MODULATORS OF MITOCHONDRIAL PROTEIN IMPORT
The present invention provides compounds that modulate protein translocation in mitochondria, compositions thereof, and methods of identifying, making and using these.
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This invention was made with Government support of Grant No. GM061721, awarded by the National Institutes of Health. The Government has certain rights in this invention.
FIELD OF THE INVENTIONThe present invention generally relates to the field of providing therapeutics.
BACKGROUND OF THE INVENTIONAll eukaryotic cells contain specialized organs called mitochondria that produce energy and house a host of metabolic processes essential for life. To achieve a fully functional state, mitochondria require proteins synthesized in the cell's cytosol to be imported into the proper location on or within them. This process is complicated because each mitochondrion is composed of two distinct compartments arising from the set of lipid membranes that surround them. Genetic, biochemical, and cellular studies have identified a complex translocation system, including translocons on the mitochondrial outer and inner membranes and intermembrane space of the mitochondrial (Scheme I).
The outer membrane contains the TOM (translocon of the outer membrane) protein complex, whereas the inner membrane contains the TIM23 (translocase of the inner membrane) and TIM22 complexes, which differ in their substrate specificity. Defects in the TIM22 import pathway lead to an inherited disease called deafness-dystonia syndrome, in which patients have deafness, blindness, and dystonia.
There is a need for compounds that are specific inhibitors of mitochondrial protein translocation. The inhibitors modulate the assembly or function of mitochondria.
There is a need for compounds effective for a disorder related to mitochondrial protein translocation.
The embodiments below address the above identified needs and issues.
SUMMARY OF THE INVENTIONIn one aspect of the present invention, it is provided a method for identifying a specific inhibitor of mitochondrial protein translocation, which method comprising culturing a tim10-1 mutant strain of yeast in a medium with a library of drug-like compounds, identifying a drug-like compound as a hit compound if the drug-like compound significantly inhibits growth of the tim10-1 mutant strain of yeast,
subjecting the hit compound to a counter screen which comprises
incubating the hit compound with the tim10-1 mutant strain and an isogenic control strain carrying an integrated version of the TIM10 gene at the leu2 locus, and
identifying the hit compound that selectively inhibits growth of the mutant strain but not the isogenic control strain as a hit compound for second counter screen where the second counter screen comprises:
-
- incubating the hit compound for second counter screen with the tim10-1 mutant strain and a tim10-1 mutant strain harboring a plasmid containing a wild-type TIM10 gene, and
- identifying the hit compound that selectively inhibits growth of the tim10-1 mutant but not the tim10-1 mutant harboring a plasmid containing the wild-type TIM10 gene; and
designating the hit compound that selectively inhibits growth of only the tim10-1 mutant in both the first counter screen and the second counter screen as the specific inhibitor of mitochondrial protein translocation (“MitoBloCk”).
In some embodiments of the method, the drug-like compound inhibits growth of the tim10-1 mutant strain of yeast by 50% or above.
In some embodiments of the method, in combination with any of the above various embodiments, the hit compound selectively inhibits growth of the mutant strain by 50% or above.
In some embodiments of the method, in combination with any of the above various embodiments, the hit compound selectively inhibits growth of the mutant strain by 80% or above.
In some embodiments of the method, in combination with any of the above various embodiments, the hit compound selectively inhibits growth of the mutant strain by 90% or above.
In some embodiments of the method, in combination with any of the above various embodiments, the hit compound selectively inhibits growth of the mutant strain by 99% or above.
In some embodiments of the method, in combination with any of the above various embodiments, the tim10-1 mutant has a concentration of about 10 μM.
In some embodiments of the method, in combination with any of the above various embodiments, the method comprises an integrated robotic system.
In another aspect of the present invention, it is provided a method for identifying a specific inhibitor or activator of mitochondrial disulfide relay pathways. The method comprises
providing a system of testing purified components of the mitochondrial oxidative folding and disulfide relay pathway including Mia40, Cmc1, Erv1, ALR, cytochrome c, and small Tim proteins in a medium with a library of drug-like compounds,
identifying a drug-like compound as a hit compound if the drug-like compound significantly inhibits or activates the activity of at least one of redox-active enzymes,
subjecting the hit compound to a counter screen which comprises
incubating the hit compound with a yeast or mammalian cell line that reports the growth inhibition of a yeast strain or mammalian cell line that had attenuated activity in its mitochondrial disulfide relay pathway and an isogenic control strain or cell line carrying a non-attenuated version of the mitochondrial disulfide relay system, and
identifying the hit compound that selectively inhibits or promotes the growth of the attenuated stain or cell line but not the strain or cell line as a hit compound for second counter screen where the second counter screen comprises:
-
- incubating the hit compound for second counter screen with the a member of a redox-active enzyme family other than ALR or Erv1, and
- identifying the hit compound that selectively inhibits or activates the activity of ALR or Erv1 but not the related redox-active enzyme; and
designating the hit compound that selectively inhibits or activates the activity of ALR or Erv in both the first counter screen and the second counter screen as the specific inhibitor of the mitochondrial disulfide relate system (“MitoBloCk”).
In some embodiments of the method, the drug-like compound inhibits or activates the activity of ALR or Erv1 by 50% or above.
In some embodiments of the method, the hit compound selectively inhibits or activates the activity of ALR or Erv1 by 50% or above.
In some embodiments of the method, the hit compound selectively inhibits or activates the activity of ALR or Erv1 by 80% or above.
In some embodiments of the method, the hit compound selectively inhibits or activates the activity of ALR or Erv1 by 90% or above.
In some embodiments of the method, the compound selectively inhibits or activates the activity of ALR or Erv1 by 99% or above.
In some embodiments of the method, in combination with any of the above various embodiments, the tim10-1 mutant has a concentration of Erv1 or ALR is at or below its Michaelis-Menten constant (Km).
In some embodiments of the method, in combination with any of the above various embodiments, the tim10-1 mutant has a concentration of Erv1 or ALR is at or below its Michaelis-Menten constant (Km).
In some embodiments of the method, in combination with any of the above various embodiments, the tim10-1 mutant has a concentration of Erv1 or ALR is about 1 μM.
In some embodiments of the method, in combination with any of the above various embodiments, the method comprises an integrated robotic system.
In another aspect of the present invention, embodiments of the invention herein provide a specific inhibitor of mitochondrial protein translocation, which the inhibitor specifically targets the protein translocation pathway thereby modulating the assembly and function of the mitochondrion with respect to protein translocation and import. In some embodiments, the inhibitors are molecules or compounds, derivatives thereof or pharmaceutically acceptable salts thereof. In some embodiments, the compound has a structure of formula I, II or III:
a derivative thereof, or pharmaceutically acceptable salt thereof, wherein each R1, R2, and R3 is independently H, C1-C10 straight-chained or branched alkyl (substituted or unsubstituted), C1-C10 cycloalkyl (substituted or unsubstituted), C1-C10 straight-chained or branched alkeynyl (substituted or unsubstituted), C1-C10 cycloalkenyl (substituted or unsubstituted), C1-C10 aryl (substituted or unsubstituted), phenyl, carboxyl, hydroxyl, amino, carbonyl, carbonate, halo (F, Cl, Br, or I), thiol, thiourea, urea, or triazole groups. In some embodiments, the compound is
a derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound has a structure of formula A-D:
derivatives thereof, or pharmaceutically acceptable salts thereof.
The molecules are effective for treating or ameliorating disorders related to mitochondrial protein import. In some embodiments, the disorder is related to deafness-dystonia syndrome (e.g., blindness, deafness, and dystonia). In some embodiments, the disorder is a disease caused by defects in mitochondrial function. Some examples of such diseases are cancer, Parkinson's disease, or Alzheimer's disease.
In some embodiments, it is provided a composition comprising the compound disclosed herein. Compositions can be formed to include an effective amount of a compound disclosed herein. In some embodiments, the composition can include a carrier, e.g., a pharmaceutically acceptable carrier.
In some embodiments, it is provided a method of using the compound. Generally, the method comprises modulating the assembly and/or function of mitochondria by applying a compound disclosed herein to a body of mitochondria or a cell. The cell can be cultured cell, or an organism dissolved in solution, or a living organism such as an animal (e.g., human being). In some embodiments, the compound can be included in a composition which optionally includes a pharmaceutically acceptable carrier.
Other embodiments of the present invention include method of making the compound disclosed herein and method of forming a composition disclosed herein.
In one aspect of the present invention, it is provided a method for identifying a specific inhibitor of mitochondrial protein translocation, which method comprising culturing a tim10-1 mutant strain of yeast in a medium with a library of drug-like compounds, identifying a drug-like compound as a hit compound if the drug-like compound significantly inhibits growth of the tim10-1 mutant strain of yeast,
subjecting the hit compound to a counter screen which comprises
incubating the hit compound with the tim10-1 mutant strain and an isogenic control strain carrying an integrated version of the TIM10 gene at the leu2 locus, and
identifying the hit compound that selectively inhibits growth of the mutant strain but not the isogenic control strain as a hit compound for second counter screen where the second counter screen comprises:
-
- incubating the hit compound for second counter screen with the tim10-1 mutant strain and a tim10-1 mutant strain harboring a plasmid containing a wild-type TIM10 gene, and
- identifying the hit compound that selectively inhibits growth of the tim10-1 mutant but not the tim10-1 mutant harboring a plasmid containing the wild-type TIM10 gene; and
designating the hit compound that selectively inhibits growth of only the tim10-1 mutant in both the first counter screen and the second counter screen as the specific inhibitor of mitochondrial protein translocation (“MitoBloCk”).
In some embodiments of the method, the drug-like compound inhibits growth of the tim10-1 mutant strain of yeast by 50% or above.
In some embodiments of the method, in combination with any of the above various embodiments, the hit compound selectively inhibits growth of the mutant strain by 50% or above.
In some embodiments of the method, in combination with any of the above various embodiments, the hit compound selectively inhibits growth of the mutant strain by 80% or above.
In some embodiments of the method, in combination with any of the above various embodiments, the hit compound selectively inhibits growth of the mutant strain by 90% or above.
In some embodiments of the method, in combination with any of the above various embodiments, the hit compound selectively inhibits growth of the mutant strain by 99% or above.
In some embodiments of the method, in combination with any of the above various embodiments, the tim10-1 mutant has a concentration of about 10 μM.
In some embodiments of the method, in combination with any of the above various embodiments, the method comprises an integrated robotic system.
In another aspect of the present invention, it is provided a method for identifying a specific inhibitor or activator of mitochondrial disulfide relay pathways. The method comprises
providing a system of testing purified components of the mitochondrial oxidative folding and disulfide relay pathway including Mia40, Cmc1, Erv1, ALR, cytochrome c, and small Tim proteins in a medium with a library of drug-like compounds,
identifying a drug-like compound as a hit compound if the drug-like compound significantly inhibits or activates the activity of at least one of redox-active enzymes,
subjecting the hit compound to a counter screen which comprises
incubating the hit compound with a yeast or mammalian cell line that reports the growth inhibition of a yeast strain or mammalian cell line that had attenuated activity in its mitochondrial disulfide relay pathway and an isogenic control strain or cell line carrying a non-attenuated version of the mitochondrial disulfide relay system, and
identifying the hit compound that selectively inhibits or promotes the growth of the attenuated stain or cell line but not the strain or cell line as a hit compound for second counter screen where the second counter screen comprises:
-
- incubating the hit compound for second counter screen with the a member of a redox-active enzyme family other than ALR or Erv1, and
- identifying the hit compound that selectively inhibits or activates the activity of ALR or Erv1 but not the related redox-active enzyme; and
designating the hit compound that selectively inhibits or activates the activity of ALR or Ery in both the first counter screen and the second counter screen as the specific inhibitor of the mitochondrial disulfide relate system (“MitoBloCk”).
In some embodiments of the method, the drug-like compound inhibits or activates the activity of ALR or Erv1 by 50% or above.
