WO2011113018A1 - Measurement and control of biological time - Google Patents

Measurement and control of biological time Download PDF

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Publication number
WO2011113018A1
WO2011113018A1 PCT/US2011/028220 US2011028220W WO2011113018A1 WO 2011113018 A1 WO2011113018 A1 WO 2011113018A1 US 2011028220 W US2011028220 W US 2011028220W WO 2011113018 A1 WO2011113018 A1 WO 2011113018A1
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organism
tissue
cell
nqol
redox
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PCT/US2011/028220
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French (fr)
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Guy M. Miller
William D. Shrader
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Ampere Life Sciences, Inc.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90209—Oxidoreductases (1.) acting on NADH or NADPH (1.6), e.g. those with a heme protein as acceptor (1.6.2) (general), Cytochrome-b5 reductase (1.6.2.2) or NADPH-cytochrome P450 reductase (1.6.2.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7042—Aging, e.g. cellular aging

Definitions

  • Living systems display many phenomena that are governed by time.
  • One prominent example is the sleeping/waking cycle in mammals, which is an example of a circadian rhythm. While many circadian rhythms are entrained to the light/dark cycle of night and day (or associated cycles, such as daily variations in temperature), the rhythms are endogenous to the organism, as animals in environments lacking a light/dark cycle will still exhibit circadian rhythms.
  • Other phenomena that are governed by time in living systems include migratory behavior in birds, puberty in humans and animals, and aging.
  • the biochemical basis for time-based biological phenomena has been the subject of much inquiry.
  • the suprachiasmatic nucleus located in the hypothalamus in the brain, plays a role in most circadian rhythms.
  • the neurons of the suprachiasmatic nucleus produce a protein product from a biological clock gene.
  • the protein product turns off production of more protein. This results in a negative feedback loop.
  • the clock gene is turned back on, thus producing more protein, which in turn again shuts off the clock gene.
  • the present invention is based on the discovery of another timekeeping system for "biological time” which is based on the state of redox-active molecules.
  • This clock system can be measured in order to determine the state of a biological system. Disorders in this clock system can lead to diseases, and alteration or modulation of the clock system can be used to treat such diseases.
  • the invention embraces a method for measuring the state of biological time in a cell, tissue, or organism, comprising measuring the concentration or absolute amount of a redox-active molecule in oxidized form, reduced form, or both oxidized form and reduced form, in the cell, tissue, or organism, and correlating the concentration or absolute amount of the redox-active molecule to the state of biological time in the cell, tissue, or organism at the time of measurement.
  • the step of correlating the concentration or absolute amount of the redox-active molecule to the state of biological time in the cell, tissue, or organism at the time of measurement can comprise diagnosing a disease in the cell, tissue or organism, including, but not limited to, Huntington's disease, Parkinson's disease and Alzheimer's disease.
  • the redox-active molecule can be selected from the group consisting of lactate, pyruvate, NADP, NADPH, NAD, NADH, reduced glutathione (GSH), oxidized glutathione (GSSG), alpha-tocopherol in oxidized form, beta-tocopherol in oxidized form, gamma-tocopherol in oxidized form, delta-tocopherol in oxidized form, alpha-tocotrienol in oxidized form, beta-tocotrienol in oxidized form, gamma-tocotrienol in oxidized form, delta- tocotrienol in oxidized form, alpha-tocopherol in reduced form, beta-tocopherol in reduced form, gamma-tocopherol in reduced form, delta-tocopherol in reduced form, alpha- tocotrienol in reduced form, beta-tocotrienol in reduced form, gamma-tocotrienol in reduced form, delta-tocopherol
  • the invention embraces a method for measuring the state of biological time in a cell, tissue, or organism, comprising measuring the concentration or absolute amount of a two-electron redox-cycling small molecule in oxidized form in the cell, tissue, or organism; measuring the concentration or absolute amount of the two-electron redox-cycling small molecule in reduced form in the cell, tissue, or organism; calculating the ratio of the concentration of the oxidized form to the concentration of the reduced form, or calculating the ratio of the absolute amount of the oxidized form to the absolute amount of the reduced form; and correlating the calculated ratio to the state of biological time in the cell, tissue, or organism at the time of measurement.
  • the step of correlating the calculated ratio to the state of biological time in the cell, tissue, or organism at the time of measurement can comprise diagnosing a disease in the cell, tissue or organism.
  • the two-electron redox- cycling small molecule can be selected from the group consisting of Coenzyme Q10, alpha- tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta-tocotrienol quinone, alpha-tocopherol quinone, beta-tocopherol quinone, gamma-tocopherol quinone, delta-tocopherol quinone, glutathione, Vitamin E, and Vitamin K.
  • the invention embraces a method for altering or modulating the state of biological time in a cell, tissue, or organism, comprising administering an effective amount of a two-electron redox-cycling small molecule to the cell, tissue, or organism.
  • the two-electron redox-cycling small molecule can be selected from the group consisting of Coenzyme Q10, alpha-tocotrienol quinone, beta-tocotrienol quinone, gamma- tocotrienol quinone, delta-tocotrienol quinone, alpha-tocopherol quinone, beta-tocopherol quinone, gamma-tocopherol quinone, delta-tocopherol quinone, glutathione, Vitamin E, and Vitamin K.
  • the invention embraces a method for measuring the state of biological time in an organism, comprising the steps of 1) obtaining a cell, tissue, or bodily fluid sample from the organism; 2) measuring the rate of NQOl activity in the cell, tissue, or bodily fluid; and 3) correlating the rate of NQOl activity to the state of biological time in the cell, tissue, bodily fluid, or organism at the time of measurement.
  • the step of correlating the rate of NQOl activity to the state of biological time in the cell, tissue, bodily fluid, or organism at the time of measurement can comprise diagnosing a disease in the cell, tissue or organism.
  • the disease is cancer.
  • the invention embraces a method for selecting a cancer treatment in an organism, comprising the steps of 1) obtaining a cell, tissue, or bodily fluid sample from the organism; 2) measuring the rate of NQOl activity in the cell, tissue, or bodily fluid; and 3) using the measured rate of NQOl activity to select a cancer treatment for the cell, tissue, or organism.
  • the invention encompasses a method of modulating biological time by altering the activity of NQOl in a cell, tissue, or organism. In one embodiment, the invention encompasses a method of modulating biological time by increasing the activity of NQOl in a cell, tissue, or organism. In another embodiment, the invention encompasses a method of modulating biological time by decreasing the activity of NQOl in a cell, tissue, or organism. In another embodiment, the invention encompasses a method of modulating biological time by administering a redox-active molecule to the cell, tissue, or organism. In one embodiment, the redox-active molecule is a two-electron redox-cycling small molecule.
  • FIGURE 1 depicts the redox coupling of the enzyme NQOl, where the enzyme oxidizes NADPH and reduces alpha-tocotrienol quinone (ATQ3); ATQ3 then subsequently reduces NBT to NBT Formazan. This reduction can be monitored by A550.
  • FIGURE 2 depicts the results of an assay for NQOl, where an increase in absorption at 550 nm commences upon addition of ATQ3 to a solution of NQOl, NADPH, and NBT. The increase terminates upon addition of the NQOl inhibitor dicumarol.
