WO2015009883A1

WO2015009883A1 - Activators of nad-dependent protein deacteylases and uses thereof

Info

Publication number: WO2015009883A1
Application number: PCT/US2014/046939
Authority: WO
Inventors: Bud M. NELSON
Original assignee: The Hamner Institutes
Priority date: 2013-07-18
Filing date: 2014-07-17
Publication date: 2015-01-22

Abstract

Provided are compositions and kits comprising a Nicotiinamide Antagonist and a sirtuin- activating compound in an effective amount to activate a NAD-dependent protein deacteylase, and methods of using the composition to treat a disease, disorder, or condition having dysregulation of NAD-dependent protein deacteylase levels or activity.

Description

ACTIVATORS OF NAD-DEPENDENT PROTEIN DEACETYLASES AND USES

THEREOF

FIELD OF THE INVENTION

The present invention relates to the discovery that certain compounds, including drugs and their metabolites, can activate NAD-dependent protein deacetylases. The invention also relates to combinations or pharmaceutical compositions comprising a compound selected from each of two or more different classes of activators of NAD-dependent protein deacetylase for modulating NAD-dependent protein deacetylase activity as well as the activity of biological pathways affected by NAD-dependent protein deacetylase activity. The invention also relates to pharmaceutical compositions and combinations comprising a compound selected from each of two or more classes of activators of NAD- dependent protein deacetylase for use in treating diseases, disorders, or conditions in which NAD-dependent protein deacetylase is otherwise modulated to promote the disease, disorder, or condition.

BACKGROUND OF THE INVENTION

Post-translational modification of cellular proteins regulates a wide variety of cellular functions. One important mechanism for post-translational modification is by reversible acetylation of proteins at lysine residues, such as by the opposing activities of protein acetyltransferases and protein deacetylases which act on both histone and nonhistone proteins. One class of protein deacetylases is known as sirtuins. Some of the sirtuins, particularly sirtuin 1 (SIRT1 ), sirtuin 2 (SIRT2), sirtuin 3 (SIRT3), sirtuin 5 (SIRT5), sirtuin 6 (SIRT6), and sirtuin 7 (SIRT7), have protein deacetylase activity. Such sirtuins are typically described as NAD-dependent protein deacteylases because, unlike other known protein deacetylases in which acetyl-lysine residues are simply hydrolyzed, the sirtuin-mediated deacetylation reaction couples lysine deacetylation to NAD (nicotine adenine dinucleotide) hydrolysis. The sirtuins acting as NAD-dependent protein deacetylases, have an NAD-binding pocket. In this reaction, NAD, the acetylated protein substrate and the sirtuin enzyme form a complex, and as a result of the enzymatic reaction (i) NAD is cleaved into nicotinamide and an ADP-ribose product; and (ii) deacetylated protein. In the case of high concentrations of free nicotinamide, nicotinamide can (i) occupy the NAD-binding pocket of the NAD-dependent protein deacetylase and may block the conformational change and subsequent cleavage of NAD, (ii) react with a substrate intermediate (O-alkyl-amidate intermediate) in a process known as nicotinamide exchange or base exchange, and, hence (iii) slow or inhibit deacetylase activity or the rate thereof. Accordingly, dependence of NAD-dependent protein deacetylases on NAD links their enzymatic activity directly to the energy status of the cell via one or more of (a) the cellular NAD:NADH ratio, and (b) the absolute levels of NAD, NADH, or nicotinamide.

Sirtuins, as NAD-dependent protein deacetylases, are found in, and regulate important biological pathways in, organisms including bacteria and eukaryotes. In humans, sirtuins are called SIRT 1 to SIRT7 ("SIRT"- silent information regulators). In humans, SIRT1 (known in the art to comprise amino acid sequences selected from SEQ ID NO:1 and SEQ ID NO:2, and isoforms thereof having most, if not all, of the amino acids in sequence as shown in either SEQ ID NO:1 or SEQ ID NO:2) is an NAD- dependent protein deacetylase localized in the cytoplasm and nucleus, and can repress transcription in the nucleus by various different mechanisms. In another example, relevant to the invention herein, an NAD-dependent protein deacetylase, Rv1 151 c (known in the art to comprise an amino acid sequence of SEQ ID NO:3, including isoforms thereof having most, if not all, of the amino acids in sequence as shown in SEQ ID NO:3), has been identified in Mycobacteria tuberculosis. Nicotinamide has been shown to be a noncompetitive inhibitor of both SIRT1 and Rv1 151 c, an indication that these two enzymes share similar chemical structure, and act under similar mechanisms and concentrations. One way SIRT1 can repress transcription is through deacteylation of transcription factors. For example, it has been shown in human cells that SIRT1 decreased acetylation of transcription factor Nrf2 (nuclear factor erythroid-related factor 2, comprising an amino acid sequence of SEQ ID NO:4, including isoforms thereof having most, if not all, of the amino acids in sequence as shown in SEQ ID NO:4). Deacetylation of Nrf2 enhanced localization of Nrf2 to the cytoplasm, as well as decreased Nrf2- dependent gene transcription. When Nrf2 is acetylated, there is increased nuclear localization of Nrf2, and promoted is binding of Nrf2 to the response element specific for Nrf2 (ARE or Antioxidant Response Element), with resultant increased Nrf2-dependent gene transcription of genes containing ARE in their promoter region.

Genes having functional ARE include cytoprotective genes, such as genes encoding antioxidant enzymes, phase II detoxification enzymes, and multidrug resistant proteins. Thus, NRF2-mediated adaptive antioxidant response plays pivotal roles against oxidative/electrophilic stress, and in chemical detoxification (including drug metabolism). For example, Nrf2 has been shown to regulate the transcription of genes that encode drug metabolizing enzymes, including UGT (UDP-glucuronosyltransferase) and NQQ1 (NAD(P)H quinone oxidoreductase 1 ), cytochrome P450 2A5 (CYP2A5), and glutathione S-transferase (GST). Nrf2 also regulates the expression of genes that are involved in direct reduction of reactive oxygen species (ROS), including superoxide dismutase, catalase, and glutathione peroxidases. Additionally, Nrf2 induces genes involved in reduction of oxidized proteins, such as thioredoxin-1 , thioredoxin reductase-1 , and sulfiredoxin, as well as genes encoding enzymes that synthesize glutathione (GSH); i.e., γ-glutamate-cysteine ligase catalyze subunit (Gclc) and the modifier subunit (Gclm).

SUMMARY OF THE INVENTION

Provided herein are novel combinations of activators of NAD-dependent protein deacetylases, and methods of use thereof. The invention is based, in part, upon the discovery of compounds which can activate NAD-dependent protein deacetylases, and the correlation between the structure of these compounds and their function to activate NAD-dependent protein deacetylases by a mechanism comprising acting as an antagonist of nicotinamide. Without being bound to any particular mechanism, it is believed that antagonists of nicotinamide inhibit or reduce the base exchange process that slows or inhibits NAD-dependent protein deacetylase activity; i.e., thereby promoting NAD-dependent protein deacetylase activity. These compounds comprise a class of activators of NAD-dependent protein deacetylases ("Nicotinamide Antagonist") distinct in chemical structure and function as compared to the class of human SIRT1 activator compounds known as sirtuin activating compounds ("STACs") comprising polyphenols (e.g., resveratrol, fisetin, chlorogenic acid) and their analogs (e.g., SRT1720, SRT1460, SRT2183), quinoxaline compounds, stilbene compounds (ester analogs of resveratrol) and the like (see, for example, Formulas ll-VI). For example, STACs are believed to work by an allosteric mechanism in which the binding of activator is enhanced for enzyme- substrate complexes or by the activator promoting a conformational change that produces enzyme-substrate complexes, in promoting NAD-dependent protein deacetylase activity.

In one aspect of the invention provided is a combination of activators of NAD- dependent protein deacetylases comprising one or more Nicotinamide Antagonists (a first class of activators of NAD-dependent protein deacetylases) and one or more STACs (a second class of activators of NAD-dependent protein deacetylases). The combination of the invention may further comprise a pharmaceutically acceptable carrier. Such combination may comprise (a) a single pharmaceutical composition comprised of an Nicotinamide Antagonist and an STAC, and may further comprise a pharmaceutically acceptable carrier; or (b) separate compositions, which can be administered simultaneously or sequentially, comprising a first composition comprising an Nicotinamide Antagonist (which may further comprise a pharmaceutically acceptable carrier); and a second composition comprising a STAC (which may further comprise a pharmaceutically acceptable carrier). Optionally, the combination of the invention may be used in combination therapy that may comprise administering additional compositions comprising other therapeutic agents used to treat the same disease (e.g., antitubercular drugs for treating tuberculosis).

Also provided is a method for activating NAD-dependent protein deacetylase, in an individual (e.g., a mammal, such as a human) in need thereof, comprising administering to the individual a Nicotinamide Antagonist and a STAC in an amount effective to induce NAD-dependent protein deacetylase activity. Such method of combination therapy may comprise administering (a) a single pharmaceutical composition comprised of an Nicotinamide Antagonist and an STAC, and may further comprise a pharmaceutically acceptable carrier; or (b) separate compositions, which can be administered simultaneously or sequentially, wherein one composition comprises an Nicotinamide Antagonist (which may further comprise a pharmaceutically acceptable carrier), and another composition comprises a STAC (which may further comprise a pharmaceutically acceptable carrier). Optionally, the method of combination therapy may comprise administering additional compositions comprising other therapeutic agents used to treat the same disease (e.g., antitubercular drugs for treating tuberculosis, etc.).

Provided is a method for inhibiting Nrf2-ARE activity, in an individual in need thereof, comprising administering to the individual a Nicotinamide Antagonist and a STAC in an amount effective to induce NAD-dependent protein deacetylase activity and inhibit one or more of Nrf2-ARE activity, and PPARy activity. Such method of combination therapy may comprise administering (a) a single pharmaceutical composition comprised of an Nicotinamide Antagonist and an STAC, and may further comprise a

pharmaceutically acceptable carrier; or (b) separate compositions, which can be administered simultaneously or sequentially, comprising a first composition comprising an Nicotinamide Antagonist (which may further comprise a pharmaceutically acceptable carrier), and a second composition comprising a STAC (which may further comprise a pharmaceutically acceptable carrier).

In another aspect of the present invention, provided is a method for treating (prophylactically and/or therapeutically) tuberculosis or mycobacterial infection in an individual in need thereof, the method comprising the step of administering to the individual a Nicotinamide Antagonist and a STAC in an amount effective to induce NAD- dependent protein deacetylase activity. The treatment may further comprise combination therapy with one or more additional known antitubercular drugs, thereby treating (preventing or treating) an active, reactivation, or inactive Mycobacterium tuberculosis infection. A known antitubercular drug may include but is not limited to isoniazid, pyrazinamide, pyrazinamine, pyrazine-2-thio carboxamide, N-hydroxymethyl pyrazine thiocarboxamide, N-substituted 3-aminopyrazine-2,5-dicarbonitriles, sparfloxacin, ethambutol dihydrochloride, ethionamide, amikacin, aminosalicylic acid, capreomycin, cycloserine, kanamycin, rifamycins (e.g., rifampicin, rifapentine and rifabutin), streptomycin, ofloxacin, ciprofloxacin, clarithromycin, azithromycin, bedaquiline, thioacetazone, SQ 109, and fluoroquinolones, or salt thereof.