In some embodiments of the method, the hit compound selectively inhibits or activates the activity of ALR or Erv1 by 50% or above.
In some embodiments of the method, the hit compound selectively inhibits or activates the activity of ALR or Erv1 by 80% or above.
In some embodiments of the method, the hit compound selectively inhibits or activates the activity of ALR or Erv1 by 90% or above.
In some embodiments of the method, the compound selectively inhibits or activates the activity of ALR or Erv1 by 99% or above.
In some embodiments of the method, in combination with any of the above various embodiments, the tim10-1 mutant has a concentration of Erv1 or ALR is at or below its Michaelis-Menten constant (Km).
In some embodiments of the method, in combination with any of the above various embodiments, the tim10-1 mutant has a concentration of Erv1 or ALR is at or below its Michaelis-Menten constant (Km).
In some embodiments of the method, in combination with any of the above various embodiments, the tim10-1 mutant has a concentration of Erv1 or ALR is about 1 μM.
In some embodiments of the method, in combination with any of the above various embodiments, the method comprises an integrated robotic system.
In another aspect of the present invention, embodiments of the invention herein provide a specific inhibitor of mitochondrial protein translocation, which the inhibitor specifically targets the protein translocation pathway thereby modulating the assembly and function of the mitochondrion with respect to protein translocation and import. In some embodiments, the inhibitors are molecules or compounds, derivatives thereof or pharmaceutically acceptable salts thereof. In some embodiments, the compound has a structure of formula I, II or III:
a derivative thereof, or pharmaceutically acceptable salt thereof, wherein each R1, R2, and R3 is independently H, C1-C10 straight-chained or branched alkyl (substituted or unsubstituted), C1-C10 cycloalkyl (substituted or unsubstituted), C1-C10 straight-chained or branched alkeynyl (substituted or unsubstituted), C1-C10 cycloalkenyl (substituted or unsubstituted), C1-C10 aryl (substituted or unsubstituted), phenyl, carboxyl, hydroxyl, amino, carbonyl, carbonate, halo (F, Cl, Br, or I), thiol, thiourea, urea, or triazole groups. In some embodiments, the compound is
a derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound has a structure of formula A-D:
derivatives thereof, or pharmaceutically acceptable salts thereof.
The molecules are effective for disorders related to mitochondrial protein import. In some embodiments, the disorder is related to deafness-dystonia syndrome (e.g., blindness, deafness, and dystonia). In some embodiments, the disorder is a disease caused by defects in mitochondrial function. Some examples of such diseases are cancer, Parkinson's disease, or Alzheimer's disease.
In some embodiments, it is provided a composition comprising the inhibitor or compound disclosed herein. Compositions can be formed to include an effective amount of a compound disclosed herein. In some embodiments, the composition can include a carrier, e.g., a pharmaceutically acceptable carrier.
In some embodiments, it is provided a method of using the compound. Generally, the method comprises modulating the assembly and/or function of mitochondria by applying a compound disclosed herein to a body of mitochondria or a cell. The cell can be cultured cell, or an organism dissolved in solution, or a living organism such as an animal (e.g., human being). In some embodiments, the compound can be included in a composition which optionally includes a pharmaceutically acceptable carrier.
Other embodiments of the present invention include method of making the compound disclosed herein and method of forming a composition disclosed herein.
The present invention represents a significant innovation. Prior to the discovery of these compounds, scientists could only modulate the biogenesis of mitochondria using genetic manipulation or non-specific drug treatments. Genetic manipulations are mostly limited to single celled organisms such as yeast and are not titratable or reversible. The available chemical tools for mitochondrial biogenesis are limited in their utility since they are either nonspecific inhibitors or metabolic poisons that block cellular respiration. Recently, Nunnari and colleagues (U.S. patent application publication No. 20050038051) discovered a drug compound that regulated the fission and fusion of mitochondria. Their discovery of mdivi-1 was the first drug that could specifically alter mitochondrial dynamics. However, at this time there are no known drugs that target the assembly of the mitochondrion with respect to protein translocation1 import. Our invention specifically targets the protein translocation pathway.
The studies disclosed in the present invention show that the inhibition by the invention compounds is specific to the protein import machinery. The following targets are the most probable ones for studying the mechanism of action:
i. MitoBloCK-1: Tim9/1 0 chaperone complex;
ii. MitoBloCK-2: Tom40 outer membrane translocation pore;
iii. MitoBloCK-3: Small Tim protein chaperones
iv. MitoBloCK-6: Redox cycling of small Tim protein chaperones
We have shown that these drugs have activity in both biochemical experiments with purified cellular components and in live cells.
As used herein, the term “specific inhibitor of mitochondrial protein translocation” refers to a small molecule that specifically targets the protein translocation pathway so as to modulate the assembly and function of the mitochondrion with respect to protein translocation and import.
The term “tim10-1 mutant” is well known to a person of ordinary skill in the art.
“Small Tim proteins” are well documented in the art and marked by their conserved ‘twin Cx(3)C’ motif separated by 11-16 residues (see, e.g., C M Koehler, Trends Biochem Sci. 29(1):1-4 (2004); Webb, C. T., et al., Mol. Cell. 21(1): 123-133 (2006); and Mesecke, N., et al., Cell 121: 1059-1069 (2005)).
“Redox-active enzymes” are enzymes involved in redox reactions in a biological system. These enzymes are well known in the art and within the general knowledge of a person of ordinary skill in the art.
Method of MakingA compound disclosed herein can be readily prepared according to established methodology in the art of organic synthesis. General methods of synthesizing the compound can be found in, e.g., Stuart Warren and Paul Wyatt, Workbook for Organic Synthesis: The Disconnection Approach, second Edition, Wiley, 2010.
Methods of UseIn a further aspect, it is provided a method of using the compound disclosed herein. The method comprises applying the compound of invention to a subject to treat, prevent, or ameliorate a medical condition. The medical condition can be any disease or disorder caused by or otherwise associated with mitochondria protein translocation.
In some embodiments, the method can be conducted in living bodies of mammals. In such a case, the compounds may be administered to the mammals.
As used herein, the term disorder and medical condition include deafness-dystonia syndrome, cancer, Parkinson's disease, or Alzheimer's disease. In some embodiments, the deafness-dystonia syndrome includes deafness, blindness, and dystonia
Pharmaceutical CompositionsIn another aspect of the present invention, a pharmaceutical composition for use in treatment or prevention of the diseases caused by or otherwise associated with mitochondria protein translocation. In some embodiments, the pharmaceutical composition comprises as an effective ingredient a compound expressed by any one of the aforementioned formulae a pharmacologically acceptable salt or prodrug thereof.
The pharmaceutical composition preferably comprises a compound described above or a pharmacologically acceptable salt or prodrug thereof.
The pharmaceutical composition more preferably comprises a compound shown in the aforementioned table.
In the aforementioned aspect of the present invention, the pharmaceutical composition may contain a pharmacologically acceptable carrier or excipients. An amount of the compound used in the pharmaceutical composition is not limited as far as it is an effective amount for treatment.
The pharmaceutical composition in the aspect of the present invention may contain, as active ingredients, the aforementioned compound and other compounds, or may contain a mixture of two or more aforementioned compounds.
The pharmacologically acceptable salt in the present specification is not specifically limited as far as it can be used in medicaments. Examples of a salt that the compound of the present invention forms with a base include the following: salts thereof with inorganic bases such as sodium, potassium, magnesium, calcium, and aluminum; salts thereof with organic bases such as methylamine, ethylamine and ethanolamine; salts thereof with basic amino acids such as lysine and ornithine; and ammonium salt. The salts may be acid addition salts, which are specifically exemplified by acid addition salts with the following: mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid:organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, and ethanesulfonic acid; acidic amino acids such as aspartic acid and glutamic acid.
Further, the compounds of the present invention include hydrates thereof, various pharmaceutically acceptable solvates thereof, and polymorphic crystals thereof.
The pharmaceutical compositions of the present invention can be formulated in various dosage forms, which are exemplified by the following: oral administration forms such as tablets, capsules, powders, granules, pills, liquids, emulsions, suspensions, solutions, spirits, syrups, extracts, and elixirs; parenteral administration forms such as injections, for example, subcutaneous injections, intravenous injections, intramuscular injections, and intraperitoneal injections; transdermal administration forms, plasters and pressure sensitive adhesives, ointments or lotions; intramouth administration forms such as sublingual forms and oral patch preparations; and nasal administration forms such as aerosols, but are not limited thereto. These preparations can be manufactured by using a known method generally used in a drug manufacturing process. In one embodiment of the present invention, the pharmaceutical composition of the present invention may be administered for treating muscular disease as an injection such as an intramuscular injection for administering directly into muscle.
The pharmaceutical compositions may contain various kind of ingredients generally used, for example, one or more pharmaceutically acceptable fillers, disintegrators, diluents, lubricants, flavoring agents, colorants, sweetening agents, corrigents, suspending agents, humectants, emulsifying agents, dispersing agents, auxiliary agents, preservatives, buffers, binders, stabilizers, and coating agents. In addition, the pharmaceutical composition of the present invention may be sustained-release dosage forms or extended-release dosage forms.
Dosage ranges of the pharmaceutical compositions are not particularly limited, and can be determined in accordance with the following: effectiveness of the ingredients contained therein; the administration form; the route of administration; the type of disease; the characteristics of the subject (e.g., body weight, age, symptomatic conditions, and whether a subject is taking other pharmaceutical agents); and the judgment of a physician in charge. In general, a suitable dosage may fall, for example, within a range of about 0.01 μg to 100 mg, per 1 kg of the body weight of the subject, and preferably within a range of about 0.1 μg to 1 mg, per 1 kg of body weight. However, the dosage may be altered using conventional experiments for optimization of a dosage that are well known in the art. The aforementioned dosage can be divided for administration once to several times a day. Alternatively, periodic administration once every few days or few weeks can be employed.
The pharmaceutical compositions may be administered to a patient whose biological sample obtained in advance is subjected to a study for presence or absence of deafness-dystonia syndrome, cancer, Parkinson's disease, or Alzheimer's disease. A biological sample may be any ones insofar as it contains nucleic acids, and is exemplified by cells, bloods, cerebrospinal fluids, bronchoalveolar lavage fluids, expectorations, or other body fluids as well as biopsy tissues. Nucleic acid samples can be prepared from the biological samples for use. The nucleic acid samples can be prepared by well known nucleic acid preparation methods. The nucleic acid samples may be DNA or RNA. The nucleic acid samples prepared may be used directly for detection, or may be subjected to enzymatic amplification of predetermined region thereof by PCR or other amplification methods in advance for analysis.
In terms of a route of administration of the pharmaceutical composition, it may be either systemic administration or local administration. The route of administration that is appropriate for a particular disease, symptomatic condition, or other factors, should be selected. For example, parenteral administration including normal intravenous injection, intra-arterial administration, subcutaneous administration, intracutaneous administration, and intramuscular administration can be employed. Oral administration can be also employed. Further, transmucosal administration or transdermal administration can be employed.
Preferably the composition is adapted for oral administration, e.g. in the form of a tablet, coated tablet, dragee, hard or soft gelatin capsule, solution, emulsion or suspension. In general the oral composition will comprise from 1 mg to 400 mg of such agent. It is convenient for the subject to swallow one or two tablets, coated tablets, dragees, or gelatin capsules per day. However, the composition can also be adapted for administration by any other conventional means of systemic administration including rectally, e.g. in the form of suppositories, parenterally, e.g. in the form of injection solutions, or nasally.
The biologically active compounds can be processed with pharmaceutically inert, inorganic or organic carriers for the production of pharmaceutical compositions. Lactose, corn starch, or derivatives thereof, talc, stearic acid or its salts and the like can be used, for example, as such carriers for tablets, coated tablets, dragees and hard gelatin capsules. Suitable carriers for soft gelatin capsules are, for example, vegetable oils, waxes, fats, semi-solid and liquid polyols and the like. Depending on the nature of the active ingredient no carriers are, however, usually required in the case of soft gelatin capsules, other than the soft gelatin itself. Suitable carriers for the production of solutions and syrups are, for example, water, polyols, glycerol, vegetable oils and the like. Suitable carriers for suppositories are, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols and the like.