  • the invention relates to methods of measuring and controlling "biological time" in a living system, such as a cell, tissue, or organism.
  • Biological time is meant a mechanism by which a living system coordinates or synchronizes biological processes.
  • “Biological time” includes, but is not limited to, circadian rhythms (i.e., cycles of approximately one day, including diurnal/nocturnal cycles), infradian rhythms (i.e., cycles shorter than one day), ultradian rhythms (i.e., cycles longer than one day), and other periodic rhythms.
  • Bio time also includes non-cyclical biological processes that have a direct time-dependent component (such as the accumulation of mutations in mitochondrial DNA in a cell; these mutations increase over time since mitochondrial DNA mutations are not repaired) or an indirect time-dependent component (such as telomere length; telomeres in, for example, human cells shorten with each successive round of cell division, and the number of rounds of cell divisions increases with time).
  • a direct time-dependent component such as the accumulation of mutations in mitochondrial DNA in a cell; these mutations increase over time since mitochondrial DNA mutations are not repaired
  • an indirect time-dependent component such as telomere length; telomeres in, for example, human cells shorten with each successive round of cell division, and the number of rounds of cell divisions increases with time.
  • small molecules are meant molecules with a molecular weight of equal to or less than 1000 Daltons, preferably equal to or less than 600 Daltons.
  • Vitamin K includes, but is not limited to, phylloquinone and menaquinone.
  • Vitamin E includes all isoforms such as alpha tocopherol, beta tocopherol, gamma tocopherol, delta tocopherol, alpha tocotrienol, beta tocotrienol, gamma tocotrienol and delta tocotrienol.
  • Alpha-tocotrienol quinone is abbreviated as aTQ3, a-TQ3, aTQ3, a-TQ3, ATQ3, or A-TQ3.
  • the cell, tissue, or organism in which biological time is measured, controlled, altered, or modulated can be, or can be derived from, any living system, including microorganisms, plants, and animals.
  • a bodily fluid obtained from an organism can be blood, plasma, serum, cerebrospinal fluid, saliva, perspiration, semen, urine, stool, sputum, tears, mucus, amniotic fluid or the like.
  • biological time is observed by measuring the electron capacity and current flow (both individual components and sum total of components) between two chronological time points of a biological system. This measurement provides a characterization of both total collection and individual component changes as a biological system alters its redox states.
  • Biological time is related to the preservation of the overall redox state of the system, and is defined by its components (such as redox active quinones) and the biological molecules (such as NQOl) which regulate the redox state of these components.
  • components such as redox active quinones
  • biological molecules such as NQOl
  • a biologically system ages as a function of its biological time. This progression of biological time is not linear like chronological time, and occurs often in non-linear steps (often as a function of disease).
  • biological time as measured by redox state can often provide information about the relative chronological state of the biological system. For example, when a system has an overall redox potential > +100 mV for its manifold of low molecular weight quinones, then the system has reached an endpoint in both chronological and biological time; that is, the biological system is defined as dead.
  • the invention encompasses a method of modulating biological time by stimulating production of NQOl in a cell, tissue, or organism.
  • the invention encompasses a method of modulating biological time by inhibiting production of NQOl in a cell, tissue, or organism. In another embodiment, the invention encompasses a method of modulating biological time by administering a redox-active molecule to the cell, tissue, or organism. In one embodiment, the redox-active molecule is a two-electron redox- cycling small molecule.
  • Placing cells under oxidative stress can cause an increase or elevation in expression of NQOl of about five-fold to fifty- fold. This places a demand on the supply of redox-active molecules, such as quinones, in the cells, which are rapidly depleted by the increased NQOl expression. Accordingly, when increased expression of NQOl is detected in a cell, tissue, or organism, administration of redox-active molecules, such as quinones, can alleviate this depletion and relieve the stress on the cell, tissue, or organism, essentially "resetting" the redox biological clock.
  • the invention embraces a method of treating an oxidative stress disorder, comprising 1) measuring the increase or elevation in expression of NQOl in a cell, tissue, or organism; and 2) if expression of NQOl is increased or elevated, administering a therapeutically effective amount of a redox-active molecule.
  • the redox-active molecule is alpha-tocotrienol quinone (ATQ3).
  • the redox-active molecule is menadione.
  • the redox-active molecule is duroquinone.
  • the redox-active molecule such as ATQ3 is administered to the cell or tissue in an amount sufficient to provide a concentration of between about 100 nanomolar to about 10 micro molar in the extracellular fluid contacting the cell or tissue, or between about 100 nM and about 1 uM, or between about 1 uM and about 10 uM.
  • the redox-active molecule such as ATQ3, is administered to the organism in an amount sufficient to provide a concentration of between about 10 nanomolar to about 10 micromolar in the plasma of the organism, or between about 10 nM and about 100 nM, or between about 10 nM and about 50 nM, or between about 100 nM and about 250 nM, or between about 100 nM and about 500 nM, or between about 100 nM and about 1 uM, or between about 1 uM and about 10 uM, or between about 1 uM to about 5 uM, or between about 1 uM to about 5 uM.
  • the redox-active molecule such as ATQ3, is administered when the expression of NQOl has increased about or at least about 25%, about or at least about 50%, about or at least about 75%, about or at least about 100%, about or at least about 200%, about or at least about 300%, about or at least about 400%, or about or at least about 500% over the normal level of expression under unstressed conditions.
  • a cell, tissue, or organism can be monitored continuously or periodically for elevated levels of expression of NQOl, and, upon increase of the level of expression of NQOl above a certain threshold amount (for example, an increase of about or at least about 25%, about or at least about 50%, about or at least about 75%, about or at least about 100%, about or at least about 200%, about or at least about 300%, about or at least about 400%, or about or at least about 500%), a redox-active molecule, such as ATQ3, can be administered to the cell, tissue, or organism, in an amount as recited above.
  • a certain threshold amount for example, an increase of about or at least about 25%, about or at least about 50%, about or at least about 75%, about or at least about 100%, about or at least about 200%, about or at least about 300%, about or at least about 400%, or about or at least about 500%
  • ATQ3 redox-active molecule
  • NQOl can be viewed as a lagging indicator of the stress on the cell, tissue, or organism.
  • the invention embraces a method of 1) measuring the increase (or elevation) in expression of NQOl in a cell, tissue, or organism; and 2) if expression of NQOl is increased (or elevated), administering a therapeutically effective amount of a redox-active molecule, wherein the redox-active molecule is administered in an amount sufficient to reduce the expression of NQOl to a level about or at least about 10%, about or at least about 25%, about or at least about 30%, about or at least about 40%, about or at least about 50%, about or at least about 60%, about or at least about 70%, about or at least about 75%, about or at least about 80%, about or at least about 90%, or about or at least about 95% of the increased (or elevated) level of NQOl expression.
  • NQOl assay An isolated human NQOl assay has been developed which can measure NQOl activity, including measurement of the different rates of reduction of small molecule quinones.
  • This assay uses the redox-active dye NBT to measure the transfer of electrons from NAD(P)H, to NQOl and then to the quinone; see Example 1. Only the electrons from a dihydroquinone will reduce the redox active dye, which is used as a proxy for electron flux through the system.
