Methods and compositions for disulfiram as an agent to inhibit tyrosine-mediated translation and transcription inhibition

Abstract

The disclosure provides disulfiram containing compositions and methods of using disulfiram for activating transcription, and / or protein synthesis, and / or tubulin tyrosination and / or histone serine- ADP-ribosylation, and / or increasing TyrRS protein levels in presence of elevated tyrosine / phenylalanine levels. Compositions may be used prophy lactically, to delay neurodegeneration or reduce the expected severity. Disulfiram-containing compositions of this disclosure may be used for treating neurodegradation that leads to constitutive aging and age-associated neurocognitive and metabolic disorders. In particular, compositions disclosed herein are neuroprotective at nanomolar concentrations against tyrosine or its metabolites-mediated transcription inhibition.

Description

METHODS AND COMPOSITIONS FOR DISULFIRAM AS AN AGENT TO INHIBIT TYROSINE-MEDIATED TRANSLATION AND TRANSCRIPTION INHIBITIONGOVERNMENT SUPPORT CLAUSE

[0001] This invention was made with government support under grant P20 GM 109091 awarded by National Institutes of Health (NIH). The government has certain rights in the invention.CROSS REFERENCE TO RELATED APPLICATION

[0002] This application claims priority to US Provisional Appl. No. 63 / 572,572, filed April 1, 2024, which is hereby incorporated by reference in its entirety.BACKGROUND

[0003] Alcohol consumption is known to increase serum tyrosine levels and inhibits protein synthesis in mice models. Moreover, in humans, alcohol consumption reduces rates of myofibrillar protein synthesis (MPS) in skeletal muscle and midlife alcohol consumption enhances the risk for cognitive impairment in late life. Similarly, alcohol-induced loss of consciousness is associated with a subsequent increased risk of dementia. We recently showed that tyrosine inhibits protein synthesis at the elongation step and induces neurodegeneration. Together, these observations suggest that an alcohol-induced increase in serum tyrosine levels might also contribute to the toxic effects of alcohol including protein synthesis inhibition, and cognitive impairment, and dementia. Tyrosine (Tyr) is not only important for protein synthesis and cellular energy production, but also acts as a substrates for tubulin tyrosine ligase (TTL), an enzyme that adds tyrosine to the C-terminal glutamic residue of a- tubulin (Tyr / Phe-tubulin). In contrast, the vasohibin-small vasohibin binding protein complexes (VASH1 / 2-SVBP) remove the C-terminal tyrosine / phenylalanine from the a - tubulin. TTL-dependent tubulin tyrosination levels decrease during aging and in age-associated neurocognitive and degenerative disorders. Also, serum tyrosine levels increase said in age- associated neurocognitive and degenerative disorders. Interestingly, Alzheimer's disease (AD)- affected brain regions have decreased tyrosyl-tRNA synthetase (TyrRS / YARS l) protein levels, and tyrosine inhibits protein synthesis at the elongation step to deplete neuronal TyrRS. De novo protein synthesis is also essential for the reactivation of the paused / stalled transcription complex to restore transcription. Stalled transcription complex is a hallmark of aging and age- associated disorders. Consistently, activation of protein synthesis restores cognitive functions and memory formation in Alzheimer’s Disease (AD) mice models. Recently, studies found that AD-affected brain regions show decreased tyrosyl-tRNA synthetase (TyrRS) and histone serine- ADP- ribosylation levels, and tyrosine inhibits protein synthesis at the elongation step to deplete neuronalTyrRS. Therefore, strategies to activate tubulin tyrosination would help to decrease serum tyrosine levels and would help to activate protein synthesis and restoration of stalled / paused transcription.

[0004] Tyrosine is circadian regulated, with the highest serum levels in the morning and with the lowest around midnight (sleep time). Intriguingly, brain protein synthesis, memory formation, and neuronal DNA repair are activated during sleep when tyrosine levels are decreased during the nadir / trough of circadian rhythm. Tyrosine levels are also modulated by the circadian activities of tyrosine hydroxylase, tyrosine aminotransferases (TAT), and gut microbiota. Genetic mutations that increase the levels of tyrosine (tyrosinemia) or its precursor L-phenylalanine (Phe, phenylketonuria [PKU]) cause multiple health problems, including cognitive deficits in children. Moreover, tyrosine and / or phenylalanine exacerbate cognitive decline in the elderly and in Alzheimer's disease (AD) patients, in addition to shortening lifespan in tyrosinemia patients. Although protein synthesis is required for long-term memory formation, and brain-derived neurotrophic factor (BDNF) stimulates the de novo synthesis of TyrRS in primary cortical neurons, recent brain proteomic analysis showed that TyrRS is decreased in the affected brain regions of AD patients through an unknown mechanism.

[0005] Increased tyrosine levels decrease TyrRS and cause neuronal oxidative DNA damage by simultaneously inhibiting protein synthesis and transcription along with stimulation of DNA repair and neurite degeneration.

[0006] Restoration of stalled transcription requires activation of protein synthesis and sustained transcription especially of long genes requires topoisomerase I (TOPl)-mediated DNA repair at the transcription promoter / enhancer sites. Therefore, TOPl-mediated DNA repair capacity would positively affect the transcription of long-genes, which are majorly associated with memory and cognition and cell identity. Disulfiram is an approved medication for treatment of alcohol addiction. However, whether disulfiram could protect against tyrosine-mediated neurotoxic effects by activating TOPl-mediated DNA repair and neuronal protein synthesis was previously unknown.

[0007] The need exists for compositions and methods to activate neuronal protein synthesis and transcription inhibition by tyrosine. Improvements in transcription may restore the function of crucial neuroinflammatory factor associated with cognitive performance and memory formation in neurodegenerant brains. In particular, a need exists for compositions and methods for inhibiting tyrosine-mediated transcription inhibition by activating protein synthesis in aging and age- associated neurocognitive and metabolic disorders including autism spectrum disorders (ASD), cardiovascular diseases (CVDs) and cancer.SUMMARY

[0008] In general, the present disclosure is directed to compositions and methods for activatingtranscription and protein synthesis along with stimulation of tubulin tyrosination. Compositions may include a prophylactic agent including disulfiram. Said compositions may be utilized in treating neurodegradation that leads to constitutive aging and age-associated neurocognitive and metabolic disorders. In particular, compositions disclosed herein are neuroprotective against tyrosine-mediated transcription inhibition or useful for activating protein synthesis and tubulin tyrosination.

[0009] A method of activating tubulin tyrosination, the method comprising determining the level of TyrRS in a cell; contacting the cell with an effective amount of disulfiram; wherein the effective amount of disulfiram is an amount sufficient to increase a TyrRS levels in the cell relative to the TyrRS level in the cell prior to contacting the cell with disulfiram.

[0010] The disclosure includes a method of activating tubulin tyrosination, the method comprising determining the level of TyrRS in a cell; contacting the cell with an effective amount of disulfiram; wherein the effective amount of disulfiram is an amount sufficient to increase a TyrRS levels in the cell relative to the TyrRS level in the cell prior to contacting the cell with disulfiram.

[0011] The disclosure includes methods in which disulfiram is administered as the only active agent as well as methods in which it is administered together with another compound useful for stimulating tubulin tyrosination or capable of protecting against tyrosine-mediated transcription inhibition or useful for activating protein synthesis and tubulin tyrosination. Compounds useful in combination with disulfiram include cis-resveratrol as well as compounds of Formula I, and pharmaceutically acceptable salts thereof.

[0012] Formula I isThe variables shown in Formula I, e.g. R1, R2, R3, W, and the A-ring are discussed in the detailed description section.

[0013] Other features and aspects of the present disclosure are discussed in greater detail below.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] A full and enabling disclosure of the present disclosure is set forth more particularly in the remainder of the specification, including reference to the accompanying figures, in which:

[0015] FIG. 1A presents primary cortical neurons (DIV 9 / 10) treated with disulfiram (0.25 - 5 nM) and changes in the levels of TyrRS, PheRSp, PheRSa, p-elF2a, and elFEa levels were determined by WB using their specific antibodies.