The pharmaceutical compositions can, moreover, contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, coating agents or antioxidants. They can also contain still other therapeutically valuable substances, particularly antidiabetic or hypolipidemic agents that act through mechanisms other than those underlying the effects of the compounds of the invention. Agents which can advantageously be combined with compounds of the invention in a single formulation include but are not limited to biguanides such as metformin, insulin releasing agents such as the sulfonylurea insulin releaser glyburide and other sulfonylurea insulin releasers, cholesterol-lowering drugs such as the “statin” HMG-CoA reductase inhibitors such as atrovastatin, lovastatin, pravastatin and simvastatin, PPAR-alpha agonists such as clofibrate and gemfibrozil, PPAR-gamma agonists such as thiazolidinediones (e.g. rosiglitazone and pioglitazone, alpha-glucosidase inhibitors such as acarbose (which inhibit starch digestion), and prandial insulin releasers such as repaglinide. The amounts of complementary agents combined with compounds of the invention in single formulations are in accord with the doses used in standard clinical practice. Established safe and effective dose ranges for certain representative compounds are set forth above.
The invention is described in more detail in the following illustrative examples. Although the examples can represent only selected embodiments of the invention, it should be understood that the following examples are illustrative and not limiting.
EXAMPLESThe following examples illustrate, but not limit, the embodiments of the invention.
Example 1 Studies on Substrate Specificity of the TIM22 Mitochondrial Import Pathway Revealed with Small Molecule Inhibitor of Protein Translocation SummaryThe TIM22 protein import pathway mediates the import of membrane proteins into the mitochondrial inner membrane and consists of two intermembrane space chaperone complexes, the Tim9-Tim10 and Tim8-Tim13 complexes. To facilitate mechanistic studies, we developed a chemical genetic approach to identify small molecule agonists that caused lethality to a tim10-1 yeast mutant at the permissive temperature. One molecule, MitoBloCK-1, attenuated the import of the carrier proteins including the ADP/ATP and phosphate carriers, but not proteins that used the TIM23 or the Mia40/Erv1 translocation pathways. MitoBloCK-1 impeded binding of the Tim9-Tim10 complex to the substrate during an early stage of translocation, when the substrate was crossing the outer membrane. As a probe to determine the substrate specificity of the small Tim proteins, MitoBloCK-1 impaired the import of Tim22 and Tafazzin, but not Tim23, indicating that the Tim9-Tim10 complex mediates the import of a subset of inner membrane proteins. MitoBloCK-1 also inhibited growth of mammalian cells and import of the ADP/ATP carrier, but not TIM23 substrates, confirming that MitoBloCK-1 can be used to understand mammalian mitochondrial import and dysfunction linked to inherited human disease. Our approach of screening chemical libraries for compounds causing synthetic genetic lethality to identify inhibitors of mitochondrial protein translocation in yeast validates the generation of new probes to facilitate mechanistic studies in yeast and mammalian mitochondria.
The mitochondrion has an outer (OM) and inner (IM) membrane that separates the matrix from the intermembrane space (IMS). The mitochondrion has developed an elaborate translocation system to orchestrate the import and subsequent sorting of proteins to the correct compartment (1). Proteins destined for the mitochondrion, termed precursors until they reach their correct location, utilize Translocase of the Outer Membrane (TOM) and Translocase of the Inner Membrane (TIM) complexes, TIM23 and TIM22, to cross the OM and IM, respectively. Proteins with a typical N-terminal targeting sequence use the TIM23 translocation system, whereas proteins destined for the IM use the TIM22 translocation system.
Components of the TIM22 translocation system include the small Tim proteins, Tim8, Tim9, Tim10, Tim12, and Tim13, and the membrane components Tim18, Tim22, and Tim54. The small Tim proteins assemble in 70-kDa hexameric complexes (referred to as small Tim complexes) in the IMS in which three Tim9 polypeptides partner with three Tim10 polypeptides, and three Tim8 polypeptides partner with three Tim13 polypeptides. Structural studies reveal that the overall structure is similar to that of the Skp and prefoldin chaperones (2), although the sequences are not conserved. The small Tim proteins function as chaperones to maintain the hydrophobic membrane proteins in an import competent state (3, 4). The 300-kDa insertion complex in the IM consists of a fraction of Tim9 and Tim10 with Tim12, Tim22, Tim18, and Tim54. The small Tim proteins escort substrates to the insertion complex, which mediates protein insertion into the membrane.
Substrates of the TIM22 complex include the carrier proteins such as the ADP/ATP carrier (AAC) and the phosphate carrier (PiC) and IM proteins Tim17, Tim22, and Tim23. In addition, the small Tim proteins facilitate the insertion of outer membrane proteins Tom40 and porin and the cardiolipin remodeling enzyme Tafazzin (5-7). The substrates cross the TOM complex as a loop in an unfolded state and then the small Tim proteins bind to the substrate at an early stage of translocation (4, 8, 9).
The Tim8-Tim13 and Tim9-Tim10 complexes display different substrate binding preferences. The Tim9-Tim10 complex can be efficiently cross-linked to carrier proteins and the import components Tim17, Tim23, and Tim22 (10-12). The Tim8-Tim13 complex can be cross-linked to Tim23 and the aspartate-glutamate carriers (10-13). Mutations in the human homolog of Tim8, DDP1, cause the X-linked disease deafness-dystonia syndrome (14, 15), and the disease may be caused by a decrease in specific IM proteins (13). Therefore, understanding the substrate specificity of the small Tim proteins is important for understanding the molecular basis of deafness-dystonia syndrome.
Mitochondrial assembly has been studied extensively using classical yeast genetics and biochemical assays with purified mitochondria. However, new strategies are needed to elucidate the details of protein translocation and its role in development and human disease. Important questions about the substrate specificity of the small Tim proteins and the mechanism by which the small Tim proteins bind substrate have not been resolved. These studies would be facilitated by drug-like inhibitors that modulate protein import. Here we report the development of a small molecule screening approach to identify inhibitors of the TIM22 import pathway. Taking advantage of our large collection of temperature-sensitive mutants for the TIM22 import pathway, we conducted a chemical genetic screen with a tim10-1 mutant to identify small molecules that caused a synthetic lethality at the permissive temperature of 25° C. (16-19). Our results indicate that a new set of tools for mechanistic studies in protein translocation can be developed and may be useful for characterizing protein translocation in mammalian mitochondria, where tools are lacking
Results A Screen to Identify Inhibitors of Mitochondrial Protein TranslocationWe exploited a large collection of temperature sensitive mutants for the TIM22 import pathway (10, 16-18) and developed a composite synthetic lethal screen to identify small molecule inhibitors that blocked the TIM22 import pathway (19). The tim10-1 mutant was used as the starting strain (16); the strains used in this study are described in Table S1. The rationale in this screen was that small molecules might be identified that target the mutant Tim10 protein or other components of the TIM22 pathway and thereby cause lethality of the tim10-1 mutant at the permissive temperature of 25° C. This approach uses the well characterized synthetic growth defects of the tim10-1 mutant to guide the design of cells genetically sensitized for inhibition of the TIM22 pathway.
To generate a suitable strain for screening, genes for the multidrug resistance pumps PDR5 and SNQ2 were disrupted to increase the steady state intracellular concentration of the drugs in yeast (19). The tim10-1 mutant grew similar to the parental strain (designated TIMID) at 25° C. but failed to grow at the restrictive temperature of 37° C. (
For subsequent testing of the compounds in biochemical assays with isolated mitochondria, a suppressor strain, designated tim10-1 tim9S, was used because growth of the tim10-1 mutant (
The screen was conducted with an integrated robotic system with plate scheduling. Briefly, diversity oriented commercial libraries of drug-like compounds from Chembridge and Asinex were screened against the tim10-1 strain at a concentration of approximately 10 μM. The screen encompassed a total of approximately 50,000 compounds dissolved in DMSO. Yeast in YPD medium was aliquoted into 384-well plates followed by compound addition with robotic pinning into the assay wells. DMSO was the vehicle for the small molecules, and several plate columns that contained only 1% DMSO were included as a control with the pinned compounds. As a negative control for growth, wells pinned with the mitochondrial uncoupler CCCP, which caused lethality, were also included. After 2 days of incubation at 25° C., cultures in each well were measured for optical density (O.D.) as a measure of growth. A typical reading for the positive control was OD600=0.8. Wells in which the growth was inhibited by >50% were deemed as potential inhibitors and chosen for further analysis. Approximately 600 inhibitors from the primary screen were selected for hit confirmation and secondary screens.
To identify possible specific inhibitors of mitochondrial protein translocation from the pool of hit compounds, two counter screens were executed. In the first round, the initial hit compounds were incubated with the tim10-1 mutant and the isogenic control strain carrying an integrated version of the TIM10 gene at the leu2 locus. Small molecules that inhibited growth of the mutant but not the control strain at 10 μM were advanced to the second counter screen. In a second round, compounds were assayed for selective growth inhibition of the tim10-1 mutant, but not the tim10-1 mutant harboring a plasmid containing the wild-type TIM10 gene. The second counterscreen was a test for chemical genetic rescue. Compounds that showed inhibition of only the tim10-1 mutant in both counter screens were dubbed “MitoBloCK” compounds based on their potential to inhibit protein translocation in mitochondria. Of 25 potential “lead” inhibitors, MitoBloCK-1 was chosen for additional analysis.