  • NQOl assay An isolated human NQOl assay has also been developed that directly measures the reduction of quinone using the 16-channel CoulArray system.
  • 10 uL samples are taken from a 1 mL reaction mixture of the following (1.5 units of NQOl, 250 uM
  • NAD(P) in a buffer of 50 mM Tris-HCl at ph 7.4, with 0.08% TX-100, at 25°C in a HPLC vial -sealed with an airtight septum).
  • a-TQ3 is added (20 uM final concentration from a ImM DMSO stock) to the reaction, and at various time points 10 uL of sample are injected and measured for amount of initial oxidized quinone and the increase of reduced quinone.
  • WO 2008/062105 and US 2010/0159458 discuss a method for selecting a cancer therapy based on a subject's genetic background, by determining the presence of a mutant or non-functional NQOl gene or gene product.
  • the NQOl assay described herein can provide an alternative procedure for carrying out the method disclosed in WO 2008/062105 and US 2010/0159458.
  • Biological time is driven, at least in part, by the reductive potential of a cellular hydride coupled through a quinone reductase, such as the human quinone reductases, for example NQOl, NQ02, PIG3, or Zeta-crystalline.
  • a quinone reductase such as the human quinone reductases, for example NQOl, NQ02, PIG3, or Zeta-crystalline.
  • the cycling can be viewed as an internal "biological clock” or "biochemical clock.”
  • Key regulator proteins such as p53, tyrosinase, p33, p73, ornithine decarboxylase, and eIF4Gl, can directly bind the quinone reductase to sample the time domain established by the two-electron redox cycle.
  • Other regulatory proteins or genes may be affected indirectly by the quinone reductase.
  • the quinone metabolites of tocopherols are essential components of this clock system.
  • Other important components include NQOl, p53, and the 20S proteasome complex.
  • the timing of this system can be modified by introducing different two- electron redox-cycling small molecules into the system, which differ by their redox potential and shape from the endogenous redox molecules comprising the clock.
  • ubiquinone or Coenzyme Q10
  • Alpha-tocotrienol quinone differs from CoQIO in the number of isoprene units in its tail and in the substituents on the quinone ring, and administration of alpha-tocotrienol quinone illustrates one method of modifying the redox-based clock.
  • This time cycling (clock) of the two-electron system can also be modified by the binding of non-redox cycling molecules which bind and compete with the quinone binding site within the quinone reductase.
  • Resveratrol is one example of such a molecule.
  • timing of this system can also be achieved for example by changing the turnover rate ("frequency") of NQOl, by genetic modification of NQOl, or by magnetic fields interacting with unpaired electrons (for example, as occurs with the magnetic compass of birds; see
  • Huntington's disease Morton et al., J. Neuroscience (2005) 25(1): 157-163; and Alzheimer's disease (van Someren et al., Biological Psychiatry (1996) 40:259-270. Re-establishment of proper biological time regulation can aid in treatment of these disorders.
  • the time domain established by this system is also involved in the regulation of biological time such as aging. For example, when the NQOl cellular hydride source is restricted, the clock function of the NQOl - quinone redox cycle is affected, which extends lifespan.
  • the present invention encompasses measurement of one or more redox-active molecules in order to assess the state of a cell, tissue or organism in terms of its biological time.
  • the redox-active molecule can be any redox-active molecule in the cell, tissue or organism, such as one or more two-electron redox-cycling small molecules (for example, lactate/pyruvate, NADP/NADPH, NAD/NADH, reduced glutathione GSH versus oxidized glutathione GSSG, Vitamin E in oxidized and reduced forms, Vitamin K in oxidized and reduced forms), or a larger molecule such as a protein having a prosthetic group (for example, cytochrome C-oxidized and cytochrome C-reduced).
  • the concentration of the oxidized and reduced form can be measured and the ratio of the oxidized to reduced form calculated (or the ratio of the reduced form to the oxidized form can be calculated).
  • the concentration of the oxidized form can be measured and may provide sufficient information in the absence of measurement of the reduced form, or the concentration of the reduced form can be measured and may provide sufficient information in the absence of measurement of the oxidized form.
  • the absolute amount of the oxidized and reduced form can be measured and the ratio of the oxidized to reduced form calculated (or the ratio of the reduced form to the oxidized form can be calculated).
  • the absolute amount of the oxidized form can be measured and may provide sufficient information in the absence of measurement of the reduced form, or the absolute amount of the reduced form can be measured and may provide sufficient information in the absence of measurement of the oxidized form.
  • the present invention encompasses the measurement of one or more redox-active molecules in order to assess the state of a cell, tissue or organism in terms of its biological time, in order to diagnose, determine, or stage the progression of, a disease associated with aging.
  • the present invention encompasses use of one or more of two- electron redox-cycling small molecules (such as alpha-tocotrienol quinone) in order to alter or modulate this cellular clock system. If measurement of the state of the cellular clock system has been performed, the one or more two-electron redox-cycling small molecules used to alter or modulate the cellular clock system can be the same as, or different from, the molecule or molecules measured to establish the state of the cellular clock system.
  • two- electron redox-cycling small molecules such as alpha-tocotrienol quinone
  • the present invention encompasses use of one or more two- electron redox-cycling small molecules (such as alpha-tocotrienol quinone) two-electron redox-cycling small molecules to treat disease states (such as Leigh syndrome) which result from cellular clock dysregulation.
  • the method involves administration of one or more two- electron redox-cycling small molecules (such as alpha-tocotrienol quinone) to cells, tissues, or organisms in an amount effective to treat a disease state.
  • the present invention encompasses use of one or more two- electron redox-cycling small molecules (such as alpha-tocotrienol quinone) to alter or modulate biological time.
  • the present invention encompasses use of one or more two-electron redox-cycling small molecules (such as alpha-tocotrienol quinone) to affect aging.
  • the method involves administration of one or more two-electron redox-cycling small molecules (such as alpha-tocotrienol quinone) to cells, tissues, or organisms in an amount effective to affect aging.
  • the one or more two-electron redox-cycling small molecules can be used to slow down biological time, thus extending life time in cells, tissues or organisms. In one embodiment, the one or more two-electron redox-cycling small molecules can be used to slow down time, thus extending biological life time in cells, tissues or organisms, and retarding aging in cells, tissues, or organisms, for example, to retard aging in healthy cells, tissues, or organisms. In one embodiment, the one or more two-electron redox-cycling small molecules can be used to slow down biological time, thus extending biological life time in cells whose mitochondria are diseased, retarding the progress of the mitochondrial disease.
  • the one or more two-electron redox-cycling small molecules can be used to accelerate biological time, in cells, tissues, or organisms, for example, to accelerate aging in cells, tissues, or organisms that are diseased or that cause disease, such as cancer cells or pathogenic microorganisms.
  • the present invention encompasses use of one or more redox- active molecules selected from the group consisting of lactate, pyruvate, NADP, NADPH, NAD, NADH, reduced glutathione (GSH), oxidized glutathione (GSSG), Vitamin E in oxidized form, Vitamin E in reduced form, Vitamin K in oxidized form, Vitamin K in reduced form, cytochrome C-oxidized, cytochrome C-reduced, Coenzyme Q10, alpha- tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta-tocotrienol quinone, alpha-tocopherol quinone, beta-tocopherol quinone, gamma-tocopherol quinone, and delta-tocopherol quinone, in order to measure or assess the state of the cellular clock system (i.e., biological time, biological clock).