[0016] FIG. IB presents primary cortical neurons treated with increasing concentrations of disulfiram (0.5 - 10 nM) for 2 hr, followed by puromycin incorporation and western blot (WB) detection using an anti-puromycin antibody.

[0017] FIG. 1C presents spectral images (scale bar, 20 pm), and quantitative IF analysis of tyrosinated tubulin (Tyr-Tub) levels in the rat cortical neurons (DIV 9 / 10) using anti-Tyr-Tub antibody after treatment with disulfiram (10 nM) for 4 hr

[0018] FIG. 2A presents primary cortical neurons (DIV 9 / 10) treated with disulfiram (0.25 - 5 nM) for 8 hr and changes in the levels of PARylation, histone serine- ADP-ribosylation (H3-Ser- ADPR), and PARP1 levels were determined by WB using their specific antibodies.

[0019] FIG. 2B presents a graph showing the quantification of EU incorporation in nascent RNA. Primary cortical neurons were treated with either tyrosine (500 mM) alone or in cultures pretreated with disulfiram (25 nM) for 16 hr followed by the addition of EU for 30 min and subsequent processing for image analysis.

[0020] FIG. 3A presents primary cortical neurons treated with the topoisomerase 1 inhibitor camptothecin (CPT) (50 mM) either alone or in combination with disulfiram (25 nM- 250 mM) for 24 hr and quantified neuronal survival using MTT assay.

[0021] FIG. 3B presents a graph showing the quantification of EU incorporation in nascent RNA. Primary cortical neurons were treated with either p-cresol (500 mM), indole-3-sulfate (3- ISP) (500 mM), 4-hydroxyphenyllactic acid (4HPA) (1 mM), or tyrosine (500 mM) alone or in cultures pre-treated with disulfiram (25 nM) for 16 hr followed by the addition of EU for 30 min and subsequent processing for image analysis.

[0022] Repeat use of reference characters in the present specification and drawings is intended to represent the same or analogous features or elements of the present invention.DETAILED DESCRIPTION

[0023] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0024] Unless specifically stated, terms and phrases used in this document, and variations thereof, unless otherwise expressly stated, should be construed as open ended as opposed tolimiting. Likewise, a group of items linked with the conjunction “and” should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as “and / or” unless expressly stated otherwise. Similarly, a group of items linked with the conjunction “or” should not be read as requiring mutual exclusivity among that group, but rather should also be read as “and / or” unless expressly stated otherwise.

[0025] Furthermore, although items, elements or components of the disclosure may be described or claimed in the singular, the plural is contemplated to be within the scope thereof unless limitation to the singular is explicitly stated. The presence of broadening words and phrases such as “one or more,” “at least,” “but not limited to” or other like phrases in some instances shall not be read to mean that the narrower case is intended or required in instances where such broadening phrases may be absent.

[0026] As used herein, the singular forms “a,” “an,” and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0027] As used herein, "about," "approximately," “substantially,” and the like, when used in connection with a measurable variable such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value including those within experimental error (which can be determined by e.g. given data set, art accepted standard, and / or with e.g. a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as variations of + / - 10% or less, + / -5% or less, + / -1% or less, and + / -0. 1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosure. As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” can mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein canalso be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0029] As will be apparent to those of skill in the art reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0030] Where a range is expressed, a further embodiment includes from the one particular value and / or to the other particular value. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y.’ The range can also be expressed as an upper limit, e.g., ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ include the specific ranges of ‘about x,’ ‘about y,’ and ‘about z’ as well as the ranges of ‘greater than x,’ greater than y,’ and ‘greater than z.’ In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0031] Ratios, concentrations, amounts, and other numerical data discussed herein can be expressed in a range format. The endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. There are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will beunderstood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0032] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or subranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0. 1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0033] Formula I includes all subformulae and compounds that fall within the scope of Formula I.

[0034] In certain situations, the compounds of Formula I may contain one or more asymmetric elements such as stereogenic centers, stereogenic axes and the like, e.g., asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms. Unless stated otherwise or clearly indicated from the context, Formula I include all stereoisomeric forms, including racemates, optically enriched, and optically pure forms. In addition, compounds with carbon-carbon double bonds may occur in Z- and E- forms, with all isomeric forms of the compounds being included in the present disclosure. In these situations, the single enantiomers, i.e., optically active forms can be obtained by asymmetric synthesis, synthesis from optically pure precursors, or by resolution of the racemates. Resolution of the racemates can also be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example, a chiral HPLC column.

[0035] Formula I includes all isotopically enriched versions of compounds of Formula I, Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example, and without limitation, isotopes of hydrogen include tritium and deuterium and isotopes of carbon includenC,13C, and14C, isotopes of fluorine include19F, isotopes of nitrogen include15N, and isotopes of oxygen include17O. Isotopically enriched versions of compounds of Formula I, include, e.g., compounds in which one or more C in a compound of Formula I is13C enriched, in which one or more hydrogen position in a compound of Formula I is deuterated or tritiated, in which one or more N in a compound of Formula I is15N enriched.

[0036] Certain compounds are described herein using a general formula that includes variables, e.g., R1, R2, R3, R, W, X, Y, and Z. Unless otherwise specified, each variable within such a formula is defined independently of other variables. Thus, if a group is said to be substituted, e.g.,with 0-2 R*, then said group may be substituted with up to two R* groups and R* at each occurrence is selected independently from the definition of R*.

[0037] Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds. A stable compound or stable structure is meant to imply a compound that is sufficiently robust to survive isolation from a reaction mixture, and subsequent formulation into an effective therapeutic agent.

[0038] The term “substituted” means that any one or more hydrogen atoms bound to the designated atom or group is replaced with a selection from the indicated group, provided that the designated atom's normal valence is not exceeded. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds or useful synthetic intermediates. A stable compound or stable structure is meant to imply a compound that is sufficiently robust to survive isolation from a reaction mixture, and subsequent formulation into an effective therapeutic agent. Unless otherwise specified substituents are named into the core structure.

[0039] Substituents are named into the ring unless otherwise indicated. A dashor a double bond (“=”) that is not between two letters or symbols indicates the point of attachment for a substituent. For example, -CONH2 is attached through the carbon atom.

[0040] The term "amino acid" as used herein refers to native amino acids, non-native amino acids, and amino acid analogs. Native amino acids include, for instance, the 20 (L)-amino acids commonly utilized during protein biosynthesis. Non-native amino acids include the 20 (D)-amino acids not utilized for protein biosynthesis, selenocysteine, and other plant-derived non-proteogenic amino acids such as pyrrolysine. Non-native amino acids include, for instance, selenocysteine, homoserine, ornithine, canvanine, N-methyl-L-alanine, L-DOPA (3,4-dihydroxyphenylalanine), Dap, and Dap. Amino acid analogs may include modified forms of naturally or non-naturally occurring amino acids, for instance, substitution or replacement of chemical groups and moieties on the amino acid or by derivatization of the amino acid. Pomaglumetad (LY-404,039) is an amino acid analog.

[0041] “Alkyl” includes both branched and straight-chain saturated aliphatic hydrocarbon groups, having the specified number of carbon atoms. Thus, the term Ci - Cealkyl includes alkyl groups having from 1 to about 6 carbon atoms. When Co-Cnalkyl is used herein in conjunction with another group, for example, (cycloalkyl)Co-C2 alkyl, the indicated group, in this case cycloalkyl, is either directly bound by a single covalent bond (Co), or attached by an alkyl chain having the specified number of carbon atoms, in this case from 1 to about 2 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and secpentyl. Ci-C4alkyl includes alkyl groups having 1, 2, 3, or 4 carbon atoms.