MitoBloCK-1 Inhibits Protein Import of TIM22 Substrates into Mitochondria
MitoBloCK-1 is a tetrahydrodibenzofuran derivative that was identified from the Chembridge library (
The ability of MitoBloCK-1 to inhibit import of mitochondrial precursors was tested using the in vitro import assay with radiolabeled substrates. For this analysis, mitochondria from the tim10-1 tim9S strain were used because MitoBloCK-1 inhibited growth of this strain (Table 1) and import of the model substrate, AAC, was restored in comparison to the tim10-1 mutant (
MitoBloCK-1 also inhibited the import of an additional carrier protein, the phosphate carrier (PiC), and the outer membrane protein Tom40, which requires the small Tim proteins for import (7) (FIG. 3B,C). However, for fusion constructs Su9-DHFR and cyt b2-DHFR as well as Hsp60 that use the TIM23 pathway, MitoBloCK-1 did not impair import (
MitoBloCK-1 does not Nonspecifically Damage Mitochondria
A potential mechanism by which MitoBloCK-1 may inhibit protein translocation indirectly is by the disruption of oxidative phosphorylation or dissipation of the membrane potential. We therefore used a battery of tests to determine if MitoBloCK-1 nonspecifically altered mitochondrial integrity or function. As a first test, the ability of MitoBloCK-1 to interfere with respiration was measured (
The membrane potential (Δψ) of mitochondria was measured with the fluorescent dye rhodamine 123, which is taken up by mitochondria and then released when the Δψ is dissipated (22, 23). The relative change of fluorescence between dye uptake and release is a relative measure of the Δψ; the dye that loads into coupled mitochondria (causing quenching and a decrease in fluorescence) is released when treated with an uncoupling agent such as CCCP (causing an increase in fluorescence). The fluorescence did not change with addition of either DMSO (
Another potential mechanism that may alter protein translocation is that the small molecules may nonspecifically permeabilize mitochondrial membranes, and proteins may be released from the mitochondrion, particularly those in the IMS. We therefore incubated mitochondria with MitoBloCK-1 for 30 min followed by centrifugation (FIGS. 10F,G). Released proteins were recovered in the supernatant fraction and analyzed by immunoblot assays for key proteins and Coomassie staining for the collective release of proteins. As a positive control, MitoBloCK-2, another compound from the screen that permeabilized mitochondrial membranes, was included. Immunoblots revealed that the release of marker proteins Tom40 (OM), cytochrome c and Tim10 (IMS), AAC (IM), and Hsp60 (matrix) was similar when mitochondria were treated with MitoBloCK-1 or DMSO (
MitoBloCK-1 can be used for mechanistic studies in protein translocation. From our previous analysis of the tim10-1 and tim12-1 mutants, we showed that Tim10 was required to mediate translocation of AAC across the outer membrane and Tim12 was required at a later step to mediate insertion of the AAC into the IM (16); this analysis was determined by monitoring protease sensitivity of the AAC precursor. We adapted this methodology to determine where MitoBloCK-1 impaired AAC translocation. In wild-type mitochondria, a small fraction of the AAC was trapped in the IMS when protease was added to mitochondria in the absence of a membrane potential (
The early obstruction in protein translocation by MitoBloCK-1 suggested that binding between the Tim9-Tim10 complex and substrate might be abrogated. We have previously used a cross-linking and immunoprecipitation approach in tim10-1 tim9S mitochondria to show that Tim9 binds to substrate during translocation (18). MitoBloCK-1 was therefore added to import assays that were subjected to cross-linking and immunoprecipitation (
A central question about the TIM22 pathway has been the specificity of the small Tim complexes. Yeast contain both the Tim8-Tim13 complex and the Tim9-Tim10 complex and a variety of studies have suggested that they might have different substrate specificities (10, 11, 13). Most precursors including the carriers, Tim22, and Tim17 require the Tim9-Tim10 complex, whereas Tim23 and the aspartate-glutamate carriers require the Tim8-Tim13 complex. In addition, the small Tim proteins facilitate the import of outer membrane proteins (5, 7). We therefore examined whether MitoBloCK-1 could be used to determine substrate specificity of the Tim9-Tim10 complex with precursors Tim22, Tim23, and Tafazzin (
Taking advantage of commercially available compounds similar to MitoBloCK-1, we purchased additional compounds for an abbreviated structure-activity relationship (SAR) study (
The long-term goal with these MitoBloCK compounds is to develop small molecules that inhibit protein translocation in mammalian systems for mechanistic studies and for developing tools to alter mitochondrial function with the objective of developing disease models. As a first step, we tested whether MitoBloCK-1 might affect general mitochondrial function in mammalian cells and measured cell viability in mammalian cells using an MTT assay (
MitoBloCK-1 is the first small molecule inhibitor that blocks the import of substrates that use the TIM22 import pathway. We started this screen with a genetic approach by developing a composite synthetic lethal screen to identify small molecules that inhibited growth of the tim10-1 mutant at the permissive temperature of 25° C. Although MitoBloCK-1 may have many potential targets within a yeast cell, we devised a battery of tests using growth analyses followed by biochemical assays to determine the specific site of inhibition by MitoBloCK-1. Because the small molecules may nonspecifically alter mitochondrial function, we determined its effect on membrane potential, respiration, and mitochondrial integrity; MitoBloCK-1 does not generally damage mitochondria. Moreover, import assays showed that import of TIM22 substrates was specifically inhibited and crosslinking and immunoprecipitation assays showed that the Tim9-Tim10 complex did not bind to substrate effectively. The combination of these assays indicated that MitoBloCK-1 inhibits an early step in protein translocation, when the Tim9-Tim10 complex binds to substrate during translocation across the outer membrane (
The characterization of MitoBloCK-1 supports that the chemical-genetic approach is important for developing probes to study assembly of mitochondrial membranes. Mechanistic studies for the assembly of outer and IM proteins still need refinement (1). Our analysis shows that Tim9-Tim10 is important for the import of Tafazzin, Tom40, the carrier proteins, and Tim22, but not Tim23, which supports that the small Tim complexes have different substrate specificity (3, 4, 10, 13). Therefore, development of these probes will yield a new set of tools for studying mitochondrial membrane biogenesis.
A potential drawback of MitoBloCK-1 is that import is inhibited in the tim10-1 tim9S mitochondria but not wild-type mitochondria. The small SAR studies suggest that particular properties of MitoBloCK-1, such as the length of the side chain and the dihydrobenzofuran ring, may be important for its function. Therefore MitoBloCK-1 may serve as a starting point for developing more potent analogs that inhibit protein import in wild-type yeast mitochondria. In addition, the overall structure of the human small Tim proteins is highly conserved with the yeast homologs (2), and we clearly show that import into isolated mammalian mitochondria is inhibited. Following the initial import assays in mammalian mitochondria with an extended SAR approach may lead to the refinement of small molecules that inhibit function of the different mammalian small Tim proteins.
Mitochondria now have been implicated in a wide array of degenerative diseases including Parkinson's and Alzheimer's (27-30). For example, a defect in import has been linked to Alzheimer's when the amyloid precursor protein arrests in the Tom40 translocon (30). These latest developments indicate that alteration of protein translocation pathways may be important for (1) mechanistic studies in these diseases and (2) to create model systems to recapitulate the disease. Thus, having new and specific tools available such as the MitoBloCK compounds may be important for broad research in understanding how mitochondrial dysfunction contributes to disease. The development of small molecule inhibitors also serves as a technological advance over general mitochondrial inhibitors (uncouplers and inhibitors of OXPHOS) that uncouple mitochondria or irreversibly inhibit respiration.
Materials and Methods Plasmids and Strains.In general, a standard set of genetic and molecular techniques were used to generate the strains in this study (31, 32). Screening strains were generated based on previously characterized temperature sensitive mutants (see supplementary table 1). The snq1 and pdr5 deletions were introduced in each strain by strain mating with MDY326 or PCR-mediated deletion (33, 34). Overexpression strains were generated by transforming 2μ yeast shuttle vectors carrying the gene of interest with the native promoter into the tim10-1 strain using a standard LiCl protocol (35). Transformed yeast was maintained on selective media appropriate for the plasmid's auxotrophic marker. Strains lacking mitochondrial DNA (rho null) were generated by two rounds of selection of the parent on YPD plates supplemented with ethidium bromide (40 μg/ml) followed by two rounds of single colony selection on YPD plates.
High-Throughput Screening.A primary screen was performed using freshly streaked tim10-1 diluted in YPD to an OD600 of approximately 0.0002 and kept on ice throughout the screening run. A Titertek multidrop (Huntsville, Ala.) was used dispense 40 μL of cell suspension to all wells of each clear 384-well plate (Greiner Bio One). After yeast suspension warmed to room temperature, a Biomek FX (Beckman Coulter) was used to pin transfer 0.5 μL of compound from 1 mM stock or DMSO to respective wells. Approximate screening concentration was 12.5 μM. All operations were performed by an automated plate scheduler to ensure consistency across the screening run. After completed compound transfer, all plates were incubated at 25° C. in a humidified incubator until the OD600 reached approximately 0.8 in the control wells; the control consisted of the tim10-1 mutant with the vehicle 1% DMSO. Each plate was shaken in a Beckman orbital shaker to resuspend settled cells, and the OD600 in each well was read by a Wallac Victor plate reader (Perkin Elmer). The top 600 growth inhibitory compounds were determined and assembled into two plates. Using a similar screening methodology, hit compounds were reconfirmed with the tim10-1 strain and growth inhibition was compared to the WT strain (TIM10) as well as the “rescued” strain (tim10-1 TIM10 that contained a copy of the wild-type TIMID genes on a centromeric plasmid) strains. Compounds reordered from Asinex and Chembridge were assayed for MIC50 using a similar automated technique in 384-well plates as previously described. Serial dilutions of purchased compounds were performed with robotic automation in 100% DMSO. Subsequently, compounds were pinned into assay plate wells containing 50 μL of the respective yeast strain in YPD medium (starting OD600=0.0002). Growth duration and conditions were similar to the original screen.
Biochemical Assays with Mitochondria and Additional Methods.
Media and Reagents. Media used in this study was purchased from EMD Biosciences and US Biological. Chemical reagents were from Chembridge, Asinex, and Sigma unless otherwise noted. YPD medium is 1% Bacto-yeast extract, 2% Bacto-peptone, dextrose added to 2% after sterilization. Yeast cultures for mitochondrial preparation and were either in YPEG (1% yeast extract, 2% peptone, 3% glycerol, 3% ethanol) or selective SEG medium (0.17% yeast nitrogen base, 0.5% ammonium sulfate, 3% glycerol, 3% ethanol) with appropriate amino acid dropout mixture. YPD and YPEG plates used in growth analysis included 2% agar. For MTT assays, cultured HeLa were grown in DMEM high glucose medium (Invitrogen) with glutamine, sodium pyruvate, 10% FBS, and penicillin-streptomycin (complete medium).
Analysis and Statistics.
Unless otherwise stated, all results reported are representative of three experimental replicates. Quantitative analysis was performed in GraphPad Prism 5 software (GraphPad Software, Inc.) unless otherwise stated. Statistical tests for significant deviation between samples were performed with unpaired, two-tailed t tests. The alpha threshold for significance was <0.05 for all tests. In graphs, error bars represent standard deviation from a given mean. Data transformation of rhodamine 123 fluorescence data was performed by setting the maximum OD=530 nm value from a particular trace to 100%. All fluorimetry data was scaled to the 0-100% range using GraphPad Prism's “normalize” function.
Purification of Mitochondria.
Mitochondria were purified from yeast cells grown in YPEG or selective SEG medium as described in previous studies (Glick B S, Pon L A (1995). Methods Enzymol 260:213-223). Yeast cultures were kept at a constant 25° C. with vigorous shaking during growth. After concentration was measured by BCA assay, mitochondria were stored in 25 mg/ml aliquots at −80° C. Mammalian mitochondria were isolated from 2-4 freshly excised mouse livers by differential centrifugation. Briefly, isolated livers were washed 3× with cold PBS and then suspended in 4-5 mL isolation buffer (70 mM Sucrose, 220 mM mannitol, 2 mM HEPES-KOH, pH 7.4) per gram of tissue. Livers were first chopped into small pieces and then dounced 5× using a teflon dounce. Homogenized material was then centrifuged at 1,000 RPM for 10 mm in a clinical centrifuge and supernatants were transferred to fresh tubes. Centrifugation at 1,000 RPM for 10 min was then repeated and supernatants were transferred to microfuge tubes. Next, supernatant material was spun for 10 min at 800×g (this process was repeated a second time). The supernatants from these steps were subjected to a high-speed (12,000×g for 20 min) spin to pellet heavy membrane fractions. Pellets were washed in isolation buffer and spun again (12,000×g for 20 min). After the final centrifugation step, supernatants were discarded and heavy membrane fraction was resuspended in mammalian import buffer (250 mM sucrose, 5 mM magnesium acetate, 80 mM potassium acetate, 10 mM sodium succinate, 1 mM dithiothreitol, 0.1 mM ADP, 20 mM Hepes-KOH, pH 7.4) as described in Johnston A J, et al. (2002), J Biol Chem 277:42197-42204 and kept on ice. All subsequent imports were performed within 1 hour of isolation of mammalian mitochondria. Mitochondrial concentrations were determined by BCA assay.
Blue Native Gel Electrophoresis.
Steady-state levels of the small Tim complexes were analyzed from mitochondria isolates from TIA110, titn10-1, and titn10-1 titn9S strains following established methods (Murphy M P, et al., (2001), Mol Cell Biol 21:6132-6138). Approximately 200 μg of mitochondria from each strain was solubilized at 5 mg/mL in 0.16% n-dodecylmaltoside (Anatrace) for 30 minutes on ice. Following removal of insoluble material (30 minute centrifugation at 14,000 RPM), solubilized protein supernatants were analyzed by blue native gel electrophoresis on a 6 to 16% linear polyacrylamide gradient (Dekker P J, et al., (1996), Biol Chem 377:535-538; Schagger H, et al., (1994)m Anal Biochem 217:220-230; and Schagger H, von Jagow G (1991), Anal Biochem 199:223-231).