  • redox- active molecules selected from the group consisting of
  • the present invention encompasses use of one or more two- electron redox-cycling small molecules selected from the group consisting of Coenzyme Q10, alpha-tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta- tocotrienol quinone, alpha-tocopherol quinone, beta-tocopherol quinone, gamma-tocopherol quinone, delta-tocopherol quinone, glutathione, Vitamin E, and Vitamin K, in order to alter or modulate the cellular clock system (i.e., biological time).
  • Coenzyme Q10 alpha-tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta- tocotrienol quinone, alpha-tocopherol quinone, beta-tocopherol quinone, gamma-tocopherol quinone, delta-to
  • NAD(P)H-quinone oxidoreductase 1 (NQOl; EC 1.6.5.2) is a flavoprotein that reversibly catalyzes the oxidation of NADH or NADPH by various quinones and oxidation- reduction dyes (see w/w/w. ncbi.nlm.nih.gov/mesh/68016660).
  • the enzyme can be inhibited by dicumarol (dicoumarol; bishydroxycoumarin).
  • Nitroblue tetrazolium (NBT; 2-[2-methoxy-4-[3-methoxy-4-[3-(4-nitrophenyl)-5- phenyltetrazol-2-ium-2- yl] phenyl] phenyl] - 3 -(4-nitrophenyl) -5 -phenyltetrazol-2-ium dichloride) can be reduced to a formazan compound (NBT Formazan) by ATQ3.
  • NBT has a measureable UV/VIS change due to its reduction, does not cross-couple with either
  • NBT NQOl oxidizes NAD(P)H to NAD(P) + , with concomitant reduction of NQOl (oxidized) to NQOl (reduced) as hydride is transferred to NQOl.
  • NQOl (reduced) then reduces ATQ3 to its hydroquinone (benzenediol) form, with concomitant regeneration of NQOl (oxidized).
  • the reduced alpha tocotrienol hydroquinone then reduces NBT to NBT Formazan, with regeneration of alpha tocotrienol quinone.
  • the formation of NBT Formazan can be monitored by A550, while the disappearance of NAD(P)H can be monitored at A 340 .
  • the graph in Figure 2 shows an assay using NQOl.

Abstract

Methods for measuring, controlling, altering and modulating biological time in living systems are provided, as well as compounds suitable for use in controlling, altering, and modulating biological time and methods for treating disorders due to defects in the system for maintaining biological time. Redox-active molecules, including two-electron redox-cycling small molecules, can be used in such methods of measuring, controlling, altering, and modulating biological time.

Description

MEASUREMENT AND CONTROL OF BIOLOGICAL TIME
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority benefit of United States Provisional Patent
Application No. 61/313,682, filed March 12, 2010. The entire content of that application is hereby incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] Living systems display many phenomena that are governed by time. One prominent example is the sleeping/waking cycle in mammals, which is an example of a circadian rhythm. While many circadian rhythms are entrained to the light/dark cycle of night and day (or associated cycles, such as daily variations in temperature), the rhythms are endogenous to the organism, as animals in environments lacking a light/dark cycle will still exhibit circadian rhythms. Other phenomena that are governed by time in living systems include migratory behavior in birds, puberty in humans and animals, and aging.
[0003] The biochemical basis for time-based biological phenomena has been the subject of much inquiry. In mammals, the suprachiasmatic nucleus, located in the hypothalamus in the brain, plays a role in most circadian rhythms. The neurons of the suprachiasmatic nucleus produce a protein product from a biological clock gene. The protein product turns off production of more protein. This results in a negative feedback loop. When levels of the protein drop below a certain level, the clock gene is turned back on, thus producing more protein, which in turn again shuts off the clock gene.
[0004] The present invention is based on the discovery of another timekeeping system for "biological time" which is based on the state of redox-active molecules. This clock system can be measured in order to determine the state of a biological system. Disorders in this clock system can lead to diseases, and alteration or modulation of the clock system can be used to treat such diseases.
BRIEF SUMMARY OF THE INVENTION
[0005] In one embodiment, the invention embraces a method for measuring the state of biological time in a cell, tissue, or organism, comprising measuring the concentration or absolute amount of a redox-active molecule in oxidized form, reduced form, or both oxidized form and reduced form, in the cell, tissue, or organism, and correlating the concentration or absolute amount of the redox-active molecule to the state of biological time in the cell, tissue, or organism at the time of measurement. The step of correlating the concentration or absolute amount of the redox-active molecule to the state of biological time in the cell, tissue, or organism at the time of measurement can comprise diagnosing a disease in the cell, tissue or organism, including, but not limited to, Huntington's disease, Parkinson's disease and Alzheimer's disease. The redox-active molecule can be selected from the group consisting of lactate, pyruvate, NADP, NADPH, NAD, NADH, reduced glutathione (GSH), oxidized glutathione (GSSG), alpha-tocopherol in oxidized form, beta-tocopherol in oxidized form, gamma-tocopherol in oxidized form, delta-tocopherol in oxidized form, alpha-tocotrienol in oxidized form, beta-tocotrienol in oxidized form, gamma-tocotrienol in oxidized form, delta- tocotrienol in oxidized form, alpha-tocopherol in reduced form, beta-tocopherol in reduced form, gamma-tocopherol in reduced form, delta-tocopherol in reduced form, alpha- tocotrienol in reduced form, beta-tocotrienol in reduced form, gamma-tocotrienol in reduced form, delta-tocotrienol in reduced form, Vitamin K in oxidized form, Vitamin K in reduced form, cytochrome C-oxidized, cytochrome C-reduced, Coenzyme Q10, alpha-tocotrienol quinone in oxidized form, beta-tocotrienol quinone in oxidized form, gamma-tocotrienol quinone in oxidized form, delta-tocotrienol quinone in oxidized form, alpha-tocopherol quinone in oxidized form, beta-tocopherol quinone in oxidized form, gamma-tocopherol quinone in oxidized form, delta-tocopherol quinone in oxidized form, alpha-tocotrienol quinone in reduced form, beta-tocotrienol quinone in reduced form, gamma-tocotrienol quinone in reduced form, delta-tocotrienol quinone in reduced form, alpha-tocopherol quinone in reduced form, beta-tocopherol quinone in reduced form, gamma-tocopherol quinone in reduced form, and delta-tocopherol quinone in reduced form.
[0006] In another embodiment, the invention embraces a method for measuring the state of biological time in a cell, tissue, or organism, comprising measuring the concentration or absolute amount of a two-electron redox-cycling small molecule in oxidized form in the cell, tissue, or organism; measuring the concentration or absolute amount of the two-electron redox-cycling small molecule in reduced form in the cell, tissue, or organism; calculating the ratio of the concentration of the oxidized form to the concentration of the reduced form, or calculating the ratio of the absolute amount of the oxidized form to the absolute amount of the reduced form; and correlating the calculated ratio to the state of biological time in the cell, tissue, or organism at the time of measurement. The step of correlating the calculated ratio to the state of biological time in the cell, tissue, or organism at the time of measurement can comprise diagnosing a disease in the cell, tissue or organism. The two-electron redox- cycling small molecule can be selected from the group consisting of Coenzyme Q10, alpha- tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta-tocotrienol quinone, alpha-tocopherol quinone, beta-tocopherol quinone, gamma-tocopherol quinone, delta-tocopherol quinone, glutathione, Vitamin E, and Vitamin K.