[0042] “Alkoxy” is an alkyl group as defined above with the indicated number of carbon atoms attached to the group it substitutes through an oxygen bridge. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, 2-butoxy, t-butoxy, n-pentoxy, 2- pentoxy, 3-pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and 3- methylpentoxy. Similarly, an “Alkylthio” or a “thioalkyl” group is an alkyl group as defined above with the indicated number of carbon atoms covalently bound to the group it substitutes by a sulfur bridge (-S-).

[0043] “Acyl” is a group of the formula alkylC(=O)-, where alkyl has the definition given above. A -G>acyl group has an alkyl group of 1 to 5 carbon covalently bound to a keto (C=O) group which is in turn covalently bound to the group it substitutes.

[0044] “Halo” or “halogen” as used herein is fluoro, chloro, bromo, or iodo.

[0045] “Haloalkyl” includes both branched and straight-chain saturated aliphatic hydrocarbon groups, having the specified number of carbon atoms, substituted with 1 or more halogen atoms, generally up to the maximum allowable number of halogen atoms. Thus, the term Ci- G, haloalky I includes haloalkyl groups having from 1 to about 6 carbon atoms. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, chloromethyl, chloroethyl, and penta-fluoroethyl. G-Galkyl includes alkyl groups having lor 2 carbon atoms, substituted with 1 or more halogen atoms, generally up to the maximum allowable number of halogen atoms.

[0046] “Haloalkoxy” is an haloalkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. Examples of haloalkoxy include, but are not limited to, chloromethoxy, chloroethoxy, bromo-n-propoxy, bromo-i-propoxy, iodo-n-butoxy, iodo-2-butoxy, or chloro-n-pentoxy.

[0047] A “heterocyclic group” is a monocyclic saturated, partially unsaturated, or aromatic ring containing from 1 to 4 heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon, or a bicyclic saturated, partially unsaturated, or aromatic heterocycle containing at least 1 heteroatom chosen from N, O, and S in one of the two rings of the two ring system and containing up to about 4 heteroatoms independently chosen from N, O, and S in each ring of the two ring system. The rings of a bicyclic heterocyclic group can be in fused, bridged, pendant, or spiro orientation. Usually, each ring of the heterocycle contains from 4-6 ring atoms but some other number of ring atoms may be specified. Unless otherwise indicated, the heterocycle may be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. When indicated the heterocycles described herein may be substituted on carbon, sulfur, or nitrogen atom if the resulting compound is stable. It is preferred that the total number of heteroatoms in a heterocycle is not more than 4 and that the total number of S and O atoms in a heterocycle is not more than 2, more preferably not more than I . Examples of heterocycles include, pyridyl, indolyl,pyrimidinyl, pyridazinyl, pyrazinyl, imidazolyl, oxazolyl, furanyl, thiophenyl, thiazolyl, triazolyl, tetrazolyl, isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl, benz[b]thiophenyl, isoquinolinyl, quinazolinyl, quinoxalinyl, thienyl, isoindolyl, dihydroisoindolyl, 5,6,7,8-tetrahydroisoquinoline, pyrazolyl, pyrrolidinyl, morpholinyl, piperazinyl, piperidinyl, and pyrrolidinyl. In certain embodiments a heterocycle is chosen from pyridinyl, pyrimidinyl, furanyl, thienyl, and pyrrolyl.

[0048] Additional examples of heterocycles include, but are not limited to, phthalazinyl, indolizinyl, indazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, benzoisoxolyl, dihydro-benzodioxinyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, oxazolopyridinyl, imidazopyridinyl, isothiazolyl, naphthyridinyl, cinnolinyl, carbazolyl, beta-carbolinyl, isochromanyl, chromanonyl, chromanyl, tetrahydroisoquinolinyl, isoindolinyl, isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isobenzothienyl, benzoxazolyl, pyridopyridinyl, benzotetrahydrofuranyl, benzo tetrahydrothienyl, purinyl, benzodioxolyl, triazinyl, phenoxazinyl, phenothiazinyl, 5 pteridinyl, benzothiazolyl, imidazopyridinyl, imidazothiazolyl, dihydrobenzisoxazinyl, benzisoxazinyl, benzoxazinyl, dihydrobenzisothiazinyl, benzopyranyl, benzothiopyranyl, coumarinyl, isocoumarinyl, chromanyl, tetrahydroquinolinyl, dihydroquinolinyl, dihydroquinolinonyl, dihydroisoquinolinonyl, dihydrocoumarinyl, dihydroisocoumarinyl, isoindolinonyl, benzodioxanyl, benzoxazolinonyl, pyrrolyl N-oxide, pyrimidinyl N-oxide, pyridazinyl N-oxide, pyrazinyl N-oxide, quinolinyl N-oxide, indolyl N-oxide, indolinyl N oxide, isoquinolyl N-oxide, quinazolinyl N-oxide, quinoxalinyl N-oxide, phthalazinyl N-oxide, imidazolyl N-oxide, isoxazolyl N-oxide, oxazolyl N- oxide, thiazolyl N-oxide, indolizinyl N oxide, indazolyl N-oxide, benzothiazolyl N-oxide, benzimidazolyl N-oxide, pyrrolyl N-oxide, oxadiazolyl N-oxide, thiadiazolyl N-oxide, tetrazolyl N- oxide, benzothiopyranyl S-oxide, and benzothiopyranyl S,S-dioxide.

[0049] “Heteroaryl” is a stable monocyclic aromatic ring having the indicated number of ring atoms which contains from 1 to 3, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon, or a stable bicyclic or tricyclic system containing at least one 5- to 7-membered aromatic ring which contains from 1 to 3, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon. Monocyclic heteroaryl groups typically have from 5 to 7 ring atoms. In some embodiments bicyclic heteroaryl groups are 9- to 10-membered heteroaryl groups, that is, groups containing 9 or 10 ring atoms in which one 5- to 7-member aromatic ring is fused to a second aromatic or non- aromatic ring. When the total number of S and O atoms in the heteroaryl group exceeds 1, these heteroatoms are not adjacent to one another. It is preferred that the total number of S and O atoms in the heteroaryl group is not more than 2. It is particularly preferred that the total number of S and O atoms in the aromatic heterocycle is not more than 1. Examples of heteroarylgroups include, but are not limited to, oxazolyl, pyranyl, pyrazinyl, pyrazolopyrimidinyl, pyrazolyl, pyridizinyl, pyridyl, pyrimidinyl, pyrrolyl, quinolinyl, tetrazolyl, thiazolyl, thienylpyrazolyl, thiophenyl, triazolyl, benzol t / |oxazoly I, benzofuranyl, benzothiazolyl, benzothiophenyl, benzoxadiazolyl, dihydrobenzodioxynyl, furanyl, imidazolyl, indolyl, and isoxazolyl.

[0050] “Heterocycloalkyl” is a stable monocyclic ring having the indicated number of ring atoms which contains from 1 to 3, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon, or a stable bicyclic or tricyclic system containing at least one 5- to 7-membered cyclic ring which contains from 1 to 3, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon. Monocyclic heterocycloalkyl groups typically have from 5 to 7 ring atoms. The stable monocyclic heterocycloalkyl may have 3-10 ring atoms which contains from 1 to 3, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon. In some embodiments bicyclic heterocycloalkyl groups are 9- to 10-membered heterocycloalkyl groups, that is, groups containing 9 or 10 ring atoms in which one 5- to 7-member cyclic ring is fused to a second aromatic or non-aromatic ring. It is preferred that the total number of S and O atoms in the heteroaryl group is not more than 2. Examples of heterocycloalkyl groups include, but are not limited to, oxiranyl, oxetanyl, tetrahydrofuranyl, azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl, or piperazinyl.

[0051] The term "administering" includes all modes and routes of administration which allow a compound to perform its intended function. In general, the compounds disclosed herein may be administered to a subject according to known methods, including injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal, etc.), oral, inhalation, and transdermal routes. The injection can be bolus injections or can be continuous infusion. Depending on the route of administration, the agent can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function. The compound may be administered alone, or in conjunction with a pharmaceutically acceptable carrier. The compound also may be administered as a prodrug, which is converted to its active form in vivo.

