhibitor. Alternatively, these compounds can be prepared by first reacting the linking group with the protease inhibitor, and then coupling the linking group to the mitochondrial targeting agent.

The appropriate route for synthesis of a given mitochondrially targeted protease inhibitor can be selected in view of the linking group desired, the mitochondrial targeting agent desired, the structure of the protease inhibitor, as well as the structure of the compound as a whole as it relates to compatibility of functional groups, protecting group strategies, and the presence of labile bonds. Mitochondrially targeted protease inhibitors may be prepared by covalent modification of commercially available Lon protease inhibitors, including, MG132, MG262, Dansyl 89-98 Abu boronate,

dichloroisocoumarin (DCI) or coumarinic derivatives or isomers thereof, and clasto-lactacystin-P-lactone.

In the case of protease inhibitors containing a peptidyl segment and an electrophilic moiety, such as MG132 or MG262, synthesis may begin by protection of the electrophilic moiety. Suitable protecting group strategies are known in the art (see, for example, Wuts, P.G.M. and Greene, T.W.

Greene 's Protective Groups in Organic Synthesis. 4^th Edition, Wiley-

Interscience Publication, New York) and can be selected in view of factors including identity of the electrophilic moiety and compatibility of the protecting group with the synthetic methodology to be employed.

Once the electrophilic moiety is protected, the linking group and/or mitochondrial targeting agent can be covalently attached to the peptidyl segment using suitable methods known in the art. See, for example, March, "Advanced Organic Chemistry," 5^th Edition, 2001, Wiley-Interscience Publication, New York). A number of synthetic methods can be used to covalently attach the linking group and/or mitochondrial targeting agent to the peptidyl segment. The appropriate route for the synthesis of a given mitochondrially targeted protease inhibitor can be selected in view of the linking group desired, targeting agent desired, inhibitor desired, as well as the structure of the compound as a whole as it relates to compatibility of functional groups, protecting group strategies, and the presence of labile bonds.

Once the linking group and mitochondrial targeting agent are attached to the protease inhibitor, the electrophilic moiety can be deprotected using methods known in the art. See, for example, Wuts, P.G.M. and Greene, T.W. Greene 's Protective Groups in Organic Synthesis. 4^th Edition, Wiley- Interscience Publication, New York. III. Screening

A. Methods for Screening for Protease Inhibitors

Methods for identifying modulators of the function, expression, or bioavailability of mitochondrial proteases, in particular Lon protease, utilize well known techniques and reagents. Preferably, the modulator inhibits or reduces mitochondrial protease activity relative to a control. Modulation of mitochondrial proteases can be direct or indirect. Direct modulation refers to a physical interaction between the modulator and the protease's mRNA, protein, or DNA. Indirect modulation of the protease can be accomplished when the modulator physically associates with a cofactor, second protein or second biological molecule that interacts with the protease's mRNA, DNA or protein either directly or indirectly. Additionally, indirect modulation includes modulators that affect the expression or the translation of RNA encoding mitochondrial proteases.

In some embodiments, the assays can include random screening of large libraries of test compounds. Alternatively, the assays may be used to focus on particular classes of compounds suspected of modulating the function or expression of mitochondrial proteases in cells, tissues, organs, or systems.

Assays can include determinations of protease gene expression, protein expression, protein activity, or protease activity. Other assays can include determinations of protease nucleic acid transcription or translation, for example mRNA levels, mRNA stability, mRNA degradation, transcription rates, and translation rates.

In one embodiment, the identification of a mitochondrial protease inhibitor occurs in the presence and absence of a test compound. The test compound or modulator can be any substance that alters or is believed to alter the function mitochondrial proteases, particular the function of Lon protease. Preferably the compound inhibits mitochondrial protease activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% relative to a control. One exemplary method includes contacting the mitochondrial protease with at least a first test compound, and assaying for proteolysis of TFAM by the mitochondrial protease in the presence of the first test compound. The assaying can include determining degradation of TFAM.

Specific assay endpoints or interactions that may be measured in the disclosed embodiments include assaying for proteolysis of TFAM, TFAM turnover, protease down or up regulation or turnover. These assay endpoints may be assayed using standard methods such as FACS, FACE, ELISA, Northern blotting and/or Western blotting. Moreover, the assays can be conducted in cell free systems, in isolated cells, genetically engineered cells, immortalized cells, or in organisms such as C. elegans and transgenic animals.