[0007] In another embodiment, the invention embraces a method for altering or modulating the state of biological time in a cell, tissue, or organism, comprising administering an effective amount of a two-electron redox-cycling small molecule to the cell, tissue, or organism. The two-electron redox-cycling small molecule can be selected from the group consisting of Coenzyme Q10, alpha-tocotrienol quinone, beta-tocotrienol quinone, gamma- tocotrienol quinone, delta-tocotrienol quinone, alpha-tocopherol quinone, beta-tocopherol quinone, gamma-tocopherol quinone, delta-tocopherol quinone, glutathione, Vitamin E, and Vitamin K.
[0008] In another embodiment, the invention embraces a method for measuring the state of biological time in an organism, comprising the steps of 1) obtaining a cell, tissue, or bodily fluid sample from the organism; 2) measuring the rate of NQOl activity in the cell, tissue, or bodily fluid; and 3) correlating the rate of NQOl activity to the state of biological time in the cell, tissue, bodily fluid, or organism at the time of measurement. The step of correlating the rate of NQOl activity to the state of biological time in the cell, tissue, bodily fluid, or organism at the time of measurement can comprise diagnosing a disease in the cell, tissue or organism. In one embodiment, the disease is cancer.
[0009] In another embodiment, the invention embraces a method for selecting a cancer treatment in an organism, comprising the steps of 1) obtaining a cell, tissue, or bodily fluid sample from the organism; 2) measuring the rate of NQOl activity in the cell, tissue, or bodily fluid; and 3) using the measured rate of NQOl activity to select a cancer treatment for the cell, tissue, or organism.
[0010] In one embodiment, the invention encompasses a method of modulating biological time by altering the activity of NQOl in a cell, tissue, or organism. In one embodiment, the invention encompasses a method of modulating biological time by increasing the activity of NQOl in a cell, tissue, or organism. In another embodiment, the invention encompasses a method of modulating biological time by decreasing the activity of NQOl in a cell, tissue, or organism. In another embodiment, the invention encompasses a method of modulating biological time by administering a redox-active molecule to the cell, tissue, or organism. In one embodiment, the redox-active molecule is a two-electron redox-cycling small molecule. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIGURE 1 depicts the redox coupling of the enzyme NQOl, where the enzyme oxidizes NADPH and reduces alpha-tocotrienol quinone (ATQ3); ATQ3 then subsequently reduces NBT to NBT Formazan. This reduction can be monitored by A550.
[0012] FIGURE 2 depicts the results of an assay for NQOl, where an increase in absorption at 550 nm commences upon addition of ATQ3 to a solution of NQOl, NADPH, and NBT. The increase terminates upon addition of the NQOl inhibitor dicumarol.
DETAILED DESCRIPTION OF THE INVENTION
[0013] The invention relates to methods of measuring and controlling "biological time" in a living system, such as a cell, tissue, or organism.
[0014] By "biological time" is meant a mechanism by which a living system coordinates or synchronizes biological processes. "Biological time" includes, but is not limited to, circadian rhythms (i.e., cycles of approximately one day, including diurnal/nocturnal cycles), infradian rhythms (i.e., cycles shorter than one day), ultradian rhythms (i.e., cycles longer than one day), and other periodic rhythms. "Biological time" also includes non-cyclical biological processes that have a direct time-dependent component (such as the accumulation of mutations in mitochondrial DNA in a cell; these mutations increase over time since mitochondrial DNA mutations are not repaired) or an indirect time-dependent component (such as telomere length; telomeres in, for example, human cells shorten with each successive round of cell division, and the number of rounds of cell divisions increases with time).
[0015] By "small molecules" are meant molecules with a molecular weight of equal to or less than 1000 Daltons, preferably equal to or less than 600 Daltons.
[0016] Vitamin K includes, but is not limited to, phylloquinone and menaquinone.
[0017] Vitamin E includes all isoforms such as alpha tocopherol, beta tocopherol, gamma tocopherol, delta tocopherol, alpha tocotrienol, beta tocotrienol, gamma tocotrienol and delta tocotrienol.
[0018] Alpha-tocotrienol quinone is abbreviated as aTQ3, a-TQ3, aTQ3, a-TQ3, ATQ3, or A-TQ3.
[0019] The cell, tissue, or organism in which biological time is measured, controlled, altered, or modulated can be, or can be derived from, any living system, including microorganisms, plants, and animals. A bodily fluid obtained from an organism can be blood, plasma, serum, cerebrospinal fluid, saliva, perspiration, semen, urine, stool, sputum, tears, mucus, amniotic fluid or the like.
[0020] In one embodiment of the invention, biological time is observed by measuring the electron capacity and current flow (both individual components and sum total of components) between two chronological time points of a biological system. This measurement provides a characterization of both total collection and individual component changes as a biological system alters its redox states.
[0021] Chronological time and biological time are not directly coupled. Biological time is related to the preservation of the overall redox state of the system, and is defined by its components (such as redox active quinones) and the biological molecules (such as NQOl) which regulate the redox state of these components. As the overall redox state of the system progresses from reductive to oxidative, a biologically system ages as a function of its biological time. This progression of biological time is not linear like chronological time, and occurs often in non-linear steps (often as a function of disease). However, biological time as measured by redox state can often provide information about the relative chronological state of the biological system. For example, when a system has an overall redox potential > +100 mV for its manifold of low molecular weight quinones, then the system has reached an endpoint in both chronological and biological time; that is, the biological system is defined as dead.
[0022] These collections of small molecules are measured in a biological sample using a 16- channel CoulArray detector after a sample has passed through a C18 RP-HPLC system. This measurement can be done in a water/methanol system with 50mM ammonium acetate as a buffer with a flow of 1 ml/min. One possible electrochemical configuration uses a pre- column cell to reduce or oxidize the analytes before separation. The main CoulArray electrochemical detector array is set in 16 steps between -750 mV to 750 mV (vs. std Pt electrode). By switching the pre-column between three states (Off, Reduction or Oxidation) a characterization is obtained of the redox state of the biological sample, and its potential capacity to undergo redox between a -750mV to 750 mV potential.
[0023] Biological time advances, and diseases of aging occur, when either there is not enough total charge in a biological system (measured as Coulombs in a sample between two redox potentials, which can be conceptualized as "lots of batteries with no charge," due, for example, to a deficiency of NQOl to "charge" the system), or there are not enough small molecular weight components to carry the charge (which can be conceptualized as "not enough batteries," due, for example, to a quinone deficiency syndrome). Thus, in one embodiment, the invention encompasses a method of modulating biological time by stimulating production of NQOl in a cell, tissue, or organism. In another embodiment, the invention encompasses a method of modulating biological time by inhibiting production of NQOl in a cell, tissue, or organism. In another embodiment, the invention encompasses a method of modulating biological time by administering a redox-active molecule to the cell, tissue, or organism. In one embodiment, the redox-active molecule is a two-electron redox- cycling small molecule.