Another aspect of the invention is pharmaceutical compositions, or medicaments, comprising at least one compound that is a Nicotinamide Antagonist and at least one compound that is a STAC, in a particular dosage or formulation for delivering an amount of the compound effective to induce NAD-dependent protein deacetylase activity (as known to a skilled medical practitioner); and may further comprise a pharmaceutically acceptable carrier. In one aspect of the aspects of the invention, of the Nicotinamide Antagonist and STAC used to produce the pharmaceutical composition or medicament of the invention, at least one of the Nicotinamide Antagonist or STAC is non-naturally occurring, or both the Nicotinamide Antagonist and STAC may be naturally occurring provided such combination of naturally occurring compounds itself is not found in or known to exist in nature.

Other aspects, objects and features of the invention will be apparent from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 A is a graph showing that isoniazid ("INH") suppresses Nrf2-ARE activity in 3T3-L1 preadipocytes in a concentration-dependent manner under basal ("Veh") conditions, and that treatment with an Nrf2 activator (iAs³⁺) modulated the inhibition of Nrf2- ARE activity by isoniazid.

FIG.1 B is a graph showing that isoniazid ("INH", 10 mM) suppresses Nrf2-ARE- dependent gene expression of glutamate-cysteine ligase catalytic subunit ("Gclc") in 3T3- L1 preadipocytes under basal ("Veh") conditions, and that treatment with an Nrf2 activator (tBHQ, 50 μΜ) modulated the inhibition of Nrf2-ARE activity by isoniazid.

FIG.1 C is a graph showing that isoniazid ("INH", 10 mM) suppresses Nrf2-ARE- dependent gene expression of NAD(P)H dehydrogenase [quinone] 1 ("Nqo1 ") activity in 3T3-L1 preadipocytes under basal ("Veh") conditions, and that treatment with an Nrf2 activator (tBHQ, 50 μΜ) modulated the inhibition of Nrf2-ARE activity by isoniazid.

FIG.1 D is a graph showing that isoniazid ("INH", 10 mM) suppresses Nrf2-ARE- dependent gene expression of Heme oxygenase ("Ho1 ") activity in 3T3-L1 preadipocytes under basal ("Veh") conditions, and that treatment with an Nrf2 activator (tBHQ, 50 μΜ) modulated the inhibition of Nrf2-ARE activity by isoniazid.

FIG 2A is a graph showing that isoniazid ("INH") inhibits Nrf2-ARE activity in human hepatocellular liver carcinoma HepG2 cells in a concentration-dependent manner under basal ("Veh") conditions, and that treatment with an Nrf2 activator (iAs³⁺) modulated the inhibition of Nrf2-ARE activity by isoniazid.

FIG. 2B is a graph showing that isoniazid ("INH") suppresses Nrf2-ARE-dependent gene expression of Heme oxygenase ("Ho1 ") activity in HepG2 cells in a concentration- dependent manner under basal ("Veh") conditions, and that treatment with an Nrf2 activator (iAs³⁺) modulated the inhibition of Nrf2-ARE activity by isoniazid.

FIG. 3 is a graph showing that ethionamide (ETH) suppresses Nrf2-ARE activity in HepG2 cells in a concentration-dependent manner under basal ("Vehicle") conditions, and that treatment with an Nrf2 activator (iAs³⁺) modulated the inhibition of Nrf2-ARE activity by ethionamide.

FIG.4A is a graph showing that ethionamide (ETH) suppresses Nrf2-ARE-dependent gene expression (mRNA expression as a percent of the Control with Vehicle) of Heme oxygenase ("HO ') activity in THP-1 cells in a concentration-dependent manner under basal ("Veh") conditions, and that treatment with an Nrf2 activator (iAs³⁺) modulated the inhibition of Nrf2-ARE activity by ethionamide.

FIG.4B is a graph showing that ethionamide (ETH) suppresses Nrf2-ARE-dependent gene expression (mRNA expression as a percent of the Control with Vehicle) of glutamate-cysteine ligase catalytic subunit ("GCLM") activity in THP-1 cells in a concentration-dependent manner under basal ("Veh") conditions, and that treatment with an Nrf2 activator (iAs³⁺) modulated the inhibition of Nrf2-ARE activity by ethionamide. FIG.4C is a graph showing that ethionamide (ETH) suppresses Nrf2-ARE-dependent gene expression (mRNA expression as a percent of the Control with Vehicle) of sulfiredoxin ("SRX") activity in THP-1 cells in a concentration-dependent manner under basal ("Veh") conditions, and that treatment with an Nrf2 activator (iAs³⁺) modulated the inhibition of Nrf2-ARE activity by ethionamide.

FIG. 5 is an illustration of chemical structures of compounds having a formula of Formula I identified as Nrf2-ARE-inhibitors. FIG. 6A is a graph showing the effects of rifampicin ("Rif"), isoniazid ("INH"), or isoniazid together with rifampicin, on PPARYI mRNA levels (expressed as percent of Control) as compared to the vehicle assay control ("Control").

FIG. 6B is a graph showing the effects of rifampicin ("Rif"), isoniazid ("INH"), or isoniazid together with rifampicin, on PPARy2 mRNA levels (expressed as percent of Control) as compared to the vehicle assay control ("Control").

FIG. 7 is a schematic showing representation of an activated SIRT1 and effects on downregulating PPARy and Nrf2 activity (black arrow), and of dysregulation of NAD- dependent protein acetylase as shown with increased activation of PPARy and effects on downregulating SI RT1 (white arrow).

DETAILED DESCRIPTION OF THE INVENTION

Provided is a combination of activators of NAD-dependent protein deacetylases comprising one or more Nicotinamide Antagonists and one or more STACs. The combination of the invention may further comprise a pharmaceutically acceptable carrier. The combinations may be used to activate a NAD-dependent protein deacetylase, including in a synergistic (more than additive) manner, and are useful in diseases, disorders, or conditions in which use of the combination to activate a NAD-dependent protein deacetylase may treat the diseases, disorder, or condition. It was unexpectedly discovered that heterocyclic compounds used in the treatment of tuberculosis can activate NAD dependent protein deacetylases by acting as nicotinamide antagonists. As shown herein, a Nicotinamide Antagonist comprises a heterocyclic compound having a hydrazide moiety or carboxamide moiety (typically, as a side chain), and is selected from a compound represented by Formula I. As illustrative, non-limiting examples, such compounds comprise ethionamide (2-ethylpyridine-4-carbothioamide), pyrazinamide (pyrazine-2-carboxamide), and isoniazid (isonicotinohydrazide). In one aspect of the invention, a Nicotinamide Antagonist is selected from compounds represented by Formula I, including Formula IA and Formula IB.

In one aspect of the invention, a Nicotinamide Antagonist is selected from compounds represented by Formula IA, and a pharmaceutically acceptable salt thereof.

Formula IA wherein:

A is N or C;

B is N or C;

R1 or R2 or R3 are each independently selected from H, (Ci-C₆)alkyl, CONH₂, CONHNH₂, CSNH₂, S0₂NH₂, NH₂, NHNH₂, CHCHCONH₂, CHCHCONHNH₂, or COCH₃;

wherein at least one of R1 , R2, and R3 is selected from CONH₂, CONHNH₂, CSNH₂, S0₂NH₂, NH₂, NHNH₂, CHCHCONH₂, or CHCHCONHNH₂; and

the dashed lines represent optional double bonds; with the proviso that the compound of Formula IA is not nicotinamide (also known as 3-pyridinecarboxamide), isonicotinamide (also known as pyridine-4-carboxamide), or nicotinamide adenine dinucleotide (NAD) (i.e., excluded from a compound represented by Formula IA, and thus excluded from being a Nicotinamide Antagonist in methods and compositions of the invention, is nicotinamide; NAD+; and isonicotinamide).

In one aspect of the invention, a Nicotinamide Antagonist is selected from compounds la IB, and a pharmaceutically acceptable salt thereof.

Formula IB

wherein:

A is O or N;

B is N or C;

R1 is selected from CONH₂, CONHNH₂, CSNH₂, S0₂NH₂, NH₂, NHNH₂, CHCHCONH₂, or

CHCHCONHNH₂;

R2 is absent if B is NH;

if B is C, R2 is absent or selected from CH₃, CH₂CH₃, NH₂, or NHNH₂; and

the dashed lines represent optional double bonds; with the proviso that the compound of Formula IB is not nicotinamide (also known as 3-pyridinecarboxamide), isonicotinamide (also known as pyridine-4-carboxamide), or nicotinamide adenine dinucleotide (NAD) (i.e., excluded from a compound represented by Formula IB, and thus excluded from being a Nicotinamide Antagonist in methods and compositions of the invention, is nicotinamide; NAD+; and isonicotinamide).

A preferred Nicotinamide Antagonist may be used as an activator of NAD-dependent protein deacetylase in accordance with the invention to the exclusion of an Nicotinamide Antagonist other than the preferred Nicotinamide Antagonist. Many compounds of Formula I are commercially available, as well as chemicals used as starting materials in their chemical syntheses. Many synthesis methods are known for preparing compounds of Formula I according to the invention such as by using standard organic chemical synthesis methods well known in the art. These and/or other well-known methods may be modified and/or adapted in known ways in order to facilitate the synthesis of additional compounds within the scope of the present invention. Compounds as described herein may be purified by any of the means known in the art, including but not limited to chromatographic means, such as high pressure liquid chromatography (HPLC), preparative thin layer chromatography, flash column chromatography and ion exchange chromatography. Any suitable stationary phase can be used, including normal and reversed phases as well as ionic resins. From chemical libraries or collections of chemical compounds, a chemical compound can be screened for identifying a Nicotinamide Antagonist by using assays for determining inhibition of NAD-dependent protein deacetylase activity as known in the art, including those described herein, as well as selecting compounds to test in the assays which have a chemical structure represented by Formula I. A compound of Formula I can be tested (in the presence of nicotinamide) for the level of activation of NAD-dependent protein deacetylase such as by measuring the rate of fluorescent-free activation substrate deacetylation, or by measuring the rate of NAD+ hydrolysis, by using methods known to those skilled in the art. In another example, and with respect to SIRT1 , the level of activation of NAD-dependent protein deacetylase may be measured by determining the effects of deacetylation of Nrf2 (e.g., by degree of inhibition of Nrf2 activity) as described herein in more detail. A sirtuin-activating compound ("STAC") has a core structure centering around a bicyclic heterocycle (as shown by Formulas II, IV, V, Va, and VI), and is typically selected from either a benzimidazole, imidazothiazole, quinoxaline, and thiazolopyridine; or a core structure comprising a stilbene (as shown by Formula III).

In one aspect of the invention, a sirtuin activating compound ("STAC") is selected from compounds of the formula of any one of Formula II, Formula III

wherein is selected from -(CH₂)₃— CH₃, and -(CH₂)CH(CH₃)₂; and R₂ is piperidine or — (CH₂)₂— NH— CH₃; or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof. Examples of compounds with this formula, and methods of making them, are known to those skilled in the art (for example, see U.S. Published Patent Appl. No. US 20130102009; the contents of which are herein incorporated by reference).

Formula I II

wherein

AT is selected from H and (CO)P ;

A₂ is selected from H and (CO)R₂;

A₃ is selected from H and (CO)R₃;

and wherein at least one of A^ A₂, and A₃ is different from another;

RT and R₂ when present are each independently selected from alkyl with at least two carbon atoms, unsubstituted aryl, and aralkyi;

R₃ when present is selected from alkyl with at least two carbon atoms, aryl, and aralkyi, and when R₃ is alkyl, it is unsubstituted straight or branched alkyl;

or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof.