Another embodiment provides a method for identifying a modulator of mitochondrial protease expression by determining the effect a test compound has on the expression of genes encoding the mitochondrial protease. For example isolated cells or whole organisms expressing a mitochondrial protease can be contacted with a test compound. Protease expression can be determined by detecting mitochondrial protease protein expression mRNA transcription or translation. Suitable cells for this assay include, but are not limited to, immortalized cell lines or primary cell culture. Compounds that modulate the expression of mitochondrial proteases, in particular that reduce or inhibit the expression or bioavailability

mitochondrial protease, can be selected.

Another embodiment provides for in vitro assays for the

identification of modulators of mitochondrial protease. Such assays generally use isolated molecules, can be run quickly and in large numbers, thereby increasing the amount of information obtainable in a short period of time. A variety of vessels may be used to run the assays, including test tubes, plates, dishes and other surfaces such as dipsticks or beads.

One example of a cell free assay is a binding assay. While not directly addressing function, the ability of a modulator to bind to a target molecule, for example a nucleic acid encoding a mitochondrial protease, in a specific fashion is strong evidence of a related biological effect. Such a molecule can bind to a nucleic acid encoding the protease and modulate expression of the protease. The binding of a molecule to a target may, in and of itself, be inhibitory, due to steric, allosteric or charge—charge interactions or may downregulate or inactivate the mitochondrial protease. The target may be either free in solution, fixed to a support, expressed in or on the surface of a cell. Either the target or the compound may be labeled, thereby permitting determining of binding. Usually, the target will be the labeled species, decreasing the chance that the labeling will interfere with or enhance binding. Competitive binding formats can be performed in which one of the agents is labeled, and one may measure the amount of free label versus bound label to determine the effect on binding.

A technique for high throughput screening of compounds is described in WO 84/03564. Large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. Bound polypeptide is detected by various methods.

In vivo assays involve the use of various animal models, including non-human transgenic animals that have been engineered to have specific defects, or carry markers that can be used to measure the ability of a test compound to reach and affect different cells within the organism. Due to their size, ease of handling, and information on their physiology and genetic make-up, mice are a preferred embodiment, especially for transgenic animals. However, other animals are suitable as well, including C. elegans, rats, rabbits, hamsters, guinea pigs, gerbils, woodchucks, cats, dogs, sheep, goats, pigs, cows, horses and monkeys (including chimps, gibbons and baboons). Assays for modulators may be conducted using an animal model derived from any of these species.

In such assays, one or more test compounds are administered to an animal, and the ability of the test compound(s) to alter one or more characteristics, as compared to a similar animal not treated with the test compound(s), identifies a modulator. Other embodiments provide methods of screening for a test compound that modulates the function of mitochondrial proteases. In these embodiments, a representative method generally includes the steps of administering a test compound to the animal and determining the ability of the test compound to increase oxidative phosphorylation.

Treatment of these animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal. Administration will be by any route that could be utilized for clinical or nonclinical purposes, including, but not limited to, oral, nasal, buccal, or even topical. Alternatively, administration may be by intratracheal instillation, bronchial instillation, intradermal, subcutaneous, intramuscular,

intraperitoneal or intravenous injection. Specifically contemplated routes are systemic intravenous injection, regional administration via blood or lymph supply, or directly to an affected site.

Determining the effectiveness of a compound in vivo may involve a variety of different criteria. Also, measuring toxicity and dose response can be performed in animals in a more meaningful fashion than in in vitro or in cyto assays.

IV. Modified TFAM

In another embodiment, TFAM is modified to be resistant to proteolysis. For example, the binding site in TFAM for Lon protease can be mutated for example by substituting, deleting, chemically modifying, inserting one or amino acids in TFAM to reduce or inhibit binding of Lon protease to TFAM. The resulting TFAM variant retains the biological activity of TFAM and is resistant to proteolysis.