[0024] Placing cells under oxidative stress can cause an increase or elevation in expression of NQOl of about five-fold to fifty- fold. This places a demand on the supply of redox-active molecules, such as quinones, in the cells, which are rapidly depleted by the increased NQOl expression. Accordingly, when increased expression of NQOl is detected in a cell, tissue, or organism, administration of redox-active molecules, such as quinones, can alleviate this depletion and relieve the stress on the cell, tissue, or organism, essentially "resetting" the redox biological clock.
[0025] Thus, in one embodiment, the invention embraces a method of treating an oxidative stress disorder, comprising 1) measuring the increase or elevation in expression of NQOl in a cell, tissue, or organism; and 2) if expression of NQOl is increased or elevated, administering a therapeutically effective amount of a redox-active molecule. In one embodiment, the redox-active molecule is alpha-tocotrienol quinone (ATQ3). In another embodiment, the redox-active molecule is menadione. In another embodiment, the redox-active molecule is duroquinone. In another embodiment, the redox-active molecule, such as ATQ3, is administered to the cell or tissue in an amount sufficient to provide a concentration of between about 100 nanomolar to about 10 micro molar in the extracellular fluid contacting the cell or tissue, or between about 100 nM and about 1 uM, or between about 1 uM and about 10 uM. In another embodiment, the redox-active molecule, such as ATQ3, is administered to the organism in an amount sufficient to provide a concentration of between about 10 nanomolar to about 10 micromolar in the plasma of the organism, or between about 10 nM and about 100 nM, or between about 10 nM and about 50 nM, or between about 100 nM and about 250 nM, or between about 100 nM and about 500 nM, or between about 100 nM and about 1 uM, or between about 1 uM and about 10 uM, or between about 1 uM to about 5 uM, or between about 1 uM to about 5 uM. In another embodiment, the redox-active molecule, such as ATQ3, is administered when the expression of NQOl has increased about or at least about 25%, about or at least about 50%, about or at least about 75%, about or at least about 100%, about or at least about 200%, about or at least about 300%, about or at least about 400%, or about or at least about 500% over the normal level of expression under unstressed conditions.
[0026] A cell, tissue, or organism can be monitored continuously or periodically for elevated levels of expression of NQOl, and, upon increase of the level of expression of NQOl above a certain threshold amount (for example, an increase of about or at least about 25%, about or at least about 50%, about or at least about 75%, about or at least about 100%, about or at least about 200%, about or at least about 300%, about or at least about 400%, or about or at least about 500%), a redox-active molecule, such as ATQ3, can be administered to the cell, tissue, or organism, in an amount as recited above.
[0027] Administration of a redox-active molecule to a cell, tissue, or organism under oxidative stress can decrease the level of expression of NQOl. Thus, NQOl can be viewed as a lagging indicator of the stress on the cell, tissue, or organism. In one embodiment, the invention embraces a method of 1) measuring the increase (or elevation) in expression of NQOl in a cell, tissue, or organism; and 2) if expression of NQOl is increased (or elevated), administering a therapeutically effective amount of a redox-active molecule, wherein the redox-active molecule is administered in an amount sufficient to reduce the expression of NQOl to a level about or at least about 10%, about or at least about 25%, about or at least about 30%, about or at least about 40%, about or at least about 50%, about or at least about 60%, about or at least about 70%, about or at least about 75%, about or at least about 80%, about or at least about 90%, or about or at least about 95% of the increased (or elevated) level of NQOl expression.
Measurement of NQOl activity
[0028] An isolated human NQOl assay has been developed which can measure NQOl activity, including measurement of the different rates of reduction of small molecule quinones. This assay uses the redox-active dye NBT to measure the transfer of electrons from NAD(P)H, to NQOl and then to the quinone; see Example 1. Only the electrons from a dihydroquinone will reduce the redox active dye, which is used as a proxy for electron flux through the system. Using the assay, it was established that electron transfer to the dye was different with different small molecule quinones, i.e. that different quinones speed up, or slow down, electron flow via NQOl. In order of speed of reduction,
menadione>duroquinone>a-TQ3> CoQ4. The difference in the rate of reaction dye reduction was 100-fold (from fastest to slowest). Through enzymes, such as NQOl and a collection of quinones, the overall redox state of a system can be maintained or reestablished to regulate biological time.
[0029] Using the human NQOl isolated enzyme assay, it has been established that: a) a-TQ3 docks and interacts with NQOl and that; b) inhibitors of NQOl block the activity of the a- TQ3-NQ01 complex.
[0030] An isolated human NQOl assay has also been developed that directly measures the reduction of quinone using the 16-channel CoulArray system. In this assay, 10 uL samples are taken from a 1 mL reaction mixture of the following (1.5 units of NQOl, 250 uM
NAD(P), in a buffer of 50 mM Tris-HCl at ph 7.4, with 0.08% TX-100, at 25°C in a HPLC vial -sealed with an airtight septum). At t=0, a-TQ3 is added (20 uM final concentration from a ImM DMSO stock) to the reaction, and at various time points 10 uL of sample are injected and measured for amount of initial oxidized quinone and the increase of reduced quinone. These rates follow what was observed indirectly with the redox active dye based assay.
[0031] WO 2008/062105 and US 2010/0159458 discuss a method for selecting a cancer therapy based on a subject's genetic background, by determining the presence of a mutant or non-functional NQOl gene or gene product. The NQOl assay described herein can provide an alternative procedure for carrying out the method disclosed in WO 2008/062105 and US 2010/0159458.
[0032] Biological time is driven, at least in part, by the reductive potential of a cellular hydride coupled through a quinone reductase, such as the human quinone reductases, for example NQOl, NQ02, PIG3, or Zeta-crystalline. This coupling produces a temporal (time domain) cycling of a two-electron redox-cycle small molecule. The cycling can be viewed as an internal "biological clock" or "biochemical clock." Key regulator proteins, such as p53, tyrosinase, p33, p73, ornithine decarboxylase, and eIF4Gl, can directly bind the quinone reductase to sample the time domain established by the two-electron redox cycle. Other regulatory proteins or genes may be affected indirectly by the quinone reductase. The quinone metabolites of tocopherols are essential components of this clock system. Other important components include NQOl, p53, and the 20S proteasome complex.
[0033] The timing of this system (the clock) can be modified by introducing different two- electron redox-cycling small molecules into the system, which differ by their redox potential and shape from the endogenous redox molecules comprising the clock. For example, ubiquinone, or Coenzyme Q10, is present in virtually all human cells. Alpha-tocotrienol quinone differs from CoQIO in the number of isoprene units in its tail and in the substituents on the quinone ring, and administration of alpha-tocotrienol quinone illustrates one method of modifying the redox-based clock.
[0034] This time cycling (clock) of the two-electron system can also be modified by the binding of non-redox cycling molecules which bind and compete with the quinone binding site within the quinone reductase. Resveratrol is one example of such a molecule.
[0035] Some other examples of modification of the timing of this system (clock) can also be achieved for example by changing the turnover rate ("frequency") of NQOl, by genetic modification of NQOl, or by magnetic fields interacting with unpaired electrons (for example, as occurs with the magnetic compass of birds; see
w/w/w.ks.uiuc.edu/Research/cryptochrome). Proteins sensitive to magnetic fields are also involved in circadian rhythms.