Import of Radiolabeled Proteins into Mitochondria and Crosslinking.
Prior to import into purified mitochondria, 35S-methionine and cysteine labeled proteins were generated with TNT Quick Coupled Transcription/Translation kits (Promega) and plasmids carrying the gene of interest. Transcription of genes was driven by either a T7 or SP6 promoter. Import reactions were conducted according to established methods. After frozen mitochondria aliquots were thawed and added to the import buffer at a final concentration of 100 μg/mL, drug or DMSO vehicle was added as indicated. A final vehicle concentration of 1% was used in all experiments. Following 15 minute incubation at 25° C., import reactions were initiated by the addition of 5-10 L of translation mix. Aliquots were removed at intervals during the reaction timecourse and import was terminated with either cold buffer, 25 μg/mL trypsin, or a combination of both. If trypsin was added to digest unimported precursor protein, soybean trypsin inhibitor (STI) was subsequently added in excess after 15 minute incubation on ice. After a final recovery of by centrifugation (8,000×g, 5 minutes), mitochondria were disrupted in Laemmli sample buffer. Imports of membrane proteins (AAC, PiC, Tom40, Tim22, and Tim23) included a carbonate extraction step to remove proteins that had not inserted into the membrane (Koehler C M, et al. (1998), Science 279:369-373). Samples from import reaction time points were resolved by SDS polyacrylamide gel electrophoresis (SDSPAGE) and gels were dried prior to exposing to film.
Crosslinking and immunoprecipitation experiments were derived from procedures previously utilized (3) with the inclusion of MitoBloCK-1 or DMSO. Following import, a portion of the reaction was subjected to crosslinking with 0.5 mM bis-maleimidohexane (BMH) for 30 minutes on ice. After quenching crosslinking reactions with 1 mM f3-mercaptoethanol, a fraction of each sample was subjected to immunoprecipitation with polyclonal antibodies against either Tim9, Tom22, or Tom40. For each immunoprecipitation, 20′IL of antisera was bound to 50 j.t1_, of protein A-sepharose slurry according to established protocols (Murphy M P, et al., (2001), Mol Cell Biol 21:6132-6138).
Membrane Potential and Oxygen Consumption.
Oxygen consumption of tim10-1 tim9S mitochondria was measured using methods previously described (8). Briefly, purified tim10-1 tim9S mitochondria aliquots (25 mg/mL) were thawed on ice and tested within 2 hours. A Clark-type oxygen electrode in a stirred, thermostatically controlled 1.5-ml chamber at 25° C. (Oxytherm; Hansatech) facilitated measurement. State II respiration was induced on a suspension of 100 μg/mL mitochondrial in 0.25 M sucrose, 20 mM KCl, 20 mM Tris-C1, 0.5 mM EDTA, 4 mM KI-l2PO4, and 3 mM MgCl2, pH 7.2 after adding 2 mM NADH. Consumption rate was monitored for approximately 2 min. Drug or DMSO was then added to a final vehicle concentration of 1% and respiration was measured for another approximately 1.5 minutes. Uncoupled respiration was achieved by adding 10 μM CCCP to the chamber.
Membrane potential measurement assays were conducted with a SPEX spectrofluorometer system (HORIBA Jobin Yvon) with a magnetically stirred cuvette held at 25° C. The quenching of rhodamine 123 fluorescence (Em, =530 nm and Ex=485 nm) was used as previously described (9) to detect changes in mitochondrial membrane potential. Purified tim10-1 tim9S mitochondrial aliquots were thawed and resuspended in respiration buffer (0.65 M mannitol, 0.3 mM EGTA, 3 mm tris-phosphate, 10 mM tris-maleate, pH 6.75). Trials were started by adding 100 nM of rhodamine 123 to respiration buffer. After a period of signal stabilization, mitochondria were added to a concentration of 100 μg/mL. Either drug or DMSO was then added to a final vehicle concentration of 1% following the establishment of baseline quenched fluorescence. Finally, mitochondria were uncoupled with 3 μM CCCP.
Cell Viability Assays.
Measurements of cell viability/toxicity were made with a MTT based toxicology assay kit (Sigma-Aldrich). HeLa cells were grown in 24-well tissue culture dishes to 80% confluency. Following this cells were either left untreated or treated with 1% DMSO or drug in complete medium for 12 hours. Following drug treatment, cells were rinsed with phosphate buffered saline and incubated with complete media with MTT solution supplement for additional 4 hours as described in manufacturer's protocols. This media was removed and 500 μL of MTT solubilization solution was added to dissolve the formazan crystals. The formazan absorbance was measured at OD=570 nm on a Wallac Victor plate reader (Perkin Elmer) along with a turbidity measurement at OD=630 nm. After turbidity subtraction, the percent viability of each cell sample was calculated as: [(absorbance of vehicle treated cells−absorbance of drug treated cells)/(absorbance of vehicle treated cells)]×100.
Miscellaneous.
Steady-state levels of mitochondrial proteins from lysed aliquots of isolated mitochondria were resolved using SDS-PAGE. Western blotting was performed using standard protocols with polyclonal antibodies raised towards highly purified antigens. Proteins were transferred to nitorocellulose membranes and immune complexes were visualized with HRP labeled Protein A in a chemiluminescence assay (Pierce). Chemiluminescent and autoradiographic imaging was performed on film unless otherwise noted. Unless otherwise stated, all results reported are representative of three experimental replicates.
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The mitochondrial disulfide relay system of Mia40 and Erv1/ALR facilitates import of the small Tim proteins and cysteine rich proteins. A chemical screen identified small molecules that inhibit Erv1 oxidase activity, thereby facilitating dissection of the disulfide relay system in yeast and vertebrate mitochondria. One molecule, MitoBloCK-6, attenuated the import of Erv1 substrates into yeast mitochondria and inhibited oxidation of Tim13 and Cmc1 in in vitro reconstitution assays. In addition, MitoBloCK-6 revealed an unexpected role for Erv1 in the carrier import pathway, namely transferring substrates from the TOM complex onto the small Tim complexes. Cardiac and somite development was impaired in MitoBloCK-6 exposed zebrafish embryos. Finally, MitoBloCK-6 induced apoptosis via cytochrome c release in human embryonic stem cells (hESCs) but not in differentiated cells, suggesting an unprecedented function for ALR in hESC homeostasis. Our target-based chemical screen validates this approach for generating newtools to dissect the mitochondrial redox system in vertebrates.
IntroductionThe mitochondrion has translocons of the outer membrane (TOM) and inner membrane (TIM) to import proteins from the cytosol. Proteins with a typical N-terminal targeting sequence are imported via the TIM23 pathway, whereas polytopic inner membrane proteins use the TIM22 import pathway (Chacinska et al., 2009; Mokranjac and Neupert, 2009; Schmidt et al., 2010). In contrast, most of the proteins imported into the intermembrane space (IMS) lack a mitochondrial targeting sequence and employ diverse routes for mitochondrial import (Herrmann and Hell, 2005).
A recently identified pathway in the IMS mediates oxidation of imported proteins that require disulfide bonds to acquire their native conformation (Deponte and Hell, 2009; Koehler and Tienson, 2009; Riemer et al., 2011; Sideris and Tokatlidis, 2010; Stojanovski et al., 2008b), such as the small Tim proteins and proteins with a twin CX9C motif (Cavallaro, 2010). In the small Tim proteins, the proximal N-terminal cysteine residues serve as internal targeting sequences that are recognized by the IMS oxidoreductase Mia40 (Milenkovic et al., 2009; Sideris et al., 2009), which functions as a receptor to mediate translocation across the outer membrane (Chacinska et al., 2004). Mia40 contains a redox-active cysteine pair that is maintained in an oxidized state by the sulfhydryl oxidase Erv1 (Tienson et al., 2009). As the imported protein substrate is oxidized, electrons are passed from Mia40 to Erv1, followed by transfer to molecular oxygen or cytochrome c (cyt c) (Bien et al., 2010; Dabir et al., 2007). Subsequently, cyt c can be reoxidized by cyt c oxidase of the respiratory chain (Bien et al.) or by cyt c peroxidase (Dabir et al., 2007). Thus, Mia40 and Erv1 constitute a mitochondrial disulfide relay system that is also evolutionarily conserved.
Erv1 belongs to the Erv/ALR sulfhydryl oxidase family and homologous proteins are found in the endoplasmic reticulum (Erv2) of yeast, in the extracellular environment (Quiescin sulfhydryl oxidase, QSOX), and in the poxvirus family (E10R) (Gerber et al., 2001; Senkevich et al., 2002; Thorpe et al., 2002). In addition to protein translocation, the role of Erv1 in various cellular pathways is exemplified by a number of defects observed in cells that lack functional Erv1 protein. For example, Erv1 is required for the maturation of cytosolic iron-sulfur cluster containing proteins (Lange et al., 2001). In ervl mutant yeast, heme maturation is impaired (Dabir et al., 2007). Also, mutations in mammalian Erv1 homolog, ALR, result in an autosomal-recessive myopathy (Di Fonzo et al., 2009), and ALR has an essential pro-survival role in the maintenance of pluripotent murine embryonic stem cells (Todd et al., 2010b).
Erv1 has several key functions in the IMS, necessitating the characterization of its homolog, ALR, to uncover basic mechanisms in mitochondrial assembly in vertebrate systems. Because Erv1 donates electrons to cyt c, Erv1/ALR may have a central role in apoptotic pathways that lead to cyt c release (Dabir et al., 2007). Classically, mitochondrial protein import has been studied using yeast genetics and biochemical assays. However, new approaches are needed to elucidate disease mechanisms and dissect essential functions in mammalian cells. Here we report a small molecule screening approach to identify Erv1 inhibitors, with the goal of developing a set of probes that can modulate the pathway quickly and recapitulate disease phenotypes. We have taken advantage of the previously developed in vitro Amplex Red assay for monitoring Erv1 activity to identify inhibitors (Dabir et al., 2007). Our results indicate that the small drug-like inhibitor characterized here is specific for Erv1/ALR and can be used to reveal normal functions and disease mechanisms in mammalian mitochondria.
Materials and Methods High-Throughput Screen for Erv1 Modulators.The primary chemical screen used fresh recombinant Erv1 (in buffer 30 mM Hepes, pH 7.4, 100 mM NaCl, 1 mM EDTA) at a concentration of 10 μM, which was expressed as described previously. A Titertek multidrop (Beckman Coulter) was used to dispense 25 μl Erv1 or 25 μl of catalytically inactive enzyme Erv1C133S into wells of a clear bottom 384-well plate (Greiner Bio One). A Biomek FX (Beckman Coulter) was used to pin transfer 0.5 μl of compound from 1 mM stock or DMSO to respective wells. Approximate screening concentration was 12.5 μM. After completed compound transfer, all plates were incubated at 25° C. in a humidified incubator for 1 hour. A Titertek multidrop was used to dispense 15 μl of Amplex Red-horseradish peroxidase (HRP) (Sigma) mix into all wells of the 384-well plate. The final concentration of Amplex Red and HRP were 46 μM and 0.092 U/ml, respectively. The Amplex Red-HRP solution was shielded from light during the entire experiment. The plates were incubated for an additional 10 min and then 15 μl of the substrate DTT (20 μM)
was added to initiate the reduction of O2 to H2O2. The plates were incubated for 12 minutes to achieve a maximal signal-to-noise ratio in the kinetic liner range. Plates were then read at an endpoint using an excitation wavelength of 545 nm and an emission wavelength of 590 nm. All operations were performed by an automated plate scheduler to ensure consistency across the screening run. We chose compounds that inhibited Erv1 activity by greater than 50%. Using a similar screening methodology as above, hit compounds were reconfirmed. Compounds that were available were ordered from Asinex and Chembridge and assayed for IC50 using a similar automated technique in 384-well plates as previously described. Serial dilutions of purchased compounds were performed with robotic automation in 100% DMSO. Subsequently, compounds were pinned into assay plate wells containing 10 μM Erv1, Erv2, or ALR.