[0052] “Pharmaceutical compositions” are compositions comprising at least one active agent, such as a compound, salt, or hydrate of a named Formulae of the disclosure (Formula I or II) and at least one other excipient. “Excipients” are any materials of a pharmaceutical composition other than the active agent or agents. Excipients include carriers and diluents, which may be added to the pharmaceutical compositions. Pharmaceutical compositions meet the U.S. FDA’s GMP (good manufacturing practice) standards for human or non-human drugs.

[0053] “Pharmaceutically acceptable salt” includes derivatives of the disclosed compounds wherein the parent compound is modified by making non-toxic acid or base salts thereof, and further refers to pharmaceutically acceptable hydrates or solvates of such compounds and such salts. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional nontoxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxylmaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC-(CH2)n- COOH where n is 0-4, and the like. Lists of additional suitable salts may be found, e.g., in G. Steffen Paulekuhn, et al., Journal of Medicinal Chemistry 2007, 50, 6665 and Handbook of Pharmaceutically Acceptable Salts: Properties, Selection and Use, P. Heinrich Stahl and Camille G. Wermuth Editors, Wiley- VCH, 2002.

[0054] A “patient” is a human or non-human animal in need of medical treatment. Medical treatment can include treatment of an existing condition, such as a disease or disorder or diagnostic treatment. In some embodiments the patient is a human patient.

[0055] “Treatment” or “treating” means providing an active compound to a patient in an amount sufficient to measurably reduce any existing condition or slow existing condition progression. In certain embodiments treatment can be preventative. Preventative treatment is treatment that reduces the likelihood of symptoms occurring, produces a statistically significant delay in symptoms occurring, or a statistically significant delay in symptoms occurring.

[0056] A significant change is any detectable change that is statistically significant in a standard parametric test of statistical significance such as Student’s T-test, where p < 0.05.

[0057] The term "therapeutically effective amount" of a compound of the disclosure, means an amount effective, when administered to a patient, to provide a therapeutic benefit such as an amelioration of symptoms, e.g., an amount effective to decrease the symptoms of a neurocognitive or neurodegenerative disorder, and including an amount sufficient to reduce or inhibit DNA repair or an amount effect to decrease neuronal tyrosine levels. Thus, a therapeutically effective amount of a compound is also an amount sufficient to significantly reduce the indicia of the disease or condition being treated. A significant reduction is any detectable negative change that isstatistically significant in a standard parametric test of statistical significance, such as Student’s t- test, in which p < 0.05.

[0058] Reference will now be made in detail to various embodiments of the presently disclosed subject matter, one or more examples of which are set forth below. Each embodiment is provided by way of explanation, not limitation, of the subject matter. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present disclosure without departing from the scope or spirit of the disclosure. For instance, features illustrated or described as part of one embodiment, may be used in another embodiment to yield a still further embodiment. Thus, the present disclosure covers such modifications and variations as come within the scope of the appended claims and their equivalents.CHEMICAL DESCRIPTION

[0059] In general, the present disclosure is directed to compositions and methods for activating tubulin tyrosination and / or transcription and / or protein synthesis and / or H3 serine- ADP- ribosylation. Compositions disclosed herein may be utilized in treating aging and age-associated neurocognitive and metabolic disorders. In particular, compositions disclosed herein are neuroprotective at nanomolar concentrations against tyrosine-mediated transcription inhibition and camptothecin (CPT)-mediated neurodegeneration. Also, advantageously, compositions disclosed herein may stimulate neuronal tubulin tyrosination (Tyr- tubulin) and H3-Ser-ADPR at nanomolar concentrations.

[0060] Compositions disclosed herein may include a prophylactic agent, such as disulfiram. Disulfiram is an FDA-approved drug for alcohol addiction. Interestingly, according to the present disclosure, disulfiram may stimulate neuronal protein synthesis, tubulin tyrosination, and PARP1- dependent H3-Ser-ADP-ribosylation and neuronal DNA repair. Further, disulfiram may provide neuro protection against tyrosine-mediated neuronal transcription inhibition and CPT-mediated neurodegeneration.

[0061] Disulfiram has the chemical name N,N-diethyldithiocarbamic acid, and is CAS Reg. No. 97-77-8.

[0062] Cis-resveratrol, of Cis-RSV, is a compound of the formulaCis-resveratrol

[0063] Compounds of Formula I, include compounds of the formulathe pharmaceutically acceptable salts thereof.Within Formula I, the variables, e.g. R1, R2, R3, A-ring, and W carry the following definitions.

[0064] The bond is a double or single bond.

[0065] The A ring (phenyl, Cs-Cecycloalky I, or a 4- to 6-membered heterocyclic group.

[0067] R1is absent or is 1 to 5 substituents independently selected from halogen, hydroxyl, amino, cyano, Ci-C4alkyl, Ci-C4alkoxy, (C3-C6Cycloalkyl)Co-C2alkyl, Ci-C2haloalkyl, and Ci- C2haloalkoxy.

[0068] R2is absent or is 1 to 5 substituents independently selected from halogen, hydroxyl, amino, cyano, Ci-C4alkyl, Ci-C4alkoxy, (C3-C6cycloalkyl)Co-C2alkyl, Ci-C2haloalkyl, and Ci- C2haloalkoxy.

[0069] R3is absent or is 1 to 4 substituents independently selected from halogen, Ci-C2alkyl, Ci-C2alkoxy. cyclopropyl, Ci-C2haloalkyl, and Ci-C2haloalkoxy.

[0070] Y is NH or O.

[0071] Z is H, hydroxyl, methyl, proline where the proline pyrrolidine ring is formed by Z and the nearest NH being joined by a -CH2CH2CH2- chain, or Z is a group -CH2X.

[0072] X is an amino acid side chain selected from H, -CH3, -CH2SH, -CH2CO2H,-CH2OH, -CH(CH3)OH, -CH2CH2CO2H, -CH2CH2CONH2,where a dash (-) indicates the point of attachment in Formula I.

[0073] This disclosure includes compounds of the following subformulae of Formula I and the pharmaceutically acceptable salts thereof.

[0074] The variables, R1, R2, R2, W, X, Y, Z, and the A-ring, can have the definitions set forth in the SUMMARY section, any of the definitions set forth in this specification. Any of the variable definitions can be combined so long as a stable compound results.(i) R1and R2are each absent or are 1 to 2 substituents independently selected from halogen, hydroxyl, amino, cyano, Ci-C4alkyl, Ci-C4alkoxy, Ci-C2haloalkyl, and Ci -Cihaloalkoxy.(ii) R3is absent.(iii) Y is NH.(iv) Y is O.(v) X is H, -CH2OH, or -CH(CH3)OH.(vi) The bond is a double bond.(vii) The bond is a single bond.

[0075] This disclosure includes the following exemplified compound of Formula I and the pharmaceutically acceptable salts thereof.

[0076] The compound 11-8 may have the following stereochemistry:

[0077] In an embodiment the compound II-8 may have the following stereochemistry:

[0078] In Formula I the variable R may be an amino acid side chain and in Formula II the variable X may be an amino acid chain. The amino acid side chain represented by variable R or X may be from an acidic amino acid. For example, the amino acid residue has a negative charge due to loss of hydrogen ion at physiological pH. Amino acids having an acidic side chain may include glutamic acid and aspartic acid.

[0079] The disclosure includes methods in which disulfiram is used as the only active agent and methods in which it is used in combination with another compound useful for stimulating tubulin tyrosination or capable of protecting against tyrosine-mediated transcription inhibition or useful for activating protein synthesis and tubulin tyrosination.