[0036] Disease states are associated with dysregulation of the time domain and/or clock established by the two-electron quinone redox cycle and the key regulator proteins which bind and sample this time domain. Disturbances in circadian rhythms have been observed in Parkinson's disease (Fertl et al., J. Neural Transm. [P-D Sect] (1993) 5:227-234;
Huntington's disease (Morton et al., J. Neuroscience (2005) 25(1): 157-163; and Alzheimer's disease (van Someren et al., Biological Psychiatry (1996) 40:259-270. Re-establishment of proper biological time regulation can aid in treatment of these disorders.
[0037] The time domain established by this system is also involved in the regulation of biological time such as aging. For example, when the NQOl cellular hydride source is restricted, the clock function of the NQOl - quinone redox cycle is affected, which extends lifespan.
[0038] In one embodiment, the present invention encompasses measurement of one or more redox-active molecules in order to assess the state of a cell, tissue or organism in terms of its biological time. The redox-active molecule can be any redox-active molecule in the cell, tissue or organism, such as one or more two-electron redox-cycling small molecules (for example, lactate/pyruvate, NADP/NADPH, NAD/NADH, reduced glutathione GSH versus oxidized glutathione GSSG, Vitamin E in oxidized and reduced forms, Vitamin K in oxidized and reduced forms), or a larger molecule such as a protein having a prosthetic group (for example, cytochrome C-oxidized and cytochrome C-reduced). The concentration of the oxidized and reduced form can be measured and the ratio of the oxidized to reduced form calculated (or the ratio of the reduced form to the oxidized form can be calculated).
Alternatively, the concentration of the oxidized form can be measured and may provide sufficient information in the absence of measurement of the reduced form, or the concentration of the reduced form can be measured and may provide sufficient information in the absence of measurement of the oxidized form. In another embodiment, the absolute amount of the oxidized and reduced form can be measured and the ratio of the oxidized to reduced form calculated (or the ratio of the reduced form to the oxidized form can be calculated). Alternatively, the absolute amount of the oxidized form can be measured and may provide sufficient information in the absence of measurement of the reduced form, or the absolute amount of the reduced form can be measured and may provide sufficient information in the absence of measurement of the oxidized form.
[0039] In one embodiment, the present invention encompasses the measurement of one or more redox-active molecules in order to assess the state of a cell, tissue or organism in terms of its biological time, in order to diagnose, determine, or stage the progression of, a disease associated with aging.
[0040] In one embodiment, the present invention encompasses use of one or more of two- electron redox-cycling small molecules (such as alpha-tocotrienol quinone) in order to alter or modulate this cellular clock system. If measurement of the state of the cellular clock system has been performed, the one or more two-electron redox-cycling small molecules used to alter or modulate the cellular clock system can be the same as, or different from, the molecule or molecules measured to establish the state of the cellular clock system.
[0041] In one embodiment, the present invention encompasses use of one or more two- electron redox-cycling small molecules (such as alpha-tocotrienol quinone) two-electron redox-cycling small molecules to treat disease states (such as Leigh syndrome) which result from cellular clock dysregulation. The method involves administration of one or more two- electron redox-cycling small molecules (such as alpha-tocotrienol quinone) to cells, tissues, or organisms in an amount effective to treat a disease state.
[0042] In one embodiment, the present invention encompasses use of one or more two- electron redox-cycling small molecules (such as alpha-tocotrienol quinone) to alter or modulate biological time. In one embodiment, the present invention encompasses use of one or more two-electron redox-cycling small molecules (such as alpha-tocotrienol quinone) to affect aging. The method involves administration of one or more two-electron redox-cycling small molecules (such as alpha-tocotrienol quinone) to cells, tissues, or organisms in an amount effective to affect aging.
[0043] In one embodiment, the one or more two-electron redox-cycling small molecules can be used to slow down biological time, thus extending life time in cells, tissues or organisms. In one embodiment, the one or more two-electron redox-cycling small molecules can be used to slow down time, thus extending biological life time in cells, tissues or organisms, and retarding aging in cells, tissues, or organisms, for example, to retard aging in healthy cells, tissues, or organisms. In one embodiment, the one or more two-electron redox-cycling small molecules can be used to slow down biological time, thus extending biological life time in cells whose mitochondria are diseased, retarding the progress of the mitochondrial disease. In another embodiment, the one or more two-electron redox-cycling small molecules can be used to accelerate biological time, in cells, tissues, or organisms, for example, to accelerate aging in cells, tissues, or organisms that are diseased or that cause disease, such as cancer cells or pathogenic microorganisms.
[0044] In one embodiment, the present invention encompasses use of one or more redox- active molecules selected from the group consisting of lactate, pyruvate, NADP, NADPH, NAD, NADH, reduced glutathione (GSH), oxidized glutathione (GSSG), Vitamin E in oxidized form, Vitamin E in reduced form, Vitamin K in oxidized form, Vitamin K in reduced form, cytochrome C-oxidized, cytochrome C-reduced, Coenzyme Q10, alpha- tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta-tocotrienol quinone, alpha-tocopherol quinone, beta-tocopherol quinone, gamma-tocopherol quinone, and delta-tocopherol quinone, in order to measure or assess the state of the cellular clock system (i.e., biological time, biological clock).
[0045] In one embodiment, the present invention encompasses use of one or more two- electron redox-cycling small molecules selected from the group consisting of Coenzyme Q10, alpha-tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta- tocotrienol quinone, alpha-tocopherol quinone, beta-tocopherol quinone, gamma-tocopherol quinone, delta-tocopherol quinone, glutathione, Vitamin E, and Vitamin K, in order to alter or modulate the cellular clock system (i.e., biological time).
EXAMPLES
Example 1
NQOl activity assay using alpha-tocotrienol quinone
[0046] NAD(P)H-quinone oxidoreductase 1 (NQOl; EC 1.6.5.2) is a flavoprotein that reversibly catalyzes the oxidation of NADH or NADPH by various quinones and oxidation- reduction dyes (see w/w/w. ncbi.nlm.nih.gov/mesh/68016660). The enzyme can be inhibited by dicumarol (dicoumarol; bishydroxycoumarin).
[0047] Nitroblue tetrazolium (NBT; 2-[2-methoxy-4-[3-methoxy-4-[3-(4-nitrophenyl)-5- phenyltetrazol-2-ium-2- yl] phenyl] phenyl] - 3 -(4-nitrophenyl) -5 -phenyltetrazol-2-ium dichloride) can be reduced to a formazan compound (NBT Formazan) by ATQ3. NBT has a measureable UV/VIS change due to its reduction, does not cross-couple with either
NAD(P)H or the reduced flavin of NQOl, and is reduced by the dihydroquinone
(benzenediol) form of ATQ3 (2-(3-hydroxy-3,7,l l,15-tetramethylhexadeca-6,10,14-trienyl)- 3,5,6-trimethylbenzene-l,4-diol) generated by NQOl, with regeneration of ATQ3 in quinone form.