V. Pharmaceutical Compositions

Pharmaceutical compositions including a mitochondrially targeted protease inhibitor are provided. Pharmaceutical compositions containing peptides or polypeptides may be for administration by parenteral

(intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration. The compositions may also be administered using bioerodible inserts and may be delivered directly to an appropriate lymphoid tissue (e.g., spleen, lymph node, or mucosal-associated lymphoid tissue) or directly to an organ or tumor. The compositions can be formulated in dosage forms appropriate for each route of administration. Compositions containing mitochondrially targeted protease inhibitor that are not or are partially peptides or

polypeptides can additionally be formulated for enteral administration.

As used herein the term "effective amount" or "therapeutically effective amount" means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated or to otherwise provide a desired pharmacologic and/or physiologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being effected. Therapeutically effective amounts of mitochondrially targeted protease inhibitors can be used to increase oxidative phosphorylation in a subject.

One of skill in the art can readily determine dosage levels and administration regimes.

A. Formulations for Parenteral Administration

In a preferred embodiment, the disclosed compositions, including those containing peptides and polypeptides, are administered in an aqueous solution, by parenteral injection. The formulation may also be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided including effective amounts of a peptide or polypeptide, and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include sterile water, buffered saline (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and optionally, additives such as detergents and solubilizing agents (e.g., TWEEN® 20, TWEEN 80, Polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may be lyophilized and redissolved/resuspended immediately before use. The formulation may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.

B. Controlled Delivery Polymeric Matrices

Compositions containing one or more mitochondrially targeted protease inhibitors can be administered in controlled release formulations. Controlled release polymeric devices can be made for long term release systemically following implantation of a polymeric device (rod, cylinder, film, disk) or injection (microparticles). The matrix can be in the form of microparticles such as microspheres, where peptides are dispersed within a solid polymeric matrix or microcapsules, where the core is of a different material than the polymeric shell, and the peptide is dispersed or suspended in the core, which may be liquid or solid in nature. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer may be cast as a thin slab or film, ranging from nanometers to four centimeters, a powder produced by grinding or other standard techniques, or even a gel such as a hydrogel. The matrix can also be incorporated into or onto a medical device to modulate an immune response, to prevent infection in an immunocompromised patient (such as an elderly person in which a catheter has been inserted or a premature child) or to aid in healing, as in the case of a matrix used to facilitate healing of pressure sores, decubitis ulcers, etc.

Either non-biodegradable or biodegradable matrices can be used for delivery of mitochondrially targeted protease inhibitors, although biodegradable matrices are preferred. These may be natural or synthetic polymers, although synthetic polymers are preferred due to the better characterization of degradation and release profiles. The polymer is selected based on the period over which release is desired. In some cases linear release may be most useful, although in others a pulse release or "bulk release" may provide more effective results. The polymer may be in the form of a hydrogel (typically in absorbing up to about 90% by weight of water), and can optionally be crosslinked with multivalent ions or polymers.

The matrices can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed for making microspheres for drug delivery, for example, as described by Mathiowitz and Langer, J. Controlled Release, 5: 13-22 (1987); Mathiowitz, et al, Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al, J. Appl. Polymer ScL, 35:755-774 (1988).

Controlled release oral formulations may be desirable.

Mitochondrially targeted protease inhibitors can be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms, e.g., films or gums. Slowly disintegrating matrices may also be incorporated into the formulation. Another form of a controlled release is one in which the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. For oral formulations, the location of release may be the stomach, the small intestine (the duodenum, the jejunem, or the ileum), or the large intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the active agent (or derivative) or by release of the active agent beyond the stomach environment, such as in the intestine. To ensure full gastric resistance an enteric coating (i.e, impermeable to at least pH 5.0) is essential. These coatings may be used as mixed films or as capsules such as those available from Banner Pharmacaps.

The devices can be formulated for local release to treat the area of implantation or injection and typically deliver a dosage that is much less than the dosage for treatment of an entire body. The devices can also be formulated for systemic delivery. These can be implanted or injected subcutaneously.

C. Formulations for Enteral Administration

Mitochondrially targeted protease inhibitors can also be formulated for oral delivery. Oral solid dosage forms are known to those skilled in the art. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders, or granules or incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 21st Ed. (2005, Lippincott, Williams & Wilins, Baltimore, Md. 21201) pages 889-964. The compositions may be prepared in liquid form, or may be in dried powder (e.g., lyophilized) form.