[0080] This disclosure provides a method of activating tubulin tyrosination, the method comprising determining the level of TyrRS in a cell; contacting the cell with an effective amount of disulfiram; wherein the effective amount of disulfiram is an amount sufficient to increase a TyrRS levels in the cell relative to the TyrRS level in the cell prior to contacting the cell with disulfiram. The cell can be contacted in vitro, e.g., in a culture of cells or in a tissue sample, or in vivo, in a living subject such as when administered to a patient. The level of TyrRS does not need to be ascertained in an individual cell, particularly when the cell is in a living subject. The amount of disulfiram administered to the subject is an effective amount if the expected in vivo concentration resulting from the disulfiram administration would be sufficient to increase TyrRS if that concentration was obtained in vitro. The tubulin tyrosination can be neuronal tubulin tyrosination. The effective amount can be a concentration of about 0.1 nM to about 10 nM, about 0.25 nM to about 5 nM, 0.75 nM to about 4 nM in vitro, in the plasma or blood of a subject administered disulfiram, in the cell, in the tissue containing the cell, or in the nucleus of the cell. The effective amount of disulfiram can also be a daily does effective to increase TyrRS in the cell. For an adult human subject, the effective amount can be a daily dose of 100 mg to 5000 mg, 100 mg to 2000 mg, 100 mg to 1000 mg, 250 mg to 500 mg, or 250 mg, or 500 mg.METHODS OF TREATMENT

[0081] The disclosure includes a method of activating tubulin tyrosination in a subject (e.g., a patient) comprising administering an effective amount of a disulfiram or salt thereof, alone, or incombination with a compound of Formula I or salt thereof, or cis-resveratrol (cis-RSV) or salt thereof. The disclosure further includes a method of treating a disease or disorder associated with reduced tubulin tyrosination in a subject (e.g., a patient) comprising administering an effective amount of a disulfiram or salt thereof, alone, or in combination with a compound of Formula I or salt thereof, or cis-resveratrol (cis-RSV) or salt thereof. A patient can be a human patient or nonhuman patient such as a companion animal or livestock animal. The disclosure includes use of a disulfiram or salt thereof, alone or in combination with a compound of Formula 1 or salt thereof, or cis-resveratrol (cis-RSV) or salt thereof for activating or increasing tubulin tyrosination in a subject (e.g., a patient). The disclosure includes use of a disulfiram or salt thereof, alone or in combination with a compound of Formula I or salt thereof, or cis-resveratrol (cis-RSV) or salt thereof for treating a disease or disorder associated with tubulin tyrosination in a subject (e.g., a patient). The disclosure includes methods of manufacturing a medicament comprising disulfiram, alone or in combination with a compound of Formula I or salt thereof, or cis-resveratrol (cis-RSV) or salt thereof, or salt thereof, for use in activating tubulin tyrosination in a subject (e.g. a patient) or for use in treating a disease or disorder associated with tyrosine / phenylalanine-mediated DNA damage and repair in a subject.

[0082] In one embodiment, compositions disclosed herein may used in methods for activating tubulin tyrosination. Methods for activating tubulin tyrosination may include administration disulfiram, alone or with another active compound disclosed herein, in vitro or ex vivo (e.g., by contacting the cell with such compounds) or, alternatively, in vivo (e.g., administering the compound to a subject). Activation of tubulin tyrosination may inferred from an increase in nuclear TyrRS levels in a cell. For instance, TyrRS levels in the cell of a subject may increase relative to a control amount by about 10% or more, such as about 20% or more, such as about 30% or more, such as about 40% or more, such as about 50% or more, or such as about 60% or more.

[0083] In one embodiment, TyrRS levels in the cell may increase by at least 2-fold after the administration of disulfiram, such as about 5-fold, such as about 10-fold, or such as about 20-fold after the administration of the composition.

[0084] When tubulin tyrosination is inactive, neuronal tyrosine levels can increase which can inhibit protein synthesis at the elongation step to deplete neuronal TyrRS. In one embodiment, activation of tubulin tyrosination may be measured by a decrease in neuronal tyrosine levels. As such, following administration of the composition disclosed herein, tyrosine levels in a cell may decrease relative to a control amount of about 10% or more, such as about 20% or more, such as about 30% or more, such as about 40% or more, such as about 50% or more, or such as about 60% or more.

[0085] Introduction of a composition disclosed herein into a cell may trigger degradation of one or more neuroinflammatory factors. For instance, the neuroinflammatory factors may include, but are not limited to, poly-ADP-ribose polymerase 1 (PARP1), signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa B (NF-KB), topoisomerase 1 (TOPI), or a combination thereof. As such, following administration of a composition disclosed herein, levels of neuroinflammatory factors in a cell may decrease relative to a control amount by about 10% or more, such as about 20% or more, such as about 30% or more, such as about 40% or more, such as about 50% or more, or such as about 60% or more.

[0086] The disclosure includes a treating a disorder in a subject associated with tyrosine- mediated transcription inhibition, or tubulin tyrosination the method comprising: administering an effective amount of disulfiram to the subject; where the effective amount is an amount sufficient to increase PARP1 -dependent neuronal DNA repair and / or to decrease CPT-mediated neurotoxicity. The method can include measuring a serine ADP ribosylation level in the subject prior to administering disulfiram. The method can include measuring a serine ADP ribosylation levels in the subject after administering disulfiram. The method can include measuring a TyrRS level in the subject prior to administering disulfiram. The method can include measuring TyrRS level in the subject after administering disulfiram.

[0087] The present disclosure provides methods for treating and / or preventing a disorder in a subject via administering a composition disclosed herein to the subject in need thereof. In one embodiment, the disorder may be mediated by increased tyrosine levels. In another embodiment, the disorder may be aging.

[0088] In one embodiment, the disorder may be an age-associated neurocognitive disorder. The age-associated neurocognitive disorder may include, but is not limited to, insomnia, hypersomnia, frontotemporal dementia, Alzheimer's disease, Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, epilepsy and seizures, learning disabilities, neuromuscular disorders, Cockayne syndrome, cerebral palsy, dystonia, spinocerebellar ataxia with axonal neuropathy- 1 (SCAN1), Angelman Syndrome, COVID-19-related neurocognitive problems, chemotherapy-associated neurocognitive problems including 'chemo brain', autism spectrum disorder (ASD), delirium, mild-cognitive impairment, traumatic brain injury, phenylketonuria, or tyrosinemia. In one embodiment, the age-associated neurocognitive disorder may be Alzheimer's disease.

[0089] In another embodiment, the disorder may be an age-associated metabolic disorder. For instance, the age-associated metabolic disorder may include, but is not limited to, heart failure, cardiovascular disease, autoimmune-related disorders, myocardial ischemia reperfusion injury,hypertension, stroke, septic encephalopathy, diabetes, obesity, sepsis, Systemic Lupus Erythematosus, or inflammation.

[0090] Similarly, in other embodiment, the disorder is cancerous growth of tissues or cells, such as breast cancer, ovarian cancer, colon cancer, pancreatic cancer, lung cancer, prostate cancer, brain tumor, leukemia, bone cancer, and cachexia.

[0091] In one embodiment, a composition disclosed herein and / or a pharmaceutically compatible carrier comprising a composition can be delivered to the targeted cells or tissue via a pharmaceutically acceptable delivery system. The term "administering" includes modes and routes of administration that allow a compound to perform its intended function. In general, the compounds disclosed herein may be administered to a subject according to known methods, including injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal, etc.), oral, inhalation, and transdermal routes. The injection can be bolus injections or can be continuous infusion. Depending on the route of administration, the agent can be coated with or retained in conjunction with a selected material to protect it from natural conditions, which may detrimentally affect its ability to perform its intended function. The compound may be administered alone or in conjunction with a pharmaceutically acceptable carrier. The compound can also be administered as a prodrug, that coverts to the active form of the compound in vivo.