Examples of compounds with this formula, and methods of making them, are known to those skilled in the art (for example, see U.S. Patent 7,714,161 ; the contents of which are herein incorporated by reference).

Formula IV

wherein

R is H or CH3;

R¹ is selected from a substituted or unsubstituted nitrogen-containing heterocyclmethyl group containing a second heteroatom selected from a nitrogen or oxygen, or a morpholine, or a morpholinomethl group, or a 1 ,2,4-triazolylmethyl group;

R² is H or CH3; and

R³ is an unsubstituted pyridyl group;

or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof. Examples of compounds with this formula, and methods of making them, are known to those skilled in the art (for example, see U.S. Patent Nos. 7,829,556 and 8,247,565; the contents of which are herein incorporated by reference).

Formula V

wherein

R¹⁹ is

wherein each of Z₁₀, Z , Z₁₂, and Z₁₃ is independently selected from CR²⁰ or CR¹ ; wherein zero to one R²⁰ is a solubilizing group; and

zero to one R¹ is an optionally substituted C1 -C3 straight or branched alkyl;

each R²⁰ is independently selected from H or a solubilizing group;

R²¹ is -NR¹-C(0)-; each R¹ is independently selected from H, or an optionally substituted C1 -C3 straight or branched alkyl;

R³¹ is selected from an optionally substituted monocyclic or bicyclic aryl, or an optionally substituted monocyclic or bicyclic heteroaryl;

or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof. Examples of compounds with this formula, and methods of making them, are known to those skilled in the art (for example, see U.S. Patent No. 7,345,178; the contents of which are herein incorporated by reference). Some examples of compounds of Formula V

Formula Va

wherein

RT is selected from a bicyclic heterocycle that is selected from a naphthalene and a quinoxaline, and a benzene ring substituted with between 2 and 3 methoxy groups; R₂ is selected from a piperazine, and a pyrrole substituted with an OH group, or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof. Examples of compounds with this formula, and methods of making them are known to those skilled in the art (for example, see U.S. Patent No. 7,345,178) and include selective SIRT1 inhibitors known as SRT1720, SRT1460, and SRT2183, one or more of which is commercially available.

Other quinoxaline compounds that are STACs include 3-benzenesulfonyl-1 -(4-fluro- phenyl)-1 H-pyrrolo[2,3-b]quinoxalin-2-ylamine; 2 -amino-1 -(2-ethyl-phenyl)-1 H- pyrrolo[2,3b]quinoxaliine-3-carboxylic acid (tetrahydro-furan-2-ylmethyl)-amine; and 2 - amino-1 -(3-methoxy-propyl)-1 H-pyrrolo[2,3b]quinoxaliine-3-carboxylic acid

cyclopentamide.

Formula VI

wherein

ring A is selected from

wherein "^*" represents a portion of ring A bound to phenyl, and

" J^⁴^^ " represents a portion of ring A bound to C=0 in the compound;

1 is selected from hydrogen, or

R³ is selected from h drogen, methoxypropyl, methox rop-1 -ynyl,

; and

at least one of R¹ or R³ comprises a nitrogen-containing saturated heterocyclyl portion. Examples of compounds with this formula, and methods of making them, are known to those skilled in the art (for example, see U.S. Patent No. 8,343,997; the contents of which are herein incorporated by reference).

Other known STACs include naturally-occurring compounds such as a polyphenol, methylxanthine, or stilbene, illustrative examples of which may include resveratrol, fisetin, chlorogenic acid, or ester analogs thereof such as 3,5,4'-trihydroxy-trans-stilbene. While the terms used in the description of the invention are believed to be well understood by one of ordinary skill in the pharmaceutical arts, definitions, where provided herein, are set forth to facilitate description of the invention, and to provide illustrative examples for use of the terms.

As used herein, the terms "a", "an", and "the" mean "one or more", unless the singular is expressly specified (e.g., singular is expressly specified, for example, in the phrase "a single formulation").

The term 'alkyl" is used herein to refer to a hydrocarbon containing normal, secondary, tertiary, or cyclic carbon atoms (e.g., linear saturated aliphatic hydrocarbon groups, branched saturated aliphatic hydrocarbon groups, or a saturated or unsaturated non- aromatic hydrocarbon mono or multi-ring system (e.g., cycloalkyl)). When the term "alkyl" is used without reference to a number of carbon atoms, it is to be understood to refer to a C(i)-C(io >alkyl ; e.g., a C(i), C^, Cpj, C(4), C(5), C(6), Cpj, C^), C(g )Or C(io >alkyl.

The term "aryl" is used herein to refer to cyclic, aromatic hydrocarbon groups which have 1 to 3 aromatic rings, for example phenyl or naphthyl. The aryl group may have fused thereto a second or third ring which is a heterocyclo, cycloalkyl, or heteroaryl ring, provided in that case the point of attachment will be to the aryl portion of the ring system. "Heteroaryl" refers to an aryl group in which at least one of the carbon atoms in the aromatic ring has been replaced by a heteroatom selected from oxygen, nitrogen and sulphur. The nitrogen and/or sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. The heteroaryl group may be a 5 to 6 membered monocyclic, 7 to 1 1 membered bicyclic, or 10 to 16 membered tricyclic ring system. The term "aralkyl" is used herein to refer to an aryl-alkyl- group in which the aryl and alkyl are as defined herein. In one aspect of the aspects of the invention, preferably an aralkyl comprises a lower alkyl group.

The terms "first", "second", and "additional", are used herein for purposes of

distinguishing between two compounds, or between two or more compositions, or between two or more steps of a method, as will be clearer from the description.

The term "individual" is used herein to mean a mammal, and more preferably, a human The term "individual having a mycobacterial infection" is herein to mean a mammal, and more preferably a human, infected with one or more strains or species of Mycobacterium; and in one aspect the infection is with M. tuberculosis. The infection may be inactive (latent, M. tuberculosis infection without manifested disease symptoms), reactivated, or active (M. tuberculosis infection with manifested disease symptoms). The infection may also comprise a multi-drug resistant strain of M. tuberculosis. "Tuberculosis" refers to the infectious disease caused by the mycobacterial infection. Diagnosis of M. tuberculosis infection, or tuberculosis, is commonly achieved using a skin test, which involves intradermal exposure to tuberculin PPD (protein-purified derivative); wherein a measurable induration at the injection site by 48-72 hours after injection indicates exposure to mycobacterial antigens. Confirmation of M. tuberculosis infection can also be achieved using one or more additional methods known in the art including, but not limited to, body fluid (sputum, gastric washings, laryngeal swab, bronchoalveolar lavage, bronchial washings) smears and cultures for acid-fast bacilli, and polymerase chain reaction or gene probe tests for detecting M. tuberculosis.

The term "known antitubercular drug" is used herein to refer to compounds that have been shown to exhibit antibacterial activity against mycobacteria as known to those skilled in the art, or approved for therapeutic use as drugs for treating mycobacterial infections or tuberculosis caused by mycobacterial infections in humans or animals. Known antitubercular drugs also include a drug that exhibits antibacterial activity against M. tuberculosis or has a primary mechanism of antibacterial action other than Nrf2-ARE- inhibition, as understood by or known to those skilled in the art without utilization of the present invention. Known antitubercular drugs include but are not Imited to isoniazid, pyrazinamide, pyrazinamine, pyrazine-2-thio carboxamide, N-hydroxymethyl pyrazine thiocarboxamide, N-substituted 3-aminopyrazine-2,5-dicarbonitriles, sparfloxacin, ethambutol dihydrochloride, ethionamide, amikacin, aminosalicylic acid, capreomycin, cycloserine, kanamycin, rifamycins (i.e., rifampin, rifapentine and rifabutin), streptomycin, ofloxacin, ciprofloxacin, clarithromycin, azithromycin, bedaquiline, SQ 109, thioacetazone, fluoroquinolones, or a salt thereof. With respect to primary known mechanism of therapeutic action, the antitubercular drugs that exhibit Nrf2-ARE-inhibitory activity are drugs that have been shown to mediate a therapeutic effect by a mechanism other than Nrf2-ARE-inhibition. For example, ethionamide is an antitubercular agent that inhibits mycolic acid synthesis; isoniazid inhibits the synthesis of mycolic acids, an essential component of the bacterial cell wall; pyrazinamide inhibits membrane transport function at acid pH in Mycobacterium tuberculosis, as well as inhibits the activity of purified FAS fatty acid synthase; rifampin is a broad spectrum antibacterial that suppresses the initiation of RNA synthesis by binding to DNA-dependent RNA polymerase and inhibiting its activity; and sparf loxacin inhibits DNA gyrase which is needed for DNA topology, replication, repair, deactivation, and transcription. Thus, the primary or known mechanisms of therapeutic action of antitubercular drugs is generally known or considered to be by a mechanism other than Nrf2-ARE-inhibitory activity.

The term "metabolic syndrome" is used herein to mean a disorder of energy utilization and storage that is believed to increase the risk of developing cardiovascular disease and diabetes. Metabolic syndrome is diagnosed by a co-occurrence of three out of five of the following medical conditions: abdominal (central) obesity, elevated blood pressure, low high-density cholesterol (HDL) levels, high serum triglycerides, and elevated fasting plasma glucose (levels of the respective measured analyte being compared to reference levels from healthy individuals, e.g., individuals lacking metabolic syndrome).

The term "NAD-dependent protein deacetylase" means a protein deacetylase in which the protein-mediated deacetylation reaction couples lysine deacetylation to NAD. In one aspect of aspects of the invention, NAD-dependent protein deacetylase is used to preferably refer to one or more of human SIRT1 (substantially comprising an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2), human SIRT2 (substantially comprising an amino acid sequence of SEQ ID NO:7), and mycobacterial Rv1 151 c (substantially comprising an amino acid sequence of SEQ ID NO:3) as in the case of mycobacterial infection or tuberculosis.

The term "activating NAD-dependent protein deacetylase" refers to the inducing effect of an activator of NAD-dependent protein deacetylase (e.g., an NAD Antagonist or sirtuin activating compounds ("STAC") or a combination thereof) on level or activity of an NAD- dependent protein deacetylase. Thus, an activator of NAD-dependent protein deacetylase has the ability to increase the level or activity of NAD-dependent protein deacetylase, resulting in increased deacetylase activity, as compared to activity of NAD- dependent protein deacetylase in the absence of such activator. A combination of an Nicotinamide Antagonist and an STAC according to the invention, for activating NAD- dependent protein deacetylase, may result in an increase in the level or activity of an NAD-dependent protein deacetylase by a factor of at least about 2, 5, 10, 20, 50, 100, or greater fold, as compared to the level or activity of the NAD-dependent protein deacetylase in the absence of such combination. Such increase can be measured in vitro such as in a cellular assay or in a cell-based assay, and compared to a basal level (e.g., measured in the absence of the activator of NAD-dependent protein deacetylase in the same assay system), using methods known to those skilled in the art. For example, a compound's ability to activate NAD-dependent protein deacetylase can be assessed and quantitated using commercially available fluorescence-based assay, such as the "SIRT1 fluorometric drug discovery assay kit" (Enzo Life Sciences), using the manufacturer's directions. In the assay, a fluorescent-labeled substrate of the NAD-dependent protein deacetylase is mixed in the presence of the NAD-dependent protein deacetylase and in the presence of the compound(s) being assessed for its ability to activate NAD- dependent protein deacetylase (e.g., STAC or Nicotinamide Antagonist). However, if assessing for a compound as a Nicotinamide Antagonist, nicotinamide is added to the reaction (also provided in the kit). The assay is run in parallel (e.g., separate microtiter well) but in the absence of the compound(s) ("control well"). Deacetylation of the substrate sensitizes the substrate to the addition of a detection reagent. The reaction is subjected to excitation at 360 nanometers (nm), and detection at 460 nm. Detection of increased NAD-dependent protein deacetylase activity in the presence of the

compound(s) as compared to the absence of the compound(s) (control well) is an indication that the compound(s) has the ability to activate NAD-dependent protein deacetylase.