Liposomal or polymeric encapsulation may be used to formulate the compositions. See also Marshall, K. In: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter 10, 1979. In general, the formulation will include the active agent and inert ingredients which protect the mitochondrially targeted protease inhibitor in the stomach environment, and release of the biologically active material in the intestine.

Liquid dosage forms for oral administration, including

pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, may contain other components including inert diluents; adjuvants such as wetting agents, emulsifying and suspending agents; and sweetening, flavoring, and perfuming agents.

Claims

I claim:

1. A mitochondrially targeted protease inhibitor represented by the following structure

A-B-(C)_n

wherein,

A is a Lon protease inhibitor

B is absent, or is a linker;

n is an integer between one a eight; and

C is a mitochondrial targeting agent.

2. The inhibitor of claim 1 , wherein A is MG132 or a derivative thereof, MG262 or a derivative thereof, Dansyl 89-98 Abu boronate or a derivative thereof, 3,4-dichloroisocoumarin (DCI) or coumarinic derivatives or isomers thereof, clasto-lactacystin-P-lactone or a derivative thereof and C is a polypeptide comprising multiple arginine residues.

3. An agent that prevents, reduces or inhibits mitochondrial protease activity.

4. The agent of claim 3, wherein the agent prevents, reduces or inhibits proteolysis of mitochondrial proteins.

5. The agent of claim 3, wherein the agent prevents, reduces or inhibits Lon protease activity.

6. The agent of claim 3, wherein the agent prevents, reduces or inhibits Lon protease activity on mitochondrial proteins

7. The agent of claim 6, wherein the agent prevents, reduces or inhibits degradation of proteins mediating mitochondrial function

8. The agent of claim 6, wherein the agent prevents, reduces or inhibits Lon protease activity on proteins mediating mitochondrial function

9. The agent of claim 6, where the agent that increases levels or activity of proteins mediating mitochondrial function

10. The agent of claim 8 that prevents, reduces or inhibits Lon protease activity on proteins involved in mitochondrial replication, transcription, or translation

11. The agent of claim 9, wherein the agent prevents, reduces or inhibits degradation of proteins involved in mitochondrial replication, transcription, or translation

12. The agent of claim 10, wherein the agent increases the levels or activity of proteins involved in mitochondrial replication, transcription, or translation.

13. The agent of claim 10, wherein the agent prevents, reduces or inhibits Lon protease activity on TFAM.

14. The agent of claim 11, wherein the agent prevents, reduces or inhibits degradation of TFAM.

15. The agent of claim 11, wherein the agent increases the levels or activity of TFAM.

16. The agent of claim 12, wherein the agent prevents, reduces or inhibits Lon protease activity on proteins of oxidative phosphorylation.

17. The agent of claim 10 that prevents, reduces or inhibits degradation of proteins involved in oxidative phosphorylation.

18. The agent of claim 11 , wherein the agent increases the levels or activity of proteins involved in oxidative phosphorylation.

19. The agent of claim 3, wherein the agent is selected from the group consisting MG132, MG262, 3,4-dichloroisocoumarin (DCI) or coumarinic derivatives or isomers thereof.

20. The agent of claim 19, wherein the agent possesses a mitochondrial targeting moiety or domain.

21. The agent of claim 20, wherein the targeting moiety is triphenyl phosphonium.

22. A method to identify molecules of claim 3 comprising

combining mitochondrial proteins with proteases in the presence of a test compound under physiological conditions,

assaying for protease activity on the mitochondrial proteins, selecting the test compound if protease activity on the mitochondrial proteins is reduced relative to a control.

23. The method of claim 22, wherein Lon protease is combined with TFAM and the test compound.

24. A method for increasing oxidative metabolism in a subject comprising administering to the subject an effective amount of a LON protease inhibitor to inhibit or reduce TFAM degradation.

25. A mitochondrially targeted Lon protease inhibitor comprising a lipophilic cation conjugated to the Lon protease inhibitor.

26. A TFAM variant having increased resistance to proteolysis relative to wildtype TFAM.