For MIC50 analysis in the yeast strains, serial dilution of MitoBloCK-6 (0.5 μl) was pinned into assay plate wells containing 50 μl of yeast (Table 2) in rich ethanol-glycerol media (starting OD600=0.0002). Plates were then incubated at 25° C. in a humidified chamber for 40 hours. Each plate was shaken in a Beckman orbital shaker to resuspend settled cells, and the OD600 in each well was read by a Wallac Victor plate reader (Perkin Elmer).
Table 2. Strains Used in Example 2.To assess the effect of MitoBloCK-6 on H2O2 production, 25 μl of buffer (30 mM Hepes, pH 7.4, 100 mM NaCl, 1 mM EDTA) containing 2 μM, 5 μM, or 10 μM MitoBloCK-6 was aliquoted into assay well plate. 15 μl of the Amplex Red/HRP was added, and the plates were incubated at room temperature for 30 minutes. The reaction was initiated with addition of 800 nM H2O2 solution and the fluorescence was measured after 10 min.
AssaysMitoBloCK-6 was analyzed using a battery of established in vitro, yeast, mammalian cell-based, and zebrafish assays. These are described in detail in the Supplemental Data.
Plasmids and StrainsRecombinant Erv1 and Mia40 were expressed and purified under native conditions as described previously (Dabir et al., 2007; Tienson et al., 2009). Recombinant Tim13 was purified under denaturing conditions as described previously (Beverly et al., 2008). Recombinant Cmc1 was generously provided by Dr. Barrientos (Univ. of Miami). Recombinant long form ALR, residues 1 to 205, was purified under native conditions (Daithankar et al., 2010). Proteins Tim13, Mia40, Erv1 and Cmc1 were detected with polyclonal antibodies and immunoblot analysis. Table 2 lists the strains used in this study.
Media and ReagentsMedia used in this study was purchased from EMD Biosciences and US Biological. Chemical reagents were from Chembridge, Asinex, and Sigma unless otherwise noted. Yeast cultures for mitochondrial preparation were grown in YPEG (1% yeast extract, 2% peptone, 3% glycerol, 3% ethanol). For MTT assays and fluorescence microscopy, cultured HeLa and HEK293 cells were grown in DMEM high glucose medium (Invitrogen) with glutamine, sodium pyruvate, 10% FBS, and penicillin-streptomycin (complete medium).
Analysis and StatisticsUnless otherwise stated, all results reported are representative of three experimental replicates. Quantitative analysis was performed in GraphPad Prism 5 software unless otherwise stated. Statistical tests for significant deviation between samples were performed with unpaired, two-tailed t-tests. The alpha threshold for significance was <0.05 for all tests. In graphs, error bars represent standard error from a given mean.
Mass SpectrometryLC-MS experiments were carried out on a Waters Acquity UPLC connected to a Waters LCT-Premier XE Time of Flight Instrument controlled by MassLynx 4.1 software. The mass spectrometer was equipped with a Multi-Mode Source operated in the electrospray mode. Briefly, samples of MitoBloCK-6, ES-1, and ES-2 were separated using an Acquity BEH C18 1.7 um column (2.1×50 mm, Waters) and were eluted with a gradient of 0.5 mL/min water/acetonitrile with 2, 80, and 95% acetonitrile at 0.5, 2.5 and 3.5 min, respectively. Mass spectra were recorded from 80 to 2000 Daltons. All solvents were LC-MS/MS Grade and purchased from Fisher Scientific.
Purification of MitochondriaMitochondria were purified from yeast cells grown in YPEG as described in previous studies (Glick and Pon, 1995). Yeast cultures were kept at 25° C. with vigorous shaking during growth. Mitochondria concentration was measured by BCA assay and stored in 25 mg/ml aliquots at −80° C.
Import of Radiolabeled Proteins into Yeast Mitochondria
Prior to import into purified mitochondria, 35S-methionine and cysteine labeled proteins were generated with TNT Quick Coupled Transcription/Translation kits (Promega) and plasmids carrying the gene of interest. Transcription of genes was driven by either a T7 or SP6 promoter. Import reactions were conducted as previously described (Hasson et al., 2010). After frozen mitochondria aliquots were thawed and added to the import buffer at a final concentration of 100 μg/ml, MitoBloCK-6 or DMSO vehicle was added as indicated. A final concentration of 1% DMSO was used in all experiments. Following incubation at 25° C. for 15 min, import reactions were initiated by the addition of 5-10 μl of translation mix. Aliquots were removed at intervals during the reaction time course and import was terminated with addition either of cold buffer or 25 μg/ml trypsin, or the combination. If trypsin was added to digest non-imported precursor protein, soybean trypsin inhibitor was subsequently added in excess after 15 min incubation on ice. After a final recovery of by centrifugation (8,000×g, 5 min), mitochondria were disrupted in Laemmli sample buffer. Imports of membrane proteins (AAC and Tim23) included a carbonate extraction step to remove proteins that had not inserted into the membrane (Koehler et al., 1998a). Samples from import reaction time points were resolved by SDS-PAGE and visualized by autoradiography. Blue-native gel analysis was performed as described previously (Koehler et al., 1998b).
Oxygen Consumption MeasurementsOxygen consumption of WT mitochondria was measured using methods previously described (Claypool et al., 2008a). Briefly, purified WT mitochondria (25 mg/ml) were thawed on ice and tested within 2 hours. Oxygen consumption assays were performed with a Clark-type oxygen electrode in a stirred thermostatically controlled 1.5-ml chamber at 25° C. (Oxytherm, Hansatech). State II respiration was induced in a suspension of 100 μg/ml mitochondria in 0.25 M sucrose, 20 mM KCl, 20 mM Tris-C1, 0.5 mM EDTA, 4 mM KH2PO4, and 3 mM MgCl2, pH 7.2 after adding 2 mM NADH. Consumption rate was monitored for approximately 2 min. MitoBloCK-6 or DMSO was then added to a final vehicle concentration of 1% and respiration was measured for another approximately 1.5 min. Uncoupled respiration was achieved by the addition of 10 μM CCCP to the chamber. For assessing succinate dehydrogenase activity, respiration was induced in a suspension of 200 μg/ml mitochondria in the buffer described above after addition of 10 mM succinate. Consumption rate was monitored for approximately 3 mins. MitoBloCK-6, SAR compounds, or DMSO was then added and respiration measured for another 3 mins. Uncoupled respiration was achieved as described above.
The effect of MitoBloCK-6 or vehicle on oxygen reduction by Erv1 was assayed with the Clark-type oxygen electrode in 1 ml of air-saturated Hepes buffer (pH 7.4) (Dabir et al., 2007) containing 100 mM NaCl and 0.5 mM EDTA. Oxygen consumption was initiated by addition of Erv1 to a final concentration of 2 μM in the reaction mixture containing 2 mM DTT. To test the effect of MitoBloCK-6 or vehicle, Erv1 was pre-incubated with the desired MitoBloCK-6 concentration for 2 min before addition of DTT.
Reconstitution StudiesReconstitution studies with reduced Tim13 were performed as described previously (Tienson et al., 2009). Briefly, 15 μM reduced Tim13 or reduced Cmc1 was incubated with 1 μM Mia40 and 1 μM Erv1 or ALR for 3 h at 25° C. Where indicated, Erv1 was pretreated with either vehicle or MitoBloCK-6 for 1 hr at 25° C. before adding to the reconstitution mix. H2O2 levels in the reconstitution assays were measured using the Amplex Red Hydrogen Peroxide/Peroxidase Assay kit according to the manufacturer's protocol (Dabir et al., 2007) (Invitrogen). In brief, Erv1 or ALR and Mia40 were mixed at concentrations mentioned above with 25 μl of the Amplex Red/horseradish peroxidase reaction mix. The reaction was initiated with the addition of reduced Tim13 or reduced Cmc1. The Erv1 or ALR-catalyzed reduction of O2 to H2O2 was measured by a FlexStation plate reader (Molecular Devices) controlled via the SoftMax Pro software package (Molecular Devices) for data acquisition. The reaction was performed at a shorter time period than the reconstitution assays because the Amplex Red assay is very sensitive (Dabir et al., 2007).
Cell ManipulationsFor microscopy experiments, HeLa or HEK293 cells were transiently transfected with Su9-EGFP (Lipofectamine, Invitrogen) at 80% confluency. 12 hours post transfection, cells were co-labeled with Mitotracker red CMXRos (Invitrogen) and visualized with a microscope (Axiovert 200M Carl Zeiss) using a Plan-Fluor 63x oil objective. Images were acquired at room temperature with a charge-coupled device camera (ORCA ER, Hamamatsu Photonics) controlled by Axiovision software (Carl Zeiss). Image files were processed by Photoshop software (Adobe). Membrane potential was disrupted with 20 μM CCCP (Sigma-Aldrich). For cyt c release assays, a cell fractionation kit (MitoSciences) was used. Briefly, HeLa or HEK293 cells were grown in 10 cm2 dishes to 80% confluency and then cells were treated with DMSO or MitoBloCK-6 in complete medium for 12-16 h. To induce apoptosis as a positive control, cells were treated with 1 μM of staurosporine for 4 h. Cells were fractionated to obtain cytosolic and mitochondrial fractions; 100 μg of each fraction was analyzed by SDS-PAGE. Blots were probed with ApoTrack cyt c apoptosis antibody cocktail (MitoSciences).
Measurements for cell viability were made with a MTT based toxicology assay kit (Sigma) as described previously (Hasson et al., 2010). Briefly, HeLa cells were grown in 24-well tissue culture dishes to 80% confluency. Cells were then treated with DMSO or MitoBloCK-6 in complete medium for 12 h and reacted with MTT solution supplement for additional 4 hr as described in manufacturer's protocols. Percentage viability of each cell sample was calculated as: [(absorbance of vehicle treated cells)−(absorbance of MitoBloCK-6 treated cells)/(absorbance of vehicle treated cells)]×100.
Assays in Embryonic Stem CellsHuman embryonic stem cell (hESCs) line hSF1 was cultured in Stem Pro SFM (Gibco) supplemented with 10 ng/ml bFGF on Matrigel (BD Biosciences) coated plates under 5% CO2, 95% air. Differentiation involved culturing cells in Stem Pro SFM with 10 μM retinoic acid (Acros Organics) for 4 days. Cells were treated with the specific concentration of the compounds or 1% DMSO as a control. For the induction of apoptosis, cells were exposed to 20 μM actinomycin D (Sigma) with or without 100 μM z-VAD-FMK (MP Biomedical). Following treatment, cells were fixed with 3.7% formaldehyde for indirect immunofluorescence study or lysed with Triton buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) for analysis by SDS-PAGE. Bright field images were acquired with Exi Blue (QImaging). Immunofluorescent images were acquired with a 63x oil immersion objective on an LSM 5 PASCAL Laser Scanning Microscope (Carl Zeiss). Antibodies against cyt c (BD Pharmingen), Tom20 (Santa Cruz), cleaved caspase-3 (Cell Signaling) and poly (ADP-ribose) polymerase (Cell Signaling) were purchased from the indicated vendors. Alkaline phosphatase activity staining was performed with the leukocyte alkaline phosphatase kit (Sigma) as per manufacturer's protocol. Coomassie brilliant blue staining was performed by staining cells with Coomassie brilliant blue solution (0.25% Coomassie brilliant blue R250, 45% methanol, 10% acetic acid) for 1 hour at room temperature. Cells were washed with phosphate-buffered saline followed by visualization as described above.