The term "non-naturally occurring" used in reference to a compound means that the compound is not known to exist in nature or that does not exist in nature. The term "naturally occurring" when used in connection with compounds refers to a compound which is found in nature. It is apparent to those skilled in the art that a naturally occurring compound can be modified or engineered by a human or by an engineered organism to be structurally or chemical different to form a non-naturally occurring compound.

Prodrugs of the compounds of Formula I, or salts thereof, are included within the scope of the invention. The term "prodrug", as used herein, refers to a compound that is transformed in vivo (e.g., by a metabolic, physiological, or chemical process) to yield a compounds of Formula I, or a pharmaceutically acceptable salt, hydrate or solvate of the compound. Prodrugs, made by synthesizing one or more prodrug moieties as part of an active compound, can serve to enhance one or more of solubility, absorption, lipophilicity, pharmacodynamics, pharmacokinetics, and efficacy, as compared to the active compound without the one or more prodrug moieties. Various forms of prodrugs are known in the art. Examples of prodrugs of the compounds of the invention include an in vivo cleavable ester of a carboxy group (e.g., lower alkyl esters, cycloalkyl esters, lower alkenyl esters, benzyl esters, mono-or di-substituted lower alkyl esters, and the like); or S-acyl and O-acyl derivatives of thiols, alcohols, or phenols. "Prodrug moiety" refers to a labile functional group, including but not limited to a protective group, which can be removed or reduced from the active compound during a process elected from one or more of metabolism, systemic circulation, intracellular, hydrolysis, or enzymatic cleavage. Enzymes which are capable of an enzymatically activating a phosphonate prodrug include, but are not limited to, amidases, esterases, phospholipases, cholinesterases, and phosphases. Prodrug moieties can serve to enhance solubility, absorption and lipophilicity to optimize drug delivery, bioavailability and efficacy. A prodrug moiety may include an active metabolite or drug itself. Exemplary prodrug moieties include the hydrolytically sensitive or labile acyloxymethyl esters -CH₂OC(=0)R³ and acyloxymethyl carbonates esters -CH₂OC(=0)OR³ where R³ is d-C₆ alkyl, d-C₆ substituted alkyl, C₆- C₂₀ aryl or C₆-C₂₀ substituted aryl. Other examples of prodrug moieties include addition of a halogen (e.g., fluoro group), carbon replacement with nitrogen ("aza" compounds), carbocyclic analogs, and chloroacetyl (2b-d) derivatives.

The terms "purified" or "isolated" for a compound or composition refers to the physical state of the compound or composition following isolation from a synthetic process or purification step described herein or well known to those in the art, and in sufficient purity to be characterizable by standard analytical methods described herein or well known in the art.

The terms "salt" or pharmaceutically acceptable salt", as used herein, refers to inorganic or organic salts of a compound. These salts can be prepared, for example, by reacting a compound of Formula I, or a compound of Formulas ll-VI disclosed herein, with an amount of acid or base, such as an equivalent amount, and in a medium such as one in which the salt formed then precipitates, or in an aqueous medium followed by lyophilization. Representative salts include bisulfate, sulfate, benzene sulfonate, camphorsulfonate, laurylsulphonate, methanesulfonate, toluenesulfonate,

naphthalenesulformate, acetate, trifluoracetate, benzoate, borate, butyrate, citrate, formate, fumarate, hydorbromide, hydrochloride, hydroiodide, lactate, laurate, maleate, malonate, mesylate, nitrate, oxalate, phosphate, hexafluorophosphate, propionate, salicylate, stearate, succinate, tartrate, thiocyanate, and the like. The salts may include base salts based on the alkali and alkaline earth metals, such as calcium, sodium, lithium, magnesium, and potassium; or with organic bases such as with organic amines (e.g., dicyclohexylamine, t-butyl amine, methylamine, dimethylamine, triethylamine, ethylamine, procaine, morpholine, N-methylpiperidine, dibenzylamine, and the like); or as an ammonium salt.

The compounds disclosed herein may exist in a solvated form or unsolvated form. Solvates of a compound disclosed in the invention may be formed in the synthetic process in which the compound becomes physically associated with one or more solvent molecules (e.g., such as by ionic and/or covalent bonding) or, optionally, may be converted to a solvate such as by dissolving the compound in desired amounts of a solvent of choice (e.g., organic solvent, water, or mixtures thereof) in forming a solution, heating the solution to a temperature higher that ambient temperature, and cooling the solution at a rate sufficient to form crystals of the solvate, which may then be further isolated using methods known the art. Examples of suitable solvents include

methanolates, ethanolates, hydrates (where the solvent molecule is water), and the like. The compounds of Formulas l-VI may contain asymmetric or chiral centers, and thus exist in different stereoisomeric forms. All stereoisomers (e.g., geometric isomers, optical isomers, and the like), enantiomeric forms, diastereomeric forms, tautomeric forms, positional isomers, of the compounds disclosed in the invention are embraced within the scope of the invention. A first conformational form of a compound can be separated from a second and different conformational form of the compound using methods well known in the chemical arts such as by chromatography, crystallization, and methods of synthesis which selectively result in a particular desired conformational form.

The term "pharmaceutically acceptable carrier" is used herein to mean any one or more of a compound or composition or carrier medium useful in any one or more of

administration, delivery, storage, stability of a composition or compound described herein. These carriers are known in the art to include, but are not limited to, a diluent, water, saline, suitable vehicle (e.g., one or more of liposome, microparticle, nanoparticle, emulsion, polymer, or capsule), buffer, medical parenteral vehicle, excipient, aqueous solution, suspension, solvent, emulsions, detergent, chelating agent, solubilizing agent, salt, colorant, polymer, hydrogel, surfactant, emulsifier, adjuvant, filler, preservative, stabilizer, oil, binder, disintegrant, absorbant, flavor agent, and the like as broadly known in the pharmaceutical art, and combinations thereof. The terms "treat", "treats", or "treating" , as used herein, embrace one or more of preventative (prophylactically) or therapeutically (palliative). "Preventative" is an art recognized term for reducing, delaying, or preventing the onset of symptoms of, or a process associated, with the particular disease, condition or disorder, or treatment, as a result of the administration of a composition, as compared to an individual whom did not receive the composition.

The phrase "medically effective amount" generally means an amount of a composition or compound that treats the particular disease, condition or disorder; ameliorates, relieves, or decreases one or more symptoms associated with the particular disease, condition or disorder, or treatment; or delays or prevents the onset of symptoms of, or a pathological process associated, with the particular disease, condition or disorder, or treatment. More specifically, a "medically effective amount" of a composition comprising a Nicotinamide Antagonist and an STAC means an amount of the composition effective to induce NAD- dependent protein deacetylase activity in a cell to which, and more preferably in an individual to whom, the composition is administered, as compared to the level of activity in the absence of the composition. In one aspect, the NAD-dependent protein

deacetylase activity induced comprises human SIRT1 . In another aspect, where the individual is an individual having an M. tuberculosis infection or tuberculosis, the NAD- dependent protein deacetylase activity induced comprises human SIRT1 , Rv1 151 c produced by Mycobacteria tuberculosis, or a combination thereof. As will be discussed in more detail herein, in some diseases, disorders, and conditions (collectively referred to as "disease"), the NAD-dependent protein deacetylase activity is reduced, and such reduction can play a role in disease pathogenesis. The reduction can be from one or more processes including, but not limited to, suppression of NAD-dependent protein deacetylase gene expression, regulation of the mRNA for NAD-dependent protein deacetylase, or modification of the NAD-dependent protein deacetylase protein itself (e.g., cleavage, or post-translational modification), caused by or occurring in the disease process. Hence, under such circumstances and with such effects, NAD-dependent protein deacetylase is dysregulated in the disease, particularly, in cells or body tissues involved in the disease. Use of a composition of the invention to induce or increase NAD- dependent protein deacetylase activity sufficiently to counteract or modulate NAD- dependent protein deacetylase dysregulation in a disease can be determined by methods known in the art (as will also be apparent from the description and figures herein).

A medically effective amount of a compound used in the invention, or a

composition of the invention, will depend on such factors as the mode of administration, the formulation for administration, disease to be modulated, the size and health of the individual to receive such a composition, and other factors which can be taken into consideration by a medical practitioner whom is skilled in the art of determining appropriate dosages for treatment. An amount of compound used in the invention in a composition to be administered may vary from 0.01 milligrams to about 500 milligrams, and more typically from about 1 milligram per day to about 300 milligram per day. One skilled in the art can apply known principles and models of drug delivery and

pharmacokinetics to ascertain a likely range of dosages to be tested in preclinical and clinical studies for determining a medically effective amount of a compound used in the invention. A pharmaceutically acceptable carrier, used in a composition according to the invention, may facilitate one or more of storage, stability, administration, and delivery, of the composition. The carrier may be particulate, so that the composition may be in, for example, powder or solid form. The carrier may be in a semi-solid, gel, or liquid formula, so that the composition may be ingested, injected, applied, or otherwise administered. The carrier may be gaseous, so that the composition may be inhaled.

For oral administration of a composition containing a compound according to the invention, suitable formulations may be presented in the form of tablets, caplets, capsules, and the like, in which typically the compound of the invention may be present in a predetermined amount as a powder, granules, solution, or suspension as the sole active agent, or in combination with an additional one or more pharmaceutical agents. As known in the art, such oral formulations typically involve one or more of a binder (e.g., syrup, sorbitol, gum, corn starch, gelatin, acacia), a filler (e.g., lactose, sugar, starch, calcium phosphate), an excipient (e.g., dicalcium phosphate), a disintegrating agent (e.g., vegetable starch, alginic acid), a lubricant (e.g., magnesium stearate), a flavoring agent (sweetening agent, natural or artificial flavors). Such oral formulations may be coated or uncoated to modify their disintegration and/or absorption. Coating may be performed using conventional coating agents and methods known in the art.

The mode of administration of a compound or composition according to the invention to an individual (such as a human) in need of such composition or compound may be any mode known in the art to be suitable for delivering a pharmaceutical composition, and particularly suitable for increasing NAD-dependent protein deacetylase activity, and may include but is not limited to, intravenously, intraperitoneally, orally, subcutaneously, intramuscularly, intranasally, transdermal^, by inhalation, by perfusion, and by peristaltic techniques. Provided herein is a combination therapy comprising administering to an individual a Nicotinamide Antagonist and an STAC (and optionally, further comprising a pharmaceutically acceptable carrier). In such combination therapy, a Nicotinamide Antagonist, and an STAC may be administered concurrently, sequentially, or in regimen alternating between a Nicotinamide Antagonist, an STAC. Such combination therapy may optionally include one or more additional therapeutic agents for treating the disease that is targeted by the composition or combination of the invention (e.g., bacterial infection, hepatic steatosis, metabolic syndrome, etc.). The structure of such additional therapeutic agents, a Nicotinamide Antagonist, an STAC, and their generic or trademark names, are readily available to those skilled in the art, such as from the standard compendium of drugs (e.g., The Merck Index) or from the applicable pharmaceutical company's web site, as well as dosages applicable for treatment (see also The Physician's Desk Reference). Alternatively, the doses and dosage regimen of an additional therapeutic agent, a Nicotinamide Antagonist, and an STAC used in accordance with the invention in combination therapy, can be determined by a physician, taking into account the medical literature, the health, age and sex of the patient, the disease or condition or disorder to be treated, the mode of administration and dosing schedule, and other relevant considerations. Generally, dosages of such compounds can range from about 0.1 mg to 1000 mg per day, with more specific dosages dependent on the aforementioned factors.