[0092] Compounds disclosed herein may be introduced to a cell at a concentration from about 1 micromolar (pM) to about 100 pM, such as from about 2 pM to about 95 pM, such as from about 10 pM to about 85 pM, such as from about 20 pM to about 75 pM, such as from about 35 pM to about 50 pM, or any range therebetween. As expected, the dosage will be dependent on the condition, size, and age of the subject.

[0093] Compounds disclosed herein may be administered, as appropriate or indicated, in a single dose as a bolus or by continuous infusion, or as multiple doses by bolus or by continuous infusion. Multiple doses may be administered, for example, multiple times per day, once daily, multiple times per week, every 2, 3, 4, 5, 6 or 7 days, weekly, every 2, 3, 4, 5 or 6 weeks, or monthly. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques.

[0094] In an embodiment methods of treatment include administering disulfiram, cis-RSV, or a compound of Formula I or salt thereof where the therapeutically effective amount of the disulfiram, cis-RSV, or compound of Formula I is 0.1 mg to 2000 mg daily, 0.5 mg to 15 g administered weekly, or 20 mg to 500 g administered monthly. The amount of disulfiram, cis-RSV, or compound of Formula I administered daily can be 0.1 mg to 2000 mg daily, 0.1 mg to 1500 mg daily, comprising 0.1 to 1000 mg daily, 0.1 to 900 mg daily, 0.1 to 700 mg daily, 0.1 to 600 mg daily, 0.1 to 500 mg daily, 0.1 to 400 mg daily, 0.1 to 300 mg daily, 0.1 to 200 mg daily, 0.1 to100 mg daily, 0.1 to 50 mg daily, 0.1 to 25 mg daily, 0.1 to 10 mg daily, 0.1 to 5 mg daily, 1.0 to 1000 mg daily, 1.0 to 900 mg daily, 1.0 to 700 mg daily, 1.0 to 600 mg daily, 1.0 to 500 mg daily, 1.0 to 400 mg daily, 1.0 to 300 mg daily, 1.0 to 200 mg daily, 1.0 to 100 mg daily, 1.0 to 50 mg daily, 1.0 to 25 mg daily, 1.0 to 10 mg daily, 1.0 to 5 mg daily, 10 to 1000 mg daily, 10 to 900 mg daily, 10 to 700 mg daily, 10 to 600 mg daily, 10 to 500 mg daily, 10 to 400 mg daily, 10 to 300 mg daily, 10 to 200 mg daily, 10 to 100 mg daily, 10 to 50 mg daily, 100 to 1000 mg daily, 100 to 900 mg daily, 100 to 700 mg daily, 100 to 600 mg daily, or 100 to 500 mg daily.

[0095] When the compound disulfiram, cis-RSV, or compound of Formula I is administered daily, it can be administered 1, 2, 3, or 4 or more times daily. Once or twice daily administration is preferred.

[0096] Methods of treatment include administering a sufficient amount disulfiram, cis-RSV, or compound of Formula I or salt thereof to provide a plasma or blood Cmax of 100 ng / ml, 90 ng / ml, 80 ng / ml, 70 ng / ml, 60 ng / ml, 50 ng / ml, 40 ng / ml, 30 ng / ml, 20 ng / ml, or 10 ng / ml. PHARMACEUTICAL COMPOSITIONS

[0097] Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, glucose in saline, etc. Solid supports, liposomes, nanoparticles, microparticles, nanospheres, or microspheres may also be used as carriers for administration of a compound disclosed herein. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, solubilizers, fillers, stabilizers, binders, absorbents, bases, buffering agents, lubricants, controlled release vehicles, diluents, emulsifying agents, humectants, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well-known in the art. Supplementary agents can also be incorporated into the compositions.

[0098] The therapeutically effective amount of the composition can vary based on factors such as the disorder stage, age, sex, and weight of the individual, as well as the ability of the compound to elicit a desired response in the subject. Further, compounds disclosed herein can be administered to a subject at one time or over a series of treatments and may be administered to the subject at any time.

[0099] Compounds disclosed herein may be introduced to a cell at a concentration less than about from about 0.1 nanomolar (nM) to about 10 nM, such as from about 0.25 nM to about 5 nM, such as from about 0.75 nM to about 4 nM, such as from about 1 nM to about 3 nM, such as from about 1.5 nM to about 2 nM, or any range therebetween. As expected, the dosage will depend on the subject's condition, size, and age.

[0100] Compounds disclosed herein may be administered, as appropriate or indicated, in a single dose as a bolus, by continuous infusion, or as multiple doses by bolus or by continuous infusion. Multiple doses may be administered, for example, multiple times per day, once daily, multiple times per week, every 2, 3, 4, 5, 6 or 7 days, weekly, every 2, 3, 4, 5 or 6 weeks, or monthly. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques.

[0101] It can be advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein includes physically discrete units suited as unitary dosages for the subject to be treated; each unit may contain a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the application is dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0102] Pharmaceutical compositions for parenteral, intradermal, or subcutaneous injection can include pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. A composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like that can enhance the effectiveness of the active ingredient. Proper fluidity may be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. A composition may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like.

[0103] For intravenous administration, suitable carriers include, without limitation, physiological saline, bacteriostatic water, Cremophor EL™ (BASF™, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, an injectable composition should be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents such as paraben,chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0104] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

[0105] Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of an orally ingestible composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel®, or com starch; a lubricant such as magnesium stearate or Stertes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0106] When administered orally in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin (e.g., peanut oil, mineral oil, soybean oil, or sesame oil), or synthetic oils may be added. A liquid form may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol. When administered in liquid form, a composition can contain from about 0.5 to 90% by weight of disulfiram.

[0107] For administration by inhalation, a compound, e.g., disulfiram, may be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0108] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, and include, for example, transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the pharmaceutical compositions are formulated into ointments, salves, gels, or creams as generally known in the art.

[0109] In certain embodiments, a pharmaceutical composition can be formulated for sustained or controlled release of the compound (e.g., disulfiram). Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, poly orthoesters, andpolylactic acid can be used. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0110] The in vivo methods of this disclosure have application for both human and veterinary use. The methods of the present invention contemplate single, as well as multiple, administration, given either simultaneously or over an extended period of time.

[0111] Interestingly, in accordance with the present disclosure, disulfiram at nanomolar concentrations (nM) stimulates neuronal tubulin tyrosination (Tyr-tubulin), protein synthesis, and increases neuronal TyrRS and PheRSP levels. Further, disulfiram protects against tyrosine- mediated transcription inhibition and camptothecin-mediated neurotoxicity. As such, the present disclosure presents a novel mechanism of neuroprotective effects of disulfiram and, therefore, therapeutic interventions using disulfiram to activate neuronal tubulin tyrosination and protein synthesis along with protection.

[0112] The pharmaceutical composition can be formulated for oral administration. The oral dosage form can comprise from 0.1 to 99% by weight (wt%) of disulfiram, cis-RSV, or compound of Formula I. Some embodiments contain from 1 to 99%, 1 to 90%, 1 to 80%, 1 to 70%, 1 to 60%, 1 to 50 %, 1 to 40%, 1 to 30%, 1 to 25%, 1 to 20%, 5 to 99%, 5 to 90%, 5 to 80%, 5 to 70%, 5 to 60%, 5 to 50 %, 5 to 40%, 5 to 30%, 5 to 25%, 5 to 20%, 10 to 99%, 10 to 90%, 10 to 80%, 10 to 70%, 10 to 60%, 10 to 50 %, 10 to 40%, 10 to 30%, 20 to 99%, 20 to 90%, 20 to 80%, 20 to 70%, 20 to 60%, 20 to 50 %, or 20 to 40% by weight (wt%) of the disulfiram, cis-RSV, or compound of Formula I.