Zebrafish ManipulationsZebrafish displaying fluorescent hearts were derived from transgenic TL fish expressing a fusion of the CoxIV targeting sequence with DsRed regulated by a cmlc2 (cardiac myocyte light chain-2) promoter (Shu et al., 2007). Zebrafish used for o-dianisidine staining of red blood cells in DMSO and MitoBloCK-6 treated fish were albino lines generated from crosses of TL and Tü fish. Line AB were injected with the ALR morpholino. Lines were maintained in a 14-hr light/10-hr dark cycle and mated for one hour to obtain synchronized embryonic development. Embryos were grown for 3 hpf in E3 buffer (5 mM sodium chloride, 0.17 mM potassium chloride, 0.33 mM calcium chloride, 0.33 mM magnesium sulfate) and then incubated with E3 buffer supplemented with 1% DMSO or MitoBloCK-6 for 3 days at 28.5° C. Following treatment, embryos were imaged using a Leica MZ16F fluorescent stereoscope (TexasRed filter set) at 5× magnification. Alternatively, 3-day embryos were stained with o-dianisidine[40% (v/v) ethanol, 0.01 M sodium acetate, 0.65% hydrogen peroxide, 0.6 mg/ml o-dianisidine] and incubated for 15 min in complete darkness. Embryos were then washed with E3 buffer to remove residual stain and stereoscopically imaged under white light using a Leica S8AP0 at 1.575× magnification. For comparison, AB embryos at the one-cell stage were micro-injected with 4 ng of an ATG morpholino targeted to zebrafish ALR protein (GAGGGTTGCCAGATCTCTGTTAAAT) (SEQ ID NO:1). Embryos were allowed to mature to 2 dpf and then imaged like the MitoBloCK-6 treated embryos; embryos were imaged at day 2 because of concerns with morpholino dilution. Images were resized to 300 dpi without resampling using Adobe Photoshop software.
Results A Chemical Screen to Identify Inhibitors of Erv1 Oxidase ActivityWe previously developed an assay to test the sulfhydryl oxidase activity of recombinant Erv1 protein based on the oxidation of a non-physiologic substrate, DTT, which produces hydrogen peroxide (H2O2) (Dabir et al., 2007). H2O2 production was measured using a standard fluorometric assay with Amplex Red and horseradish peroxidase (HRP). The assay was adapted in high throughput format and a chemical screen was conducted on an integrated robotic system with plate scheduling (
To eliminate false positives, a counter screen was used to test whether the small molecule compounds directly inhibited the Amplex Red-HRP assay. H2O2 (800 nM) was reacted with Amplex Red-HRP in the presence of the small molecules; this is the approximate amount of H2O2 that was produced by Erv1 during the assay. Those compounds that did not inhibit the Amplex Red assay directly and showed >50% inhibition of Erv1 activity (˜29 compounds) were selected for additional characterization and designated as “MitoBloCK” compounds based on their potential to inhibit Erv1 activity. Of these potential “lead” inhibitors, MitoBloCK-6 was chosen for additional analysis.
MitoBloCK-6 is 2,4-dichloro-6-((((phenylamino)phenyl)imino)methyl)phenol) from the Chembridge library (
To determine whether MitoBloCK-6 generally impaired redox active enzymes, we investigated the oxidiative folding properties of protein disulfide isomerase (PDI). MitoBloCK-6 did not inhibit the ability of PDI to reduce insulin (
The import of Erv1 substrates was tested with an in organello import assay. Substrates included twin CX9C proteins (Mia40, Cmc1, Cox19, and Cox17), twin CX3C protein Tim8, and Erv1 (
To confirm specificity of MitoBloCK-6, we purchased two additional compounds, termed ES-1 and ES-2 (Erv1-SAR), for an abbreviated structure-activity relationship (SAR) study (
To confirm that mitochondrial Erv1 is the target of MitoBloCK-6, an increased abundance of Erv1 should require an increased MitoBloCK-6 concentration to inhibit protein import. Previously, we designed a yeast strain in which Erv1 with a C-terminal hexahistidine tag (designated ↑Erv1) was expressed from a high copy plasmid (Dabir et al., 2007). This strain contained an approximate 5-fold increase in Erv1 with no aberrant phenotypes detected. The import of Mia40, Cmc1, and AAC proteins was tested in isolated WT and ↑Erv1 mitochondria. For Mia40 and Cmc1, the concentration of MitoBloCK-6 that was required to inhibit import increased from 10 μM to 50 μM (FIGS. 19A,B). A similar trend was detected for AAC import, with a concentration increase from 15 μM to 30 μM (
To evaluate the cell-based activity of MitoBloCK-6, we also determined the MIC50 with the Δpdr5Δsnq2 yeast strain in which the genes for the multi-drug resistance pumps PDR5 and SNQ2 were disrupted in the wild-type strain (Duncan et al., 2007; Hasson et al., 2010). Deletion of these pumps increases the steady state intracellular concentration of drugs in yeast. The MIC50 was 15.2 μM (
A potential mechanism by which MitoBloCK-6 could alter protein translocation is to nonspecifically permeabilize membranes, resulting in the release of mitochondrial proteins, particularly from the IMS. We have previously shown that MitoBloCK-2, an inhibitor of the TIM22 import pathway, nonspecifically permeabilizes mitochondrial membranes (Hasson et al., 2010). We incubated energized mitochondria with 1% DMSO or MitoBloCK-6 followed by centrifugation. Released proteins were recovered in the supernatant fraction and analyzed by Coomassie staining for the collective release of proteins (
Another potential mechanism by which MitoBloCK-6 may disrupt protein translocation is indirect, by dissipation of the membrane potential (Δψ) or disruption of oxidative phosphorylation, both of which can be measured with a Clark-type oxygen sensing electrode (
To determine how MitoBloCK-6 inhibited Erv1 function, we investigated whether MitoBloCK-6 altered Erv1 interactions with partner proteins in isolated mitochondria (
If MitoBloCK-6 interferes with Mia40-Erv1 binding, then the oxidation of substrates may be inhibited in vitro. We therefore evaluated Tim13 oxidation and subsequent production of H2O2 in vitro (
As an additional test for MitoBloCK-6 inhibition of Erv1 oxidase activity, we measured the oxygen consumption rate by Erv1 with an oxygen electrode in the presence of excess DTT (Dabir et al., 2007). When Erv1 was added alone or with DMSO, the oxygen consumption rate was similar (
The long-term goal in developing the MitoBloCK compounds is to adapt them for studies in vertebrate mitochondria, such as recapitulating biochemical phenotypes similar to those in cells derived from patients with mutations in ALR (Di Fonzo et al., 2009). In addition, MitoBloCK-6 may be useful for studies of apoptosis, iron sulfur cluster and heme export (Dabir et al., 2007), and cell differentiation (Todd et al., 2010b), because ALR has been implicated in these pathways. Since MitoBloCK-6 inhibits ALR oxidase activity in vitro, we asked whether MitoBloCK-6 disrupts mitochondrial function in mammalian cells by investigating mitochondrial morphology, a general readout for mitochondrial defects. HeLa cells were transiently transfected with mitochondrial matrix targeted Su9-EGFP and co-labeled with Mitotracker-Red (
ALR was identified in a set of common genes that are enriched in embryonic, neuronal, and hematopoietic stem cells (Ivanova et al., 2002; Ramalho-Santos et al., 2002), and ALR has a pro-survival role in maintaining pluripotent embryonic stem cells (Todd et al., 2010a). Thus, ALR may have a specific and different role in pluripotent stem cells than in differentiated cells, such as HeLa and HEK293 cells. Therefore, we determined whether MitoBloCK-6 affected hESC survival. HSF1 hESCs and normal human dermal fibroblasts (NHDFs), which represent a differentiated cell type, were exposed with 20 μM MitoBloCK-6 or 0.1% DMSO and visualized using brightfield microscopy (
To confirm that MitoBloCK-6 specifically inhibited the survival of hESCs and not of differentiated cells, HSF1 cells were induced to differentiate with 10 μM retinoic acid followed by MitoBloCK-6 exposure (
Having characterized the effects of MitoBloCK-6 in vitro and in primary cell culture systems, we applied MitoBloCK-6 to developing zebrafish embryos, which is a useful in vivo vertebrate model. The effect of MitoBlock-6 on mitochondrial function and zebrafish development was tested using previously established parameters (Mendelsohn et al., 2006; Murphey and Zon, 2006). Zebrafish embryos were placed in either 1% DMSO or 2.5 μM MitoBloCK-6 at 3 h post fertilization (hpf) and allowed to develop until 72 hpf. Higher concentrations of MitoBloCK-6 were toxic to the fish. MitoBloCK-6 but not DMSO incubated embryos displayed ventral curvature of the body and cardiac edema (FIGS. 25A,B). Furthermore, we also treated fish with MitoBloCK-6 from 3 to 24 hpf, followed by removal of MitoBloCK-6, and the zebrafish embryos were identical to those exposed to DMSO at 72 hpf, indicating that the effects of MitoBloCK-6 are reversible (unpublished data). Because ALR may play a role in FeS cluster assembly and export (Lange et al., 2001), erythropoiesis may be defective (Shaw et al., 2006). Therefore, embryos were stained with o-dianisidine, which binds to heme (Lumsden et al., 2007), as a method to visualize hematopoietic development. Whereas embryos exposed to 1% DMSO or MitoBloCK-6 showed normal hematopoiesis, embryos treated with MitoBloCK-6 showed erythrocyte pooling along the yolk sac prior to entering the lower chamber of the heart and an absence of red blood cells in the tail (FIG. 25D,E). To confirm that the observed phenotypes were specifically caused by ALR inhibition via MitoBloCK-6, 1-cell embryos were also injected with 4 ng of an ATG morpholino targeted to ALR (FIG. 25C,F). This morpholino prevents ALR translation in embryos. The phenotypes observed from the morpholino-injected embryos were identical to that of MitoBlock-6 exposure, strongly suggesting that ALR is specifically targeted. Cardiac development was also investigated in a transgenic zebrafish line in which DsRed is targeted to mitochondria under control of the heart specific cardiac myosin light chain promoter cmlc2 (
We have identified MitoBloCK-6 as the first selective inhibitor of the Mia40/Erv1 redox-mediated import pathway. Based on the assay in which oxidation of substrate DTT by Erv1 was inhibited, the mechanism by which MitoBloCK-6 may attenuate Erv1 activity is to potentially interfere with binding or electron transfer between Mia40, cyt c, and/or oxygen. Because the inhibitors were identified in an in vitro assay, it is possible that the small molecules might lack specificity in vivo and generally inhibit mitochondrial function. However, we have used a variety of approaches to show that MitoBloCK-6 specifically inhibits Erv1 function. MitoBloCK-6 is a stable compound and the potential breakdown product, 3,5-dichlorosalicyclaldehyde, does not inhibit Erv1 function. Instead, the hydroxyl group at the ortho-position likely stabilizes the compound (Crugeiras et al., 2009), and a similar class of molecules has been identified in a small molecule screen for inhibitors of Type III secretion (Nordfelth et al., 2005). A small SAR study also supports that similar compound ES-2 inhibits Erv1 function, but ES-1 does not. In addition, MitoBloCK-6 did not alter mitochondrial integrity and respiration; therefore, MitoBloCK-6 does not generally damage mitochondria. Instead, our biochemical assays show that MitoBloCK-6 inhibits oxidation of physiologic substrates Tim13 and Cmc1 and inhibits hydrogen peroxide production.