Accordingly, also provided herein is a pharmaceutical composition or medicament comprising a medically effective amount of a Nicotinamide Antagonist, an STAC, and optionally administered with one or more additional therapeutic agents; and optionally further comprising a pharmaceutically acceptable carrier.

EXAMPLE 1

Discovered and identified is a novel class of compounds with strong inhibitory effects on Nrf2 activity (including downstream genes having an ARE ). These compounds include a panel of antitubercular agents, such as isoniazid, ethionamide, ethambutol dihydrochloride, rifampicin, ethionamide, and sparfloxacin; and other chemicals, including 4-aminobenzoic hydrazide, aminopyrazine, cyclohexanecarboxamide, 2-furoic hydrazide, phenylhydrazine, phenylacetic hydrazide, pyrazinecarboxamide, p-toluic hydrazide, and 4-(aminomethyl)piperidine (see, e.g., Table 1 ). These compounds decrease ARE- luciferase activity, in a concentration-dependent manner in treated cells, under basal and arsenite-treated conditions. These Nrf2 inhibitors suppress Nrf2-ARE activity, and suppress induction of ARE-driven gene expression. However, no change in Nrf2 protein level was observed. These chemical modulators of Nrf2-ARE activity were identified by a series of chemical screenings using an assay in which an ARE-luciferase reporter is stably expressed in cells in which there is confirmed constitutive activation of Nrf2-ARE activity. These cells include mouse preadipocyte 3T3-L1 cell line; mouse insulinoma MIN6 cell line; human keratinocyte HaCaT cell line; and human hepatocellular cancer cell line, HepG2 cells. A commercially available ARE-luciferase reporter, in ready-to-transduce lentiviral particles, was used for assessing when the Nrf2 pathway is activated or inhibited by a drug or chemical, via detection of any modulation of luciferase reporter activity which can then be measured quantitatively. This type of assay has been validated in the art to measure and represent induction or inhibition of Nrf2-ARE activity taking place in cells in the body.

Lentiviral transduction of 3T3-L1 , HaCaT and HepG2 cells was performed based on manufacturer's protocol. Briefly, 24 hours before transduction, the cells to be transduced were plated in 6-well plates at 40-50% confluency in complete cell culture medium. The following day, hexadimethrine bromide, a transduction enhancer, was added to each well at a concentration of 8 \g/m\, and viral particles were added to each well at a concentration of 2 x 10⁵ transducing units/ml. After overnight incubation, medium containing viral particles was removed and replaced with fresh medium containing 2 μg/ml puromycin. Cells were grown to -90% confluence and sub-cultured in medium containing puromycin. The 3T3-L1 cells, HaCaT cells, MIN6 cells, and HepG2 cells, with stable expression of ARE-luciferase reporter, were used to identify Nrf2-ARE activators and inhibitors, and more particularly, Nrf2 inhibitors.

Assessed was the ability of a chemical compound (including drugs) to inhibit Nrf2- ARE activity under basal conditions (i.e., no added exogenous Nrf2 activator; absence of exogenous Nrf2 activator). For those chemical compounds identified as having Nrf2- ARE-inhibitory activity, also assessed was the ability of an Nrf2 activator to modulate the Nrf2-ARE-inhibitory activity of the compound (e.g., induce Nrf2 activation which then lessens, prevents or inhibits (in "modulating") Nrf2-ARE-inhibition). Briefly, chemical compounds were individually added to the cells, and incubated for 24 hours under basal conditions (no added Nrf2 activator), or with tBHQ-treated or sodium arsenite-treated cells (6 hour treatment, 5-10 μΜ iAs³⁺ or 50 μΜ tBHQ, each a known Nrf2 activator), and measured was luciferase activity, as compared to assay controls. The luciferase activity was measured by a commercially available luciferase reporter assay system according to the manufacturer's protocol. The luciferase activity was normalized to protein content or cell viability. To confirm an inhibitory effect on ARE as observed by a decrease in luciferase activity (as compared to the assay control), the compound was also tested for its ability to inhibit, in a concentration dependent manner, cell expression of multiple ARE- dependent genes, including one or more of H01 (Heme oxygenase 1), GCLC

(Glutamate— cysteine ligase catalytic subunit), GCLM (Glutamate— cysteine ligase modifier subunit), Nqo1 (NAD(P)H dehydrogenase [quinone] 1) and SRX (sulfiredoxin 1) by using real-time quantitative reverse transcription polymerase chain reaction (RT- qPCR) and commercially available primers. An inhibitory effect on expression of multiple ARE-dependent genes was used as an indication of inhibition of Nrf2-ARE activity.

First, cytotoxicity of isoniazid (a widely used antitubercular drug) in 3T3-L1 cells and HepG2 cells was determined by exposing the cells to various concentrations of isoniazid, ranging from 1 mM to 200 mM, for 24 hours, and subsequently determining cell viability by a commercially available MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay. From this determination, non-cytotoxic

concentrations of isoniazid ranging from 1 mM to 50 mM were tested, as was an equal volume of cell culture without isoniazid ("Vehicle") for comparison, in the ARE-luciferase reporter assay. As shown in FIGs. 1 A and 2A, isoniazid ("INH") exhibited a

concentration-dependent inhibitory effect on ARE-luciferase activity in 3T3-L1 cells and HepG2 cells, respectively, under basal conditions. However, as shown in FIGs. 1 A and 2A, cells treated with both isoniazid and Nrf2 activator (iAs³⁺-treated) exhibited substantial ARE-luciferase activity, indication that the Nrf2 activator was capable of modulating the Nrf2-ARE-inhibitory effect of isoniazid. The Nrf2-ARE-inhibitory effect of isoniazid was also observed in HaCaT cells stably expressing the same ARE-luciferase reporter assay. The inhibitory activity of isoniazid was confirmed by decreased expression of multiple ARE-dependent genes, including GCLC (FIG. 1 B), NQ01 (FIG. 1 C) and H01 (FIG. 1 D, and FIG. 2B) under basal conditions. The ability of an Nrf2 activator (as illustrated by fert-butylhydroquinone (tBHQ) or iAs³⁺) to modulate the Nrf2-ARE-inhibitory activity of isoniazid was confirmed by an increased expression of multiple ARE-dependent genes, including GCLC (FIG. 1 B), NQ01 (FIG. 1 C) and H01 (FIG. 1 D, and FIG. 2B) in cells treated with both the Nrf2 activator and the compound having Nrf2-ARE-inhibitory activity, as compared to expression of the ARE-dependent genes in cells treated with the compound having Nrf2-ARE-inhibitor activity alone (e.g., under basal conditions).

Using these methods and the ARE-luciferase reporter assay described herein, another antitubercular agent ethionamide (ETH), in non-cytotoxic concentrations, also displayed a concentration-dependent inhibitory effect on ARE-luciferase activity under basal conditions in HepG2 cells (FIG. 3). Additionally, as shown in FIG. 3, HepG2 cells treated with both ethionamide (ETH) and an Nrf2 activator ("iAs ) exhibited substantial ARE-luciferase activity, indicating that the Nrf2 activator was capable of modulating the Nrf2-ARE-inhibitory effect of the compound having Nrf2-ARE-inhibitory activity.

Confirmation of the Nrf2-ARE-inhibitory effect mediated by ETH was shown by the ability of ETH treatment to significantly decrease the expression of multiple ARE-dependent genes, including H01 (FIG. 4A), GCLM (FIG. 4B), and sulfiredoxin (SRX, FIG. 4C) in THP-1 cells under basal conditions. Likewise, confirmation of the ability of an Nrf2 activator to modulate the Nrf2-ARE-inhibitory activity of a compound having Nrf2-ARE- inhibitory activity (e.g., ETH) was shown by an increased expression of multiple ARE- dependent genes, including H01 (FIG. 4A), GCLM (FIG. 4B), and sulfiredoxin 1 (SRX, FIG. 4C) in THP-1 cells treated with both the Nrf2 activator and the drug having Nrf2- ARE-inhibitory activity, as compared to expression of the ARE-dependent genes in cells treated with the drug having Nrf2-ARE-inhibitory activity alone (e.g., under basal conditions).

By using the same methods, it was demonstrated that (a) antitubercular agents other than isoniazid and ethionamide, including ethambutol dihydrochloride, rifampicin, and sparfloxacin, are Nrf2-ARE-inhibitors as demonstrated by the concentration- dependent inhibitory effect on ARE-luciferase activity under basal conditions (Table 1 ); (b) that an Nrf2 activator can modulate the Nrf2-ARE-inhibitory activity of a compound having Nrf2-ARE-inhibitory activity, including antitubercular agents; and (c) additionally, a number of other compounds represented by either Formula I, particularly heterocyclic compounds having a hydrazide side chain or carboxamide side chain, including 4- aminobenzoic hydrazide, aminopyrazine, 2-furoic hydrazide, cyclohexane-carboxamide, phenylhydrazine, phenylacetic hydrazide, pyrazinecarboxamide, and p-toluic hydrazide, were discovered inhibit Nrf2 activity as demonstrated by the concentration-dependent inhibitory effect on ARE-luciferase activity under basal conditions and iAs³⁺-treated or tBHQ-treated conditions (Table 1 ). As apparent from FIG. 5 showing the chemical structure of these newly discovered Nrf2 inhibitors, many of these compounds are represented by Formula I, particularly heterocyclic compounds having a hydrazide side chain or carboxamide side chain, suggesting a structure-function relationship between such chemical representation and the ability to inhibit Nrf2-ARE activity. Based on this discovery, and structure-function relationship, additional compounds represented by Formula I may be screened for activity for Nrf2 inhibitory activity. Illustrative examples of such compounds may include pyrrole-2 carboxamide, and pyrrole-2 hydrazide, or other compounds consisting of five- or six-membered rings that comprise a hydrazide side chain or carboxamide side chain. As apparent from FIG. 5 showing the chemical structure of compounds that have Nrf2-ARE-inhibitory activity, these compounds comprise a formula of Formula I, an indication of a structure-function relationship between compounds of Formula I, and the ability to inhibit Nrf2-ARE activity.