[0113] In an embodiment the dosage form disulfiram, cis-RSV, or compound of Formula I is an immediate or extended release dosage from comprising 0.1 to 1000 mg, 0.1 to 900 mg, 0.1 to 700 mg, 0.1 to 600 mg, 0.1 to 500 mg, 0.1 to 400 mg, 0.1 to 300 mg, 0.1 to 200 mg, 0.1 to 100 mg, 0.1 to 50 mg, 0.1 to 25 mg, 0.1 to 10 mg, 0.1 to 5 mg, 1.0 to 1000 mg, 1.0 to 900 mg, 1.0 to 700 mg, 1.0 to 600 mg, 1.0 to 500 mg, 1.0 to 400 mg, 1.0 to 300 mg, 1.0 to 200 mg, 1.0 to 100 mg, 1.0 to 50 mg, 1.0 to 25 mg, 1.0 to 10 mg, 1.0 to 5 mg, 10 to 1000 mg, 10 to 900 mg, 10 to 700 mg, 10 to 600 mg, 10 to 500 mg, 10 to 400 mg, 10 to 300 mg, 10 to 200 mg, 10 to 100 mg, 10 to 50 mg, 100 to 1000 mg, 100 to 900 mg, 100 to 700 mg, 100 to 600 mg, or 100 to 500 mg of the disulfiram, cis-RSV, or compound of Formula I per unit dosage form.

[0114] In an embodiment the dosage form comprises a disulfiram, cis-RSV, or compound of Formula I and is an extended release dosage form formulated for once weekly to once monthlyadministration that provides a plasma Cmax of 0.1 to 100 nM of the compound of Formula I to the patient. The form can be an extended release dosage form.

[0115] The present disclosure may be better understood with reference to the following examples.EXAMPLESMaterials and MethodsPrimary Neuronal Culture

[0116] Primary cortical neurons were harvested from 18-day-old Sprague Dawley rat pups using Hibernate E (BrainBits) and dissociated with the Neural Tissue Dissociation kit (Miltenyi Biotec). Briefly, the cortices were minced cortices and incubated in a pre-heated enzyme mix at 37 °C for 15 minutes. The tissues were then strained using a 40 m cell strainer, washed, and centrifuged. The neurons were then cultured on tissue culture plates coated with 50 pg / ml poly-D- Lysine (Sigma Aldrich). The culture medium consisted of NBActive-1 medium (BrainBits) supplemented with 100 U / ml of Penicillin-Streptomycin (Life Technologies), 2 mM L-Glutamine (Life Technologies), and IX N21 supplement (R&D Systems) was used as culture medium. Control (non-targeting), TyrRS, and PARP1 siRNAs were obtained from Invitrogen (# AM4635, s443, and si 30207, respectively). 5 DIV neurons were transfected with 75 nM control or TyrRS siRNA using Dharmafect 3 Transfection Reagent. A second transfection was done two days later using 75 nM of TyrRS siRNA, followed by cell collection or assays after another 48 hr. For PARP1 siRNA, 7 DIV neurons were transfected with either 75 nM control or PARP1 siRNA.Western Blotting

[0117] Cultured primary rat cortical neurons (DIV 9 / 10) were prepared for analysis by washing with cold lx PBS and lysing in cell lysis buffer. The lysates were then centrifuged at 15,000g for 15 min at 4 °C to separate the chromatin-bound and soluble fractions and equal amounts of protein were loaded onto a 4% to 12% gradient gel (NuPAGE-Invitrogen) for electrophoresis. The protein was transferred to a 0.2 pm NC membrane and the membrane was blocked with 5% non-fat milk in TBST. Primary antibodies were applied to the membrane and incubated overnight at 4 °C, followed by incubation with secondary antibodies for 1 hour at room temperature. The immobilon ECL Ultra Western HRP Substrate was used to detect the proteins, and the luminescent image analyzer (ChemiDoc Imaging System, Bio-Rad) was used for quantification. The western blots were quantified using Imaged software (Version 1.53t).Immunofluorescence (IF)

[0118] Cultured cortical neurons at DIV 9-10 were fixed in 4% formaldehyde for 15 minutes, permeabilized, and blocked with 5% BSA (PBS) and 0.1% Tween20 for 30 minutes at room temperature. Primary antibodies were added and incubated overnight at 4 °C, followed by secondary antibody incubation for 1 hour at room temperature. Alexa Fluor 647 (anti-chicken), Alexa Fluor 555 (anti-mouse), and Alexa Fluor 488 (anti- rabbit) from Invitrogen were used as secondary antibodies at a dilution of 1:1000. Coverslips were mounted with D API-supplemented mounting medium, Prolong Gold Antifade (Invitrogen), and imaged with a Leica DMI6000 epifluorescent microscope using an oil immersion 63x / NA 1.4 objective. The quantification of total protein levels in neurons was performed using ImageJ (Version 1.53c), with imaging parameters matched for exposure, gain, and offset.Statistical Analysis

[0119] The data was analyzed for statistical significance and differences between groups were determined. Depending on the number of groups being compared, either one-way ANOVA with multiple comparisons without correction or two-way ANOVA with multiple comparisons without correction were used. When comparing two groups, either paired or unpaired t-tests were used. The data analysis was performed using GraphPad statistical analysis software.EXAMPLE 1. DISULFIRAM INCREASES NEURONAL TYRRS AND PHERSB PROTEIN LEVELS BY ACTIVATING PROTEIN SYNTHESIS AND TUBULIN TYROSINATION IN RAT CORTICAL NEURONS.

[0120] Alcohol consumption increases serum tyrosine levels and inhibits brain protein synthesis in mice models. Moreover, in humans, alcohol consumption reduces rates of myofibrillar protein synthesis (MPS) in skeletal muscle and midlife alcohol consumption enhances the risk for cognitive impairment in late life. Similarly, alcohol-induced loss of consciousness is associated with a subsequent increase in the risk of dementia. We recently showed that tyrosine inhibits protein synthesis at the elongation step and induces neurodegeneration. Although disulfiram is used against alcohol addiction, whether disulfiram affects neuronal TyrRS levels is not yet known. Therefore, we treated rat cortical neurons (DIV 9 / 10) with increasing concentrations (0.5-5 nM) of disulfiram for 2 hr and determined the changes in the TyrRS and PheRSa / b levels and phosphorylated eIF2a (p-eIF2a) levels. We found that low nanomolar concentrations of disulfiram increased both TyrRS and PheRSb levels (PheRSa levels did not change) while p-eIF2a levels decreased (FIG. 1A). Further, to confirm the effect of decreased p-eIF2a levels in global protein synthesis, we treated rat cortical neurons with increasing concentrations of disulfiram (0.5-10 nM) for 2 hr flowed by addition of puromycin (50 mM) for 30 min and detected the levels of puromycin incorporation by its specific antibody (FIG. IB). Our data showed that low nanomolar concentration of disulfiram is a strong stimulator of neuronal protein synthesis. Since tyrosine is an inhibitor of protein synthesis at the elongation step that depletes neuronal TyrRS, we determined the effect of disulfiram on tubulin tyrosination, a major modulator of cellular tyrosination levels. As expected, we found that treatment with 25 nM disulfiram strongly activates tubulin tyrosination (FIG. 1C). Together, these data provide novel insights into the mechanism of action of disulfiram- mediated activation of neuronal protein synthesis and upregulation of TyrRS.EXAMPLE 2. DISULFIRAM STIMULATES PARP- DEPENDENT ACTIVATION OF HISTONE SERINE- ADP-RIBOSYLATION AND PROTECTS AGAINST TYROSINE-MEDIATED TRANSCRIPTION INHIBITION.