[0048] Identification of NBT permitted development of an assay for NQOl activity, where NQOl oxidizes NAD(P)H to NAD(P)+, with concomitant reduction of NQOl (oxidized) to NQOl (reduced) as hydride is transferred to NQOl. NQOl (reduced) then reduces ATQ3 to its hydroquinone (benzenediol) form, with concomitant regeneration of NQOl (oxidized). The reduced alpha tocotrienol hydroquinone then reduces NBT to NBT Formazan, with regeneration of alpha tocotrienol quinone. The formation of NBT Formazan can be monitored by A550, while the disappearance of NAD(P)H can be monitored at A340.
[0049] The graph in Figure 2 shows an assay using NQOl. A solution of 120 uM NAD(P)H, 120 uM NBT, and 1.5 units NQOl in assay buffer (50 mM Tris'HCl, pH= 7.4, 0.08% TX- 100, 2% DMSO) is monitored at 550 nm (appearance of NBT Formazan) and 340 nm
(disappearance of NAD(P)H).
[0050] Absorbance at 550 nm remains steady in the absence of ATQ3. Upon addition of ATQ3 (1 uM), the absorbance steadily increases. Addition of dicumarol (1 uM), an inhibitor of NQOl, terminates the increase in A550. The NQOl assay is linear in both the rate of formation of NBT Formazan (Figure 2) and the rate of disappearance of NAD(P)H (not shown).
[0051] Although the foregoing invention has been described in some detail by way of illustration and examples for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced without departing from the spirit and scope of the invention. Therefore, the description should not be construed as limiting the scope of the invention.
[0052] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. Links to World-Wide Web sites have been deactivated by including forward slashes in their Web addresses as follows: w/w/w. They can be accessed by removing the forward slashes to regenerate the leading www in the address.

Claims

CLAIMS What is claimed is:
1. A method for measuring the state of biological time in a cell, tissue, or organism, comprising:
measuring the concentration or absolute amount of a redox-active molecule in oxidized form, reduced form, or both oxidized form and reduced form, in the cell, tissue, or organism;
correlating the concentration or absolute amount of the redox-active molecule to the state of biological time in the cell, tissue, or organism at the time of measurement.
2. The method of claim 1, wherein the step of correlating the concentration or absolute amount of the redox-active molecule to the state of biological time in the cell, tissue, or organism at the time of measurement comprises diagnosing a disease in the cell, tissue or organism.
3. A method for measuring the state of biological time in a cell, tissue, or organism, comprising:
measuring the concentration or absolute amount of a two-electron redox- cycling small molecule in oxidized form in the cell, tissue, or organism;
measuring the concentration or absolute amount of the two-electron redox- cycling small molecule in reduced form in the cell, tissue, or organism;
calculating the ratio of the concentration of the oxidized form to the concentration of the reduced form, or calculating the ratio of the absolute amount of the oxidized form to the absolute amount of the reduced form; and correlating the calculated ratio to the state of biological time in the cell, tissue, or organism at the time of measurement.
4. The method of claim 1, wherein the step of correlating the calculated ratio to the state of biological time in the cell, tissue, or organism at the time of measurement comprises diagnosing a disease in the cell, tissue or organism.
5. The method of any of claims 1 or 2, wherein the redox-active molecule is selected from the group consisting of lactate, pyruvate, NADP, NADPH,
NAD, NADH, reduced glutathione (GSH), oxidized glutathione (GSSG),
Vitamin E in oxidized form, Vitamin E in reduced form, Vitamin K in
oxidized form, Vitamin K in reduced form, cytochrome C-oxidized,
cytochrome C-reduced, Coenzyme Q10, alpha- tocotrienol quinone, beta- tocotrienol quinone, gamma-tocotrienol quinone, delta-tocotrienol quinone,
alpha-tocopherol quinone, beta-tocopherol quinone, gamma-tocopherol
quinone, and delta-tocopherol quinone.
6. The method of any of claims 3 or 4, wherein the two-electron redox- cycling small molecule is selected from the group consisting of Coenzyme
Q10, alpha-tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol
quinone, delta-tocotrienol quinone, alpha-tocopherol quinone, beta-tocopherol
quinone, gamma-tocopherol quinone, delta-tocopherol quinone, glutathione,
Vitamin E, and Vitamin K.
7. A method for altering or modulating the state of biological time in a cell,
tissue, or organism, comprising:
measuring the state of biological time in a cell, tissue, or organism; and
administering an amount of a two-electron redox-cycling small molecule to
the cell, tissue, or organism effective to alter the state of biological time in the
cell, tissue, or organism.
8. The method of claim 7, wherein the two-electron redox-cycling small
molecule is selected from the group consisting of Coenzyme Q10, alpha- tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone,
delta-tocotrienol quinone, alpha-tocopherol quinone, beta-tocopherol quinone,
gamma-tocopherol quinone, delta-tocopherol quinone, glutathione, Vitamin E,
and Vitamin K.
9. A method for measuring the state of biological time in an organism, comprising the steps of:
obtaining a cell, tissue, or bodily fluid sample from the organism; measuring the rate of NQOl activity in the cell, tissue, or bodily fluid; and correlating the rate of NQOl activity to the state of biological time in the cell, tissue, bodily fluid, or organism at the time of measurement.
10. The method of claim 9, wherein the step of correlating the rate of NQOl activity to the state of biological time in the cell, tissue, bodily fluid, or organism at the time of measurement comprises diagnosing a disease in the cell, tissue or organism.
11. The method of claim 10, wherein the disease is cancer.
12. A method for selecting a cancer treatment in an organism, comprising the steps of: obtaining a cell, tissue, or bodily fluid sample from the organism;
measuring the rate of NQOl activity in the cell, tissue, or bodily fluid; and
using the measured rate of NQOl activity to select a cancer treatment for the cell, tissue, or organism.
13. A method of modulating biological time in a cell, tissue, or organism,
comprising altering the activity of NQOl in the cell, tissue, or organism.
14. The method of claim 13, wherein altering the activity of NQOl in the cell,
tissue, or organism comprises increasing the activity of NQOl in the cell,
tissue, or organism.
15. The method of claim 13, wherein altering the activity of NQOl in the cell,
tissue, or organism comprises decreasing the activity of NQOl in the cell,
tissue, or organism.
16. A method of modulating biological time in a cell, tissue, or organism,
comprising the steps of:
measuring the expression of NQOl in the cell, tissue, or organism;
comparing the level of expression of NQOl in the cell, tissue, or organism to a
normal or average value established for said cell, tissue, or organism; and
administering a redox-active molecule to the cell, tissue, or organism if the
level of expression of NQOl is elevated above a normal or average value.
17. The method of claim 16, wherein the elevation of the level of expression of NQOl is at least 100%.
18. The method of claim 16, wherein the redox-active molecule is alpha- tocotrienol quinone.
19. The method of claim 16, wherein the redox-active molecule is administered in an amount sufficient to provide a concentration of redox- active molecule between about 1 uM and 10 uM in the extracelluar fluid of a cell or tissue, or a concentration of redox-active molecule between about 10 nM and 10 uM in the plasma of the organism.
20. The method of claim 16, wherein the redox-active molecule is administered in an amount sufficient to lower the expression level of NQOl to about 50% below its elevated value.
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