Table 1

EXAMPLE 2

In this Example, the mechanism of action by which compounds of Formula I inhibit Nrf2-ARE activity was further explored. Unexpectedly, compounds of Formula I inhibited Nrf2-ARE activity. It is known that Nrf2 undergoes acetylation to enhance the binding of Nrf2 to the ARE in promoting Nrf2-induced transcription of genes containing an ARE in their promoter region. Human NAD-dependent protein deacetylase SIRT1 has been shown to deacetylate Nrf2, resulting in decreased Nrf2-dependent gene transcription. Deacetylation of Nrf2 by SIRT1 resulted in primarily cytoplasmic localization of Nrf2 with a resultant decrease in Nrf2 transcription in the nucleus of the cell; i.e., decreased Nrf2- ARE activity. The mechanisms by which NAD-dependent protein deacetylases can be activated (e.g., induction of NAD-dependent protein deacetylase activity) was examined. As previously described herein, changes in nicotinamide and NAD+ concentrations occur in response to altered cell physiology or stress factors. Nicotinamide is believed to be involved in a chemical competition between base-exchange and deacetylation involving NAD-dependent protein deacetylases, thereby inhibiting deacetylation. Isonicotinamide has been described as binding to the site at which nicotinamide binds in the enzyme complex, and inhibits the base exchange reaction, thereby increasing the deacetylation rate (e.g., relieves nicotinamide inhibition of deacetylation by acting as a nicotinamide antagonist). Thus, for example, SIRT1 is an NAD-dependent protein deacetylase that can be inhibited by nicotinamide, but activated by isonicotinamide. Exemplary compounds of Formula I, discovered to inhibit Nrf2-ARE activity as described herein, were compared to isonicotinamide in a structural analysis. As shown below in Table 2, exemplary compounds of Formula I, discovered to inhibit Nrf2-ARE activity as described herein, are structurally similar to isonicotinamide, an indication that these compounds can also serve as nicotinamide antagonists in increasing the rate of deacetylation by NAD-dependent protein deacetylases.

Table 2

Based on the discovery of the structural likeness between compounds of Formula I discovered to have Nrf2-ARE inhibitory activity and that of isonicotinamide,

isonicotinamide was tested in the ARE-luciferase reporter assay using the methods described herein in Example 1 . As shown in Table 3, surprisingly, isonicotinamide shared the same ability (e.g., at the same concentrations) as isoniazid to inhibit Nrf2-ARE activity. The structural resemblances of compounds of Formula I discovered to have

Nrf2-ARE inhibitory activity with isonicotinamide, the shared ability of isonicotinamide and compounds of Formula I to inhibit Nrf2-ARE activity, and that NAD-dependent protein deacetylase can act to deacetylate Nrf2 in reducing Nrf2-mediated transcription, are all indications that compounds of Formula I act as nicotinamide antagonists which increase NAD-dependent protein deacetylase activity (e.g., are activators of NAD-dependent protein deacetylase) that can subsequently modulate Nrf2-mediated transcription.

Table 3

In addition to human NAD-dependent protein deacetylase SIRT1 having the ability to regulate Nrf2-dependent gene transcription by deacetylation, SIRT1 also represses peroxisome proliferator-activated receptor gamma (PPARy), a protein known by those skilled in the art to comprise an amino acid sequence selected from SEQ ID NO:5

(PPARy isoform 1 , or "PPARyl "), or SEQ ID NO:6 (PPARy isoform 2, or "PPARY2"), and isoforms thereof. PPARyl is mostly expressed in hematopoietic cells; and PPARv2 is primarily expressed in adipose tissue, and hence is the most important isoform in adipogenesis. Thus, a Nicotinamide Antagonist, represented by Formula I, which activates NAD-dependent protein deacetylase SIRT1 should also repress PPARy.

PPARy is a transcription factor known to regulate adipogenesis; i.e., the differentiation of preadipocytes to adipocytes. Thus, repression of PPARy can be demonstrated by impairment of adipogenesis. It has been demonstrated that upregulation (activation) of SIRT1 attenuates adipogenesis, RNA interference of SIRT enhances adipogeneis, and these observations can be demonstrated in differentiation of 3T3-L1 preadipocytes. In this experiment, 3T3-L1 preadipocytes were treated with differentiation medium (e.g., cell culture medium with 10% fetal bovine serum containing 1 μΜ dexamethasone, 0.5 mM IBMX (3-isobutyl- 1 -methylxanthine), and 1 Mg/ml insulin). After 48 hours, the medium was changed to cell culture medium with 10% fetal bovine serum containing 1 μg/ml insulin, followed by additional 3 days of culture. Differentiation of preadipocytes to adipocytes was confirmed by Oil Red O staining (a fat soluble dye that stains neutral trigylcerides and lipids a red color), and detection was quantitated using spectral and software analysis using standard methods known in the art. Also, total RNA was isolated from the treated cells, and standard reverse transcription, quantitative real-time polymerase chain reaction was performed to amplify mRNA levels of PPARy isoforms PPARyl and PPARy2, followed by fluorescent detection for quantitation. For illustration purposes, isoniazid (in non-cytotoxic concentrations) was used as the Nicotinamide Antagonist, and was added to 4 hours prior to the addition of the differentiation medium, and kept in the medium during the differentiation process. A vehicle control was used in the assay as a negative control (i.e., would expect no effect on adipogenesis nor PPARy mRNA levels) for comparison purposes ("Control"). Oil Red O staining showed a decrease (approximately a one third reduction, as compared to the Control) in adipogenesis in the presence of 2.5 mM isoniazid. As shown in Figures 6 A and 6B, this correlates with an observed, similar reduction mediated by 2.5 mM isoniazid ("INH") in the level of mRNA (expressed as percent of Control) for both PPARyl (FIG. 6A) and PPARy2 (FIG. 6B). Similar studies using human adipose tissue-derived stem cells also showed that isoniazid and isonicotinamide each inhibited adipogenesis. These results showing repression of PPARy are further indications that isoniazid and other compounds represented by Formula I are activators of NAD-dependent protein deacetylase SIRT1 (See also FIG. 7).

As noted above, a composition of the invention comprises a combination comprising an Nicotinamide Antagonist and a STAC. The unexpected discovery that compounds of Formula I can activate NAD-dependent protein deacetylase as

Nicotinamide Antgaonists provides a method by which an NAD-dependent protein deacetylase can now be dually activated by a Nicotinamide Antagonist represented by Formula I and a STAC, since a Nicotinamide Antagonist and a STAC each work by a different mechanism to activate NAD-dependent protein deacetylase, as compared to the other. In one aspect of the invention, a resultant effect of such combined or dual activation (i.e., activation by a Nicotinamide Antagonist, and activation by an STAC, each a separate class of activator based on different structure and function relationships) of NAD-dependent protein deacetylase may be a synergistic effect or an amplified effect (e.g., a greater effect than what is expected to be the total effect from addition of the separate effects of each class of activator of NAD-dependent protein deacetylase) which can potentiate the ability of such dually activated NAD-dependent protein deacetylase to regulate cell processes, physiological processes and disease by increased deacetylase activity. In this regard, in screening antitubercular drugs for Nrf2-ARE activity, it was noted that both rifampicin and sparfloxacin also demonstrate Nrf2-ARE inhibitory activity. Further, rifampicin demonstrates the ability to inhibit PPARy. Interestingly, rifampicin and sparfloxacin more resemble the structure of an STAC (compare, for example, to the chemical structure of fisetin, a known STAC). EXAMPLE 3

In this example, illustrated is activation of NAD-dependent protein deacetylase activity by a Nicotinamide Antagonist represented by Formula I. In this demonstration, a commercially available fluorescent assay system was used to measure the lysyl deacetylase activity of human SIRT1 (as a representative NAD-dependent protein deacetylase). In this assay system, a peptide having an acetylated lysine residue is mixed with the NAD-dependent protein deacetylase enzyme, NAD, and an activator of NAD-dependent protein deacetylase activity (resveratrol). Deacetylation of the substrate by the enzyme can be detected and quantitated by subsequent addition of a developer which produces a fluorophore. The assay system also includes nicotinamide, which can be added to the substrate-enzyme reaction to inhibit NAD-dependent protein deacetylase activity. Using this assay system, experimental conditions were worked out to allow detection of antagonism of the nicotinamide inhibition of NAD-dependent protein deacetylase activity by a Nicotinamide Antagonist. These conditions included a 60 minute incubation time, a positive control (the substrate, SIRT1 , 100 μΜ ΝΑϋ+, 100 μΜ resveratrol, with no nicotinamide), a test condition with nicotinamide inhibition of NAD- dependent protein deacetylase activity (the substrate, SIRT1 , 100 μΜ NAD+, 100 μΜ resveratrol, 1 mM nicotinamide), and a negative control (the substrate, SIRT1 , 100 μΜ NAD+, no resveratrol, and 1 mM nicotinamide). Using this system, a compound being assayed as a Nicotinamide Antagonist is added to the test condition to observe for antagonism of the nicotinamide inhibition of NAD-dependent protein deacetylase activity. Thus, the presence of a Nicotinamide Antagonist in the test condition would result in an increase in NAD-dependent protein deacetylase activity as compared to the NAD- dependent protein deacetylase activity in the test condition in the absence of a

Nicotinamide Antagonist. Isonicotinamide, a known antagonist of nicotinamide, and isoniazid (a representative Nicotinamide Antagonist represented by Formula I) were evaluated in this assay system at concentrations of 10 μΜ and 100 μΜ. Isoniazid, a representative Nicotinamide Antagonist represented by Formula I, showed a dose dependent antagonism of nicotinamide inhibition of NAD-dependent protein deacetylase activity which was at least 2 fold more potent than the antagonism of nicotinamide demonstrated by isonicotinamide in this assay system.

EXAMPLE 4

In some diseases, disorders, and conditions (collectively referred to as "disease"), the NAD-dependent protein deacetylase activity is dysregulated (e.g., reduced as compared to the activity in the same cells or tissues in absence of the disease), and such reduction can play a role in disease pathogenesis. While in a disease there may be more than one process which leads to dysregulation of NAD-dependent protein deacetylase activity (e.g., suppression of NAD-dependent protein deacetylase gene expression, regulation of the mRNA for NAD-dependent protein deacetylase, or modification of the NAD-dependent protein deacetylase protein itself (e.g., cleavage, or post-translational modification)), in one aspect of the invention the dysregulation of NAD-dependent protein deacetylase activity relates to an increase in PPARy (one or more of level of expression and activity) (see, e.g., FIG. 7, white arrow). SIRT1 deacetylates a wide range of nonhistone proteins, including PPARy in modulating PPARy activity. SIRT1 has also been shown to suppress PPARy activity by docking with cofactors needed for PPARy activity. It has been demonstrated that both PPARy and SIRT1 can bind to the promoter of the gene encoding SIRT1 , and hence PPARy can inhibit SIRT1 expression at the transcriptional level in providing a negative feedback loop. In diseases in which PPARy activity is upregulated, SIRT activity can be down regulated via a negative feedback loop (see, e.g., FIG. 7, as a representation of the interaction between, and effects on the respective levels of activity of, PPARy and SIRT1 ). Thus, a composition or method of the invention comprising a combination of a Nicotinamide Antagonist and a STAC to activate an NAD-dependent protein deacetylase is also useful in treating diseases in which there is a dysregulation of NAD-dependent protein deacetylase comprising an increase in PPARy (one or more of level of expression and activity) and a decrease in NAD- dependent protein deacetylase (one or more of level of expression and activity) (see, e.g., FIG. 7). As described herein, a composition and method according to the invention, comprising a combination of a Nicotinamide Antagonist and STAC, can activate an NAD- dependent protein deacetylase which can result in a decrease in PPARy (one or more of level of expression and activity) in overcoming or correcting the dysregulation of the NAD- dependent protein deacetylase activity present in the disease (e.g., in one or more body tissues and cell types (e.g., lineages) involved in the disease process, as can be determined by or known to one skilled in the art). Dysregulation of NAD-dependent protein deacetylase activity is known to occur in diseases including, but not limited to the following diseases. When used in a method to treat dysregulation of NAD-dependent protein deacetylase activity in a disease other than tuberculosis or M. tuberculosis infection, either or both of a Nicotinamide Antagonist and STAC can comprise a known antitubercular drug that can activate an NAD-dependent protein deacetylase. However, when used in a method to treat dysregulation of NAD-dependent protein deacetylase activity in a disease comprising tuberculosis or M. tuberculosis infection, at least one of a Nicotinamide Antagonist and STAC is not a known antitubercular drug.