MitoBloCK-6 also inhibits Erv1 in isolated mitochondria, suggesting that small molecules are valuable for mechanistic studies. As expected, import assays showed that import of substrates of the Erv1-Mia40 pathway was specifically inhibited and this inhibition was dependent on steady-state Erv1 levels, because increased Erv1 expression correlated with increased MitoBloCK-6 addition. Import of CX9C proteins was reduced more than CX3C protein Tim8. Pfanner and colleagues have shown that a ternary complex is formed by the substrate, Mia40, and Erv1 (Stojanovski et al., 2008a); MitoBloCK-6 may potentially interfere with the formation of this ternary complex in a substrate-specific manner. Strong inhibition of Mia40 import by MitoBloCK-6 was also unexpected, because full-length Mia40 in yeast uses the TIM23 pathway (as in
ALR has a Key Function in hESC Maintenance and Zebrafish Development
Our strategy of screening with the yeast protein Erv1 was also constructive because MitoBloCK-6 inhibited the human homolog ALR with an improved IC50 of 700 nM. Our biochemical assays also support that MitoBloCK-6 is an effective ALR inhibitor, because the oxidation of Cmc1 was inhibited (
Whereas MitoBloCK-6 inhibits activity of the Erv1 family in vitro, a surprising finding was that MitoBloCK-6 did not inhibit growth or function of differentiated cells such as HEK293, HeLa and COST cells in vivo. An initial reason may be that a factor in the media inhibited MitoBloCK-6 action. However, several types of media were tested, including the permissive hESC media with differentiated cells, and MitoBloCK-6 remained inactive. In contrast, MitoBloCK-6 specifically induced apoptosis in hESCs, suggesting ALR may have a distinct role in pluripotent stem cell maintenance. Published studies support a role for ALR in stem cells, because ALR expression is enriched in embryonic, neuronal, and hematopoietic stem cells (Ivanova et al., 2002; Ramalho-Santos et al., 2002). ALR has been reported to have a pro-survival role in maintaining mouse pluripotent embryonic stem cells by interacting with Drp1 (Todd et al., 2010a). However, Drp1 is a cytosolic protein mediating mitochondrial fission and it is not apparent how IMS-localized ALR associates with Drp1; our data supports the model that ALR inactivation by MitoBlock-6 results in cyt c release and the mitochondrial network collapses as a consequence of apoptosis (Parone et al., 2006). We and others have shown that Erv1 and ALR shuttle electrons to cyt c (Bihlmaier et al., 2007; Dabir et al., 2007; Farrell and Thorpe, 2005). In differentiated cells, approximately 85% of the cyt c population is distributed in the cristae in association with the respiratory complexes and 15% is located in the IMS in the region between the inner and outer membrane (Bernardi and Azzone, 1981); this 85% population of cyt c is released from the cristae during apoptosis in differentiated cells (Scorrano et al., 2002). However, hESC mitochondria lack numerous cristae and display decreased respiration compared to differentiated cells (Zhang et al., 2011), so the population of cyt c that associates with ALR may be the critical pool that is released during apoptosis. As a result of our preliminary finding, MitoBloCK-6 is an excellent tool to understand the contribution of mitochondrial to pluripotent stem cell function and differentiation. Additional studies are ongoing to understand how MitoBloCK-6 induces apoptosis in hESCs.
In contrast to differentiated culture cells, zebrafish provide a powerful model system for characterizing ALR function because cells are not transformed and are in their normal physiologic setting of cell-cell and cell-extracellular matrix interactions (Murphey and Zon, 2006). The embryos are also in simple buffered water, so MitoBloCK-6 uptake may be enhanced. Defects in mitochondrial biogenesis in zebrafish display varied phenotypes. Mutations in the Tomm22 import component result in defects in liver development (Curado et al., 2010) and mutations in Fe—S cluster biogenesis typically impact erythropoiesis (Shaw et al., 2006; Wingert et al., 2005). Indeed, MitoBloCK-6 also elicited gross morphologic and cardiac defects in zebrafish that were akin to ALR downregulation. Overall, characterization of MitoBloCK-6 supports that the chemical approach is valid for developing probes to study protein translocation and understand the role of protein import in development.
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While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications can be made without departing from the embodiments of this invention in its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as fall within the true spirit and scope of the embodiments of this invention.
Claims
1. A specific inhibitor of mitochondrial protein translocation, which inhibitor modulates the assembly or function of mitochondria, wherein the inhibitor specifically targets the protein translocation pathway thereby modulating the assembly and function of the mitochondrion with respect to protein translocation and import.
2. The inhibitor of claim 1, which is a compound comprising a structure of formula I, II or III: a derivative thereof, or pharmaceutically acceptable salt thereof, wherein each R1, R2, and R3 is independently H, C1-C10 straight-chained or branched alkyl (substituted or unsubstituted), C1-C10 cycloalkyl (substituted or unsubstituted), C1-C10 straight-chained or branched alkeynyl (substituted or unsubstituted), C1-C10 cycloalkenyl (substituted or unsubstituted), C1-C10 aryl (substituted or unsubstituted), phenyl, carboxyl, hydroxyl, amino, carbonyl, carbonate, halo (F, Cl, Br, or I), thiol, thiourea, urea, or triazole groups.
3. The inhibitor of claim 1, which is MitoBlock-1, MitoBlock-6, or a formula according to any of A-D: derivatives thereof, or pharmaceutically acceptable salts thereof.
4. A composition comprising a specific inhibitor of mitochondrial protein translocation, which inhibitor specifically targets the protein translocation pathway thereby modulating the assembly and function of the mitochondrion with respect to protein translocation and import.
5. The composition of claim 4, wherein the inhibitor is a compound of a structure of formula I, II or III: a derivative thereof, or pharmaceutically acceptable salt thereof, wherein each R1, R2, and R3 is independently H, C1-C10 straight-chained or branched alkyl (substituted or unsubstituted), C1-C10 cycloalkyl (substituted or unsubstituted), C1-C10 straight-chained or branched alkeynyl (substituted or unsubstituted), C1-C10 cycloalkenyl (substituted or unsubstituted), C1-C10 aryl (substituted or unsubstituted), phenyl, carboxyl, hydroxyl, amino, carbonyl, carbonate, halo (F, Cl, Br, or I), thiol, thiourea, urea, or triazole groups.
6. The composition of claim 4, wherein the inhibitor is MitoBlock-1, MitoBlock-6 or a compound of a formula according to any of A-D: derivatives thereof, or pharmaceutically acceptable salts thereof.
7. The composition of claim 4, further comprising a carrier.
8. The composition of claim 6, further comprising a carrier.
9. A method, comprising modulate the assembly or function of mitochondria by applying to a body of mitochondria or a cell a specific inhibitor of mitochondria protein translocation, wherein the inhibitor specifically targets the protein translocation pathway thereby modulating the assembly and function of the mitochondrion with respect to protein translocation and import.
10. The method of claim 9, wherein the specific inhibitor is according to claim 1.
11. The method of claim 9, wherein the specific inhibitor is included in a composition.
12. The method of claim 11, wherein the composition further comprises a carrier.
13. A method of treating or ameliorating a disorder, comprising administering to a patient in need thereof a specific inhibitor of mitochondria protein translocation, wherein the inhibitor specifically targets the protein translocation pathway thereby modulating the assembly and function of the mitochondrion with respect to protein translocation and import.
14. The method of claim 13, wherein the specific inhibitor is according to claim 1.
15. The method of claim 13, wherein the specific inhibitor is included in a composition.
16. The method of claim 15, wherein the composition further comprises a carrier.
17. The method of claim 11, wherein the disorder is a disease of deafness-dystonia syndrome, cancer, Parkinson's disease, or Alzheimer's disease.
18. A method for identifying a specific inhibitor of mitochondrial protein translocation, comprising
- culturing a tim10-1 mutant strain of yeast in a medium with a library of drug-like compounds, identifying a drug-like compound as a hit compound if the drug-like compound significantly inhibits growth of the tim10-1 mutant strain of yeast,
- subjecting the hit compound to a counter screen which comprises incubating the hit compound with the tim10-1 mutant strain and an isogenic control strain carrying an integrated version of the TIM10 gene at the leu2 locus, and identifying the hit compound that selectively inhibits growth of the mutant strain but not the isogenic control strain as a hit compound for second counter screen where the second counter screen comprises: incubating the hit compound for second counter screen with the tim10-1 mutant strain and a tim10-1 mutant strain harboring a plasmid containing a wild-type TIM10 gene, and identifying the hit compound that selectively inhibits growth of the tim10-1 mutant but not the tim10-1 mutant harboring a plasmid containing the wild-type TIM10 gene; and
- designating the hit compound that selectively inhibits growth of only the tim10-1 mutant in both the first counter screen and the second counter screen as the specific inhibitor of mitochondrial protein translocation (“MitoBloCk”).
19. The method of claim 18, wherein the drug-like compound inhibits growth of the tim10-1 mutant strain of yeast by 50% or above.
20. The method of claim 18, wherein the hit compound selectively inhibits growth of the mutant strain by 50% or above.
21. The method of claim 18, wherein the hit compound selectively inhibits growth of the mutant strain by 80% or above.
22. The method of claim 18, wherein the hit compound selectively inhibits growth of the mutant strain by 90% or above.
23. The method of claim 18, wherein the hit compound selectively inhibits growth of the mutant strain by 99% or above.
24. The method of claim 18, wherein the tim10-1 mutant has a concentration of about 10 μM.
25. The method of claim 18, comprising using an integrated robotic system to perform any or all of the acts.
26. A method for identifying a specific inhibitor or activator of mitochondrial disulfide relay pathways, comprising
- providing a system of testing purified components of the mitochondrial oxidative folding and disulfide relay pathway including Mia40, Cmc1, Erv1, ALR, cytochrome c, and small Tim proteins in a medium with a library of drug-like compounds,
- identifying a drug-like compound as a hit compound if the drug-like compound significantly inhibits or activates the activity of at least one of redox-active enzymes,
- subjecting the hit compound to a counter screen which comprises incubating the hit compound with a yeast or mammalian cell line that reports the growth inhibition of a yeast strain or mammalian cell line that had attenuated activity in its mitochondrial disulfide relay pathway and an isogenic control strain or cell line carrying a non-attenuated version of the mitochondrial disulfide relay system, and identifying the hit compound that selectively inhibits or promotes the growth of the attenuated stain or cell line but not the strain or cell line as a hit compound for second counter screen where the second counter screen comprises: incubating the hit compound for second counter screen with the a member of a redox-active enzyme family other than ALR or Erv1, and identifying the hit compound that selectively inhibits or activates the activity of ALR or Erv1 but not the related redox-active enzyme; and
- designating the hit compound that selectively inhibits or activates the activity of ALR or Ery in both the first counter screen and the second counter screen as the specific inhibitor of the mitochondrial disulfide relate system (“MitoBloCk”).
27. The method of claim 26, wherein the drug-like compound inhibits or activates the activity of ALR or Erv1 by 50% or above.
28. The method of claim 26, wherein the hit compound selectively inhibits or activates the activity of ALR or Erv1 by 50% or above.
29. The method of claim 26, wherein the hit compound selectively inhibits or activates the activity of ALR or Erv1 by 80% or above.
30. The method of claim 26, wherein the hit compound selectively inhibits or activates the activity of ALR or Erv1 by 90% or above.
31. The method of claim 26, wherein the hit compound selectively inhibits or activates the activity of ALR or Erv1 by 99% or above.
32. The method of claim 26, wherein the tim10-1 mutant has a concentration of Erv1 or ALR is at or below its Michaelis-Menten constant (Km).
33. The method of claim 18, wherein the tim10-1 mutant has a concentration of Erv1 or ALR is at or below its Michaelis-Menten constant (Km).
34. The method of claim 18, wherein the tim10-1 mutant has a concentration of Erv1 or ALR is about 1 μM.
35. The method of claim 26, comprising using an integrated robotic system to perform any or all of the acts.
Type: Application
Filed: Apr 12, 2012
Publication Date: Sep 4, 2014
Applicant: The Regents of the University of California (Oakland, CA)
Inventors: Carla M. Koehler (Los Angeles, CA), Samuel A. Hasson (Los Angeles, CA), Kiyoko Miyata (Los Angeles, CA), Micheal A. Teitell (Tarzana, CA), Deepa Dabir (Los Angeles, CA)
Application Number: 14/111,265
International Classification: A61K 31/343 (20060101); C12Q 1/18 (20060101); A61K 31/404 (20060101); A61K 31/15 (20060101); A61K 31/4196 (20060101);