A. Tuberculosis and infection with M. tuberculosis

Virulent M. tuberculosis and its cell wall lipids (e.g., mannose-capped

lipoarabinomannan) have been demonstrated to induce PPARy expression in

macrophages (infected with M. tuberculosis or present in the granulomas formed during infection by M. tuberculosis). Thus, PPARy expression is highly upregulated (increased) during mycobacterial infection (as compared to the absence of mycobacterial infection). The contribution of increased PPARy levels to the pathogenesis in tuberculosis is evident by the appearance and function of macrophages having increased PPARy levels. For example, mycobacterial- induced PPARy plays roles in lipid metabolism leading to increased lipid droplet formation, and the formation of "foamy macrophages"

characterized by lipid body biogenesis. It is believed that this lipid accumulation provides lipids that serve as a nutrient source to M. tuberculosis, leading to an enhanced ability to survive and replicate in such macrophages. Additionally, it has been shown that PPARy contributes to (a) induction of macrophages into "M2" macrophages which are poorly microbicidal, (b) downregulation of antimicrobial products (reactive oxygen and nitrogen species) in macrophages, and (c) block of phagolysosome maturation; all of which promote the intracellular growth and survival of M. tuberculosis. It has been demonstrated that a reduction in PPARy levels (e.g., by PPARy gene knockdown) in human

macrophages results in a restored ability of the macrophages to control the intracellular growth of M. tuberculosis; and enhances mycobacterial killing of macrophages in reducing or inhibiting M. tuberculosis infection and tuberculosis. Thus, use of a composition or combination of the invention comprising a Nicotinamide Antagonist and a STAC to activate an NAD-dependent protein deacetylase, in an individual infected M. tuberculosis or having tuberculosis having increased PPARy levels (e.g., in

macrophages, such as in infected tissue or formed granulomas) can act to modulate the increase in PPARy levels observed in M. tuberculosis infection and tuberculosis and, hence, treat one or more of M. tuberculosis infection and tuberculosis. It has also been reported that Nrf2-deficient mice infected with M. tuberculosis have a significant reduction in granuloma formation and tubercule bacilli counts, as compared with M. tuberculosis- infected mice that are not Nrf2-deficient, suggesting that Nrf2 activation is important in M. tuberculosis-induced granuloma formation, and that decreased Nrf2 activity could contribute to inhibition of M. tuberculosis infection. As described herein, increase in NAD- dependent protein deacetylase activity leads to inhibition of Nrf2 activity. Thus, use of a composition or combination of the invention comprising a Nicotinamide Antagonist and a STAC to activate an NAD-dependent protein deacetylase, in an individual infected M. tuberculosis or having tuberculosis, may inhibit Nrf2-ARE activity in treating one or more of M. tuberculosis infection and tuberculosis in an individual.

Provided herein is a method for treating M. tuberculosis infection or tuberculosis comprising administering to an individual infected with M. tuberculosis, or other mycobacterial species as a causative agent of tuberculosis or mycobacterial, a composition comprising a Nicotinamide Antagonist and a STAC in an amount effective (or in a medically effective amount) to activate an NAD-dependent protein deacetylase in an individual infected M. tuberculosis or having tuberculosis (particularly, in cells or body tissues involved in the disease or promotion of infection). Thus, a method for increasing the level of activation of an NAD-dependent protein deacetylase, such as SIRT1 , with subsequent modulation (e.g., down-regulation) of the level or activity of Nrf2 activity and PPARy, may inhibit or reduce one or more of M. tuberculosis infection or tuberculosis. The method and compositions may be used to treat drug-resistant strains of M.

tuberculosis. The composition used in the method may further comprise a

pharmaceutically acceptable carrier. The method and composition may further comprise use of a known antitubercular drug in combination therapy with the composition of the invention, wherein the composition and the known antitubercular drug may be

administered concurrently, sequentially, or in regimen alternating between a composition according to the invention and one or more known antitubercular drugs. Such combination therapy may optionally include one or more additional therapeutic agents for treating the disease caused by mycobacterial infection in an individual, with the dosages applicable being well known in the art to a medical care giver, or can be determined by a physician, taking into account the medical literature, the health, age and sex of the patient, the disease or condition or disorder to be treated, the mode of administration and dosing schedule, and other relevant considerations. As described herein, known antitubercular drugs may include amikacin, aminosalicylic acid, capreomycin, cycloserine, kanamycin, streptomycin, thioacetazone, ofloxacin, ciprofloxacin, clarithromycin, azithromycin, bedaquiline, SQ 109, and fluoroquinolones or a salt thereof. As noted above, a proviso to the composition or combination of the invention, or a method of using a composition or combination of the invention in treating one or more of M. tuberculosis infection, mycobacteria infection, or tuberculosis in an individual, is that at least one of a Nicotinamide Antagonist and STAC is not a known antitubercular drug. For example, if the STAC in a composition or combination according to the invention is not a known antitubercular drug, the Nicotinamide Antagonist can be a known antitubercular drug (e.g., isoniazide, ethionamide, pyrazinamide). If there is more than one Nicotinamide Antagonist in such combination, and one Nicotinamide Antagonist is a known

antitubercular drug, the other Nicotinamide Antagonist may be a compound represented by Formula I which is not a known antitubercular drug (e.g., a proviso that the compound is not isoniazid, pyrazinamide, ethionamide, pyrazinamine, pyrazine-2-thio carboxamide, N-hydroxymethyl pyrazine thiocarboxamide, N-substituted 3-aminopyrazine-2,5- dicarbonitriles). Mycobacterial infection or tuberculosis can be caused by several mycobacterial species such as M. tuberculosis; M. bovis; or M. africanum; or a

Mycobacterium species that is environmental or opportunistic and that causes opportunistic infections such as lung infections in immune compromised hosts (e.g., patients with AIDS), e.g., M. avium, M. intracellular, M. celatum, M. genavense, M. haemophilum, M. kansasii, M. simiae, M. vaccae, M. fortuitum, and M. scrofulaceum.

In another aspect of the invention, in treating one or more of M. tuberculosis infection or tuberculosis in an individual with a composition of the invention, a NAD- dependent protein deacetylase that is activated comprises Rv1 151 c produced by M. tuberculosis. Since bacteria lack histones, an NAD-dependent protein deacetylase in bacteria has more general functions relating to metabolism. One mechanism by which M. tuberculosis adjusts to changes in the environment of the infected cell and granuloma is by changing between acetylation (inactivation) and deacetylation (activation) of acetyl- CoA synthetase. It has been shown that NAD-dependent protein deacetylase Rv1 151 c, produced by M. tuberculosis, reactivates acetyl-CoA synthetase. Disruption of this mechanism and a balance between deacetylation and acetylation, such as by increasing activity of Rv1 151c in infected cells and the microenvironment of a granuloma, by treatment with a composition according to the invention, can compromise the ability of M. tuberculosis to adjust to changes in the host microenvironment.

B. Hepatic steatosis

Nonalcoholic hepatic steatosis, or fatty liver, is the abnormal accumulation of triglycerides in the cytoplasm of hepatocytes. Hepatic steatosis is a condition which increases the vulnerability of the liver to progress to steatohepatitis and to more advanced stages of liver disease. In contrast to healthy livers, up-regulation of PPARy expression is a general property of steatotic livers. Hepatic PPARy overexpression has been linked to exacerbated hepatic steatosis through mechanisms that may include activation of lipogenic genes and de novo lipogenesis, and increased hepatic triglyceride concentrations. Decrease of PPARy in hepatocytes, and to a lesser extent in macrophages, protected mice fed a high fat diet from induction of hepatic steatosis. Similarly, a high level of Nrf2 activation has been observed in obese individuals, and associated with this appears to be hyperlipidemia and an elevated incidence of hepatic steatosis. Additionally, hepatic overexpression of SIRT1 reduces steatosis and glucose intolerance in obese individuals. Data suggests that a reduction in SIRT1 activity increases the risk of fatty liver in response to dietary fat. Pharmacological activation of SIRT1 may be important for the prevention of obesity-associated metabolic diseases including, but not limited to, hepatic steatosis. Hepatic steatosis may be diagnosed by abnormal liver function tests during routine blood tests.

Provided herein is a method for treating hepatic steatosis comprising

administering to an individual in need thereof a composition comprising a Nicotinamide Antagonist and a STAC in an amount effective (or in a medically effective amount) to activate an NAD-dependent protein deacetylase in the individual. Thus, a method for increasing the level of activation of an NAD-dependent protein deacetylase, such as SIRT1 , with subsequent modulation (e.g., downregulation) of the level or activity of Nrf2 activity and PPAR, may inhibit or reduce hepatic steatosis and its symptoms. The composition used in the method may further comprise a pharmaceutically acceptable carrier. The method and composition may further comprise use of an agent for treating hepatic steatosis in combination therapy with the composition of the invention, wherein the composition and the agent for treating hepatic steatosis may be administered concurrently, sequentially, or in regimen alternating between a composition according to the invention and one or more agents for treating hepatic steatosis. Appropriate dosages for such combination therapy are well known in the art to a medical care giver, or can be determined by a physician, taking into account the medical literature, the health, age and sex of the patient, the disease or condition or disorder to be treated, the mode of administration and dosing schedule, and other relevant considerations. There is a need for new treatments for hepatic steatosis, as there are few agents currently being used for treating hepatic steatosis, such as high doses of vitamin E (e.g., about 800 lU/day), and statins.

B. Metabolic Syndrome

A major medical problem worldwide is metabolic syndrome, the two major components of which are obesity and type 2 diabetes mellitus (insulin resistance). It estimated that 34% of the adult population in the U.S. has metabolic syndrome. SIRT1 expression is progressively downregulated when the number of metabolic syndrome conditions increases. For example, it has been demonstrated in v/Vo that insulin resistance and metabolic syndrome were associated with decreased SIRT1 gene and protein expression. Studies in standard animal models for metabolic syndrome and obesity showed that SIRT1 deacetylase activity protected against the symptoms of metabolic syndrome. In a study of obese individuals (body mass index (BMI)≥30), it was found that there is correlation between increased PPARy levels and body composition in the obese population; i.e., the risk of metabolic syndrome in individuals with higher concen-tration of PPARy was almost 2 fold in compared with individuals having a lower concentration of PPARy, after adjustment for age, sex and body mass index. Studies involving a standard animal model in which Nrf2 expression and activity is significantly decreased, showed that decreased Nrf2 activity inhibits or reduces the incidence of obesity and insulin resistance when challenged with a long-term high fat diet. Provided herein is a method for treating metabolic syndrome comprising administering to an individual in need thereof a composition comprising a Nicotinamide Antagonist and a STAC in an amount effective (or in a medically effective amount) to activate an NAD- dependent protein deacetylase in the individual. Thus, a method for increasing the level of activation of an NAD-dependent protein deacetylase, such as SIRT1 , with subsequent modulation (e.g., downregulation) of the level or activity of Nrf2 activity and PPARy, may inhibit or reduce metabolic syndrome. The method may further comprise use of, and the composition may further comprise a pharmaceutically acceptable carrier. The method and composition may further comprise use of an agent for treating metabolic syndrome in combination therapy with the composition of the invention, wherein the composition and the agent for treating metabolic syndrome may be administered concurrently, sequentially, or in regimen alternating between a composition according to the invention and one or more agents for treating metabolic syndrome. Appropriate dosages for such combination therapy are well known in the art to a medical care giver, or can be determined by a physician, taking into account the medical literature, the health, age and sex of the patient, the disease or condition or disorder to be treated, the mode of administration and dosing schedule, and other relevant considerations. There is a need for new treatments for metabolic syndrome, as there are few agents currently being used for treating metabolic syndrome. Lifestyle changes are the preferred method for treating metabolic syndrome. Agents that can be used to treat metabolic syndrome include agents for treating symptoms of metabolic syndrome including drugs to control high blood pressure (e.g., diuretics such as chlorthalidone, chlorothiazide, furosemide,

hydrochlorothiazide, amiloride hydrochloride, and bumetanide), drugs to lower high cholesterol (e.g., statins, bile acid sequestrants, cholesterol absorption inhibitors, nicotinic acid, fibric acid derivatives), and drugs to lower high blood glucose levels (e.g., insulin, dapagliflozin, metformin, sulphonylureas, glitazones, meglitinides, gliptins, and acarbose).

Provided herein is a method for treating a disease characterized by dysregulation of an NAD-dependent protein deacetylase in an individual in need of such treatment, comprising administering to an individual in need thereof a composition comprising a Nicotinamide Antagonist and a STAC in an amount effective (or in a medically effective amount) to activate an NAD-dependent protein deacetylase in the individual (see, e.g., FIG. 7, black arrow). Dysregulation of an NAD-dependent protein deacetylase can be characterized by (as compared to the levels or activity in absence of the disease): (a) downregulation or decreased levels and/or activity of an NAD-dependent protein deacetylase, such as SIRT1 , in the individual (particularly in hematopoietic cells or body tissue affected by the disease); and (b) upregulation or increased levels and/or activity of one or more Nrf2 and PPARy (see, e.g., FIG. 7, white arrow). Illustrative examples of a disease characterized by dysregulation of an NAD-dependent protein deacetylase that have been provided herein include, but are not limited to, metabolic syndrome, hepatic steatosis, and M. tuberculosis infection or tuberculosis.

Where combination therapy according to the invention comprises administration of a single pharmaceutical dosage formulation comprising a Nicotinamide Antagonist and a STAC (and optionally further comprising a pharmaceutically acceptable carrier), and where the single pharmaceutical dosage formulation is administered orally, the single pharmaceutical dosage formulation can be administered to an individual in one oral composition, such as a tablet or capsule; or in one inhaled composition (e.g., propellant- based inhalation, and nasal aerosols). As previously described herein, the tablet or capsule may be formulated with inactive ingredients including, but not limited to, colloidal silicon dioxide, lactose monohydrate, pregelatinized starch, stearic acid, sodium, microcrystalline cellulose, silicified microcrystalline cellulose, croscarmellose, talc, silica colloidal silicon dioxide, magnesium stearate, triethyl citrate, methacrylic acid copolymer- Type A, methacrylic acid copolymer dispersion, simethicone emulsion, sodium lauryl sulphate, polysorbate 80, and combinations thereof.

Also provided herein is a kit including a composition according to the invention. A representative kit comprises a Nicotinamide Antagonist and a STAC, together with packaging for same. The kit can include one or more separate containers, dividers or compartments and, optionally, informational material such as instructions for

administration. For example, each of a Nicotinamide Antagonist and a STAC, or various combinations thereof, can be contained in a container comprising a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet or provided in a label. In some embodiments, the kit may comprise a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms of a composition comprising a Nicotinamide Antagonist, a STAC, or a combination thereof. For example, the kit can include a plurality of foil packets, or blister packs, each containing a single unit dose of a compound or composition described herein or any of the various combinations thereof.

What is claimed is:

Claims

1. Use of a composition comprising a compound represented by Formula I and a sirtuin- activating compound ("STAC") in an effective amount to activate a NAD-dependent protein deacteylase wherein:

(a) Formula I is represented by Formula IA and Formula IB;

(i) with Formula IA as

Formula IA

wherein:

A is N or C;

B is N or C;

R1 or R2 or R3 are each independently selected from H, (C₁-C₆)alkyl, CONH₂,

CONHNH₂, CSNH₂, S0₂NH₂, NH₂, NHNH₂, CHCHCONH₂, CHCHCONHNH₂, or COCH₃; wherein at least one of R1 , R2, and R3 is selected from CONH₂, CONHNH₂, CSNH₂, S0₂NH₂, NH₂, NHNH₂, CHCHCONH₂, or CHCHCONHNH₂; a pharmaceutically acceptable salt thereof, and the dashed lines represent optional double bonds;

(ii) with Formula IB as

Formula IB

wherein:

A is O or N;

B is N or C;

CHCHCONHNH₂;

R2 is absent if B is NH;

if B is C, R2 is absent or selected from CH₃, CH₂CH₃, NH₂, or NHNH₂; a pharmaceutically acceptable salt thereof; and the dashed lines represent optional double bonds;

(iii) with the proviso that the compound represented by Formula I is not nicotinamide, isonicotinamide, or nicotinamide adenine dinucleotide (NAD); (b) the STAC comprises a structure comprising a benzimidazole, imidazothiazole, quinoxaline, thiazolopyridine, stilbene, polyphenol, or a methylxanthine; and

(c) the use is to treat a disease, disorder or condition other than tuberculosis,

mycobacterial infection or Mycobacteria tuberculosis infection.

2. The use of the composition of claim 1 to treat a disease, disorder or condition comprising one or more of metabolic syndrome and hepatic steatosis.

3. The use of the composition of claim 1 , wherein the composition further comprises a pharmaceutically acceptable carrier.

4. Use of a composition comprising a compound represented by Formula I and a sirtuin- activating compound ("STAC") in an effective amount to activate a NAD-dependent protein deacteylase in treating a disease, disorder or condition comprising one or more of tuberculosis, mycobacterial infection or Mycobacteria tuberculosis infection, wherein: (a) Formula I is represented by Formula IA and Formula IB;

(i) with Formula IA as

Formula IA

wherein:

A is N or C;

B is N or C;

(ii) with Formula IB as

Formula IB

wherein:

A is O or N;

B is N or C;

R1 is selected from CONH₂, CONHNH₂, CSNH₂, S0₂NH₂, NH₂, NHNH₂, CHCHCONH₂, or CHCHCONHNH₂;

R2 is absent if B is NH;

(iii) with the proviso that the compound represented by Formula I is not nicotinamide, isonicotinamide, or nicotinamide adenine dinucleotide (NAD);

(b) the STAC comprises a structure comprising a benzimidazole, imidazothiazole, quinoxaline, thiazolopyridine, stilbene, polyphenol, or a methylxanthine; and

(c) at least one of the compound represented by Formula I or an STAC is not a known antitubercular drug.

5. The use of the composition of claim 5, wherein the composition further comprises a pharmaceutically acceptable carrier.

6. A pharmaceutical composition or medicament comprising a compound represented by Formula I and a sirtuin-activating compound ("STAC") in a medically effective amount to activate a NAD-dependent protein deacteylase wherein:

a) Formula I comprises Formula IA and Formula IB;

(i) with Formula IA as

Formula IA

wherein:

A is N or C; B is N or C;

as

Formula IB

wherein:

A is O or N;

B is N or C;

CHCHCONHNH₂;

R2 is absent if B is NH;

7. The pharmaceutical composition or medicament according to claim 6, further comprising a pharmaceutically acceptable carrier.

8. A kit for combination therapy comprising a composition comprising a compound represented by Formula I and a sirtuin-activating compound ("STAC") in a medically effective amount to activate a NAD-dependent protein deacteylase, together with packaging and one or more separate containers, wherein:

a) Formula I is represented by Formula IA and Formula IB;

(i) with Formula IA as

Formula IA

wherein:

A is N or C;

B is N or C;

(ii) with Formula IB as

Formula IB

wherein:

A is O or N;

B is N or C;

CHCHCONHNH₂;

R2 is absent if B is NH;

9. The kit of claim 8, further comprising a pharmaceutically acceptable carrier.

10. A method for treating a mycobacterial infection or tuberculosis comprising

administering to an individual in need thereof a composition comprising a compound represented by Formula I and a sirtuin-activating compound ("STAC") in a medically effective amount to activate a NAD-dependent protein deacteylase wherein:

a) Formula I is represented by Formula IA and Formula IB;

(i) with Formula IA as

Formula IA

wherein:

A is N or C;

B is N or C;

(ii) with Formula IB as

Formula IB

wherein:

A is O or N;

B is N or C;

CHCHCONHNH₂;

R2 is absent if B is NH;

if B is C, R2 is absent or selected from CH₃, CH₂CH₃, NH₂, or NHNH₂; a pharmaceutically acceptable salt thereof; and the dashed lines represent optional double bonds; (iii) with the proviso that the compound represented by Formula I is not nicotinamide, isonicotinamide, or nicotinamide adenine dinucleotide (NAD);

11 . The method according to claim 10, wherein the mycobacterial infection is by M. tuberculosis.

12. The method according to claim 10, wherein the compound represented by Formula I is administered sequentially or simultaneously with administration of the STAC.

13. The method according to claim 10, wherein the composition further comprises a pharmaceutically acceptable carrier.

14. A method for treating hepatic steatosis comprising administering to an individual in need thereof a composition comprising a compound represented by Formula I and a sirtuin-activating compound ("STAC") in a medically effective amount to activate a NAD- dependent protein deacteylase wherein:

a) Formula I is represented by Formula IA and Formula IB;

(i) with Formula IA as

Formula IA

wherein:

A is N or C;

B is N or C;

CONHNH₂, CSNH₂, S0₂NH₂, NH₂, NHNH₂, CHCHCONH₂, CHCHCONHNH₂, or COCH₃; wherein at least one of R1 , R2, and R3 is selected from CONH₂, CONHNH₂, CSNH₂, S0₂NH₂, NH₂, NHNH₂, CHCHCONH₂, or CHCHCONHNH₂; a pharmaceutically acceptable salt thereof, and the dashed lines represent optional double bonds; as

Formula IB

wherein:

A is O or N;

B is N or C;

CHCHCONHNH₂;

R2 is absent if B is NH;

(iii) with the proviso that the compound represented by Formula I is not nicotinamide, isonicotinamide, or nicotinamide adenine dinucleotide (NAD); and

(b) the STAC comprises a structure comprising a benzimidazole, imidazothiazole, quinoxaline, thiazolopyridine, stilbene, polyphenol, or a methylxanthine.

15. The method according to claim 14, wherein the compound represented by Formula I is administered sequentially or simultaneously with administration of the STAC.

16. The method according to claim 14, wherein the composition further comprises a pharmaceutically acceptable carrier.

17. A method for treating metabolic syndrome comprising administering to an individual in need thereof a composition comprising a compound represented by Formula I and a sirtuin-activating compound ("STAC") in a medically effective amount to activate a NAD- dependent protein deacteylase wherein:

a) Formula I is represented by Formula IA and Formula IB;

(i) with Formula IA as

Formula IA

wherein:

A is N or C;

B is N or C;

(ii) with Formula IB as

Formula IB

wherein:

A is O or N;

B is N or C;

CHCHCONHNH₂;

R2 is absent if B is NH;

18. The method according to claim 17, wherein the compound represented by Formula I is administered sequentially or simultaneously with administration of the STAC.

19. The method according to claim 17, wherein the composition further comprises a pharmaceutically acceptable carrier.