[0121] We previously showed that PARPl-dependent H3 serine ADP-ribosylation levels are decreased in the brains of Alzheimer’s disease patients, suggesting that neuronal H3-Ser- ADP- ribosylation levels may correlate with cognitive performance and memory formation. Moreover, cA-RSV-mediated nuclear localization of TyrRS also stimulates PARPl-dependent H3-Ser-ADP-ribosylation. To determine if disulfiram would protect against alcohol-mediated neurotoxic effects by activating PARP1 -dependent H3 serine- ADP-ribosylation, we treated rat cortical neurons (DIV 9 / 10) with increasing concentrations of disulfiram (0.5-5 nM) and found that disulfiram is a strong inducer of H3-Ser- ADP-ribosylation and PARylation (FIG. 2A). PARP1 activation facilitates transcription elongation and oxidative DNA damage inhibits transcription. We previously observed that tyrosine induces oxidative DNA damage, potentially by depleting TyrRS. Therefore, we hypothesized that tyrosine inhibits global transcription. Therefore, we treated rat cortical neurons with tyrosine for 16 hr and measure the levels of transcription using EU incorporation and found that tyrosine inhibits global transcription (FIG. 2B). Since tyrosine inhibited transcription, without wishing to be bound by theory, it was hypothesized that disulfiram would rescue tyrosine-mediated transcription inhibition by activating the repair of DNA damage. To evaluate this hypothesis, the effect of tyrosine alone or in combination with disulfiram on transcription was analyzed using a 5- ethynyluridine (EU) incorporation assay. To do so, primary cortical neurons were treated with disulfiram for up to 2 hr and then treated with EU for 30 min in neuronal cultures pre-treated with tyrosine that inhibits transcription. The extent of EU incorporation in nascent RNA was quantified and found disulfiram-treated neurons show increased incorporation of EU, tyrosine decreased the incorporation of EU and co-treatment with disulfiram rescued tyrosine-mediated transcription inhibition (FIG. 2B).EXAMPLE 3. DISULFIRAM STIMULATES NEURONAL DNA REPAIR AND PROTECTS AGAINST TYROSINEMETABOLITES -MEDIATED TRANSCRIPTION INHIBITION.

[0122] PARP1 is essential for the repair of topoisomerase 1 -mediated neuronal single strand DNA breaks (SSBs). Topoisomerase 1 / CPT-mediated DNA damage is an inhibitor of transcription associated with synapse-associated genes, and long-genes, and cell identity genes. Moreover, CPT- treatment also known to stimulate neurodegeneration. Since disulfiram protects against alcohol- mediated neurotoxicity, stimulates PARP1 (FIG. 2A), and tyrosine-mediated transcription inhibition (FIG. 2B), we hypothesized that disulfiram would protect against CPT-mediated neurotoxicity as well. To determine the effect of disulfiram against CPT-mediated neurotoxicity, we treated rat cortical neurons with CPT (50mM) alone or in combination with disulfiram (25 nM- 250 mM) for 24 hr and subjected them to MTT assay to determine their effects on neuronal survival. We found that treatment lower concentrations of disulfiram (<50 mM) protected against CPT whereas higher concentrations of disulfiram (>100 mM) exacerbated CPT-mediated neurotoxicity (FIG. 3A). Beyond tyrosine, the metabolites of tyrosine and phenylalanine such as p-cresol and 4HPA are increased in the serum of patients with autism spectrum disorders (ASD) as well, suggesting that p- cresol and 4HPA may also inhibit global transcription. Therefore, we determined the effect of disulfiram against p-cresol and 4HPA and found that disulfiram protects against p-cresol, 3-ISP, and4HPA-mediated transcription inhibition (FIG. 3B). Together, these data suggest that disulfiram protects against alcohol-induced neurotoxicity by activating PARP1 -dependent DNA repair, increasing neuronal TyrRS, and by stimulating neuronal tubulin tyrosination, and neuronal protein synthesis and by protecting against tyrosine / its metabolites-mediated transcription inhibition.

[0123] These and other modifications and variations to the present invention may be practiced by those of ordinary skill in the art, without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. In addition, it should be understood that aspects of the various embodiments may be interchanged both in whole, and in part. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention so further described in such appended claims.EXAMPLE 4. SYNTHESIS SCHE E OF II-7 AND II-8 : SYNTHESIS OF 4-((2,3-DIPHENYLALLYL)AMINO)-N-(2-HYDROXYETHYL)BENZAMIDE (II-7) AND 4-((2,3-DIPHENYLALLYL)AMINO)-N-(2-HYDROXYPROPYL)BENZAMIDE (II- 8)

[0124] To connect a serine amino acide side chain to II-3, we initially synthesized the ethanolamine connected benzamide which upon reductive amination should provide the desired product. Nevertheless, our efforts with the treatment of 9 with the benzamide 11 was unsuccessful under reductive amination reaction conditions. Therefore, we have choosen the acid-amine coupling approach by converting the estre to acid chloride, and then treating the acid chloride with 2-methoxyethylamine under basic conditions to provide the desired II-7 in good yield over four steps.

[0125] A similar approach with 2-methoxypropan-l-amine was ineffective to provide the respective amide derivative II-8. However, the TBS-protected l-aminopropan-2-ol (12) under acid- amine coupling reaction conditions furnished the desired threonyl side chain linker of the benzamide II-8 as depicted in the Scheme. Interestingly, the II-8 (EC50 = < 25 nM) is more potent than the II-3 (ECso = > 25 pM) as shown in MTT assay.

[0126] ’H NMR for compound II-8 (400 MHz, Chloroform-d) 6 7.61-7.52 (m, 2H), 7.30-7.19 (m, 3H), 7.14-7.06 (m, 2H), 7.01 (dd, J = 4.9, 1.9 Hz, 3H), 6.85 (dd, J = 6.8, 3.0 Hz, 2H), 6.55-6.51 (m, 3H), 4.30 (t, J = 6.0 Hz, 1H), 4.05 (d, J = 4.5 Hz, 2H), 3.90 (ddq, J = 12.5, 6.2, 2.9 Hz, 1H), 3.50 (ddd, J = 14.0, 6.4, 2.9 Hz, 1H), 3.27 (s, 1H), 3.19 (ddd, 7 = 14.0, 7.6, 5.2 Hz, 1H), 1.12 (d, 7 = 6.3 Hz, 3H).nC NMR (101 MHz, Chloroform-7) 5 168.59, 150.72, 139.09, 138.38, 136.31, 129.18, 128.91, 128.80, 128.56, 127.98, 127.64, 127.50, 126.87, 122.42, 112.17, 67.82, 51.60, 47.58, 20.99. HRMS (ESI) calcd for C25H27N2O2, 387.2073; found, 387.2069.

Claims

CLAIMSWhat Is Claimed:

1. A method of activating tubulin tyrosination, the method comprising determining the level of TyrRS in a cell; contacting the cell with an effective amount of disulfiram; wherein the effective amount of disulfiram is an amount sufficient to increase a TyrRS levels in the cell relative to the TyrRS level in the cell prior to contacting the cell with disulfiram.

2. The method of claim 1, wherein the cell is contacted in vitro.

3. The method of claim 1 , wherein the cell is present in a living subject.

4. The method of claim 1 , wherein tubulin tyrosination comprises tubulin tyrosine ligase (TTL)-dependent tubulin tyrosination.

5. The method of claim 1, wherein tubulin tyrosination comprises neuronal tubulin tyrosination.

6. The method of any one of claims 1 to 5, wherein the effective amount of the disulfiram is an amount effective to provide a concentration of 0.1 nM to about 10 nM disulfiram at the cell surface.

7. The method of any one of claims 1 to 5, wherein the effective amount of the disulfiram is an amount effective to provide a concentration of about 0.25 nM to about 5 nM at the cell surface.

8. The method of any one of claims 1 to 5, wherein the effective amount of the disulfiram is an amount effective to provide a concentration of about 0.75 nM to about 4 nM at the cell surface.

9. The method of any one of claims 1 to 5, wherein the effective amount of disulfiram is a daily dose of from 100 mg to 1000 mg.

10. The method of any one of claims 1 to 5, wherein the effective amount of disulfiram is a daily dose of 250 mg or 500 mg.

11. The method of any one of claims 1 to 5, wherein the effective amount of disulfiram is an amount effective to increase the TyrRS levels in the cell at least 2-fold.

12. The method of any one of claims 1 to 5, wherein the effective amount of disulfiram is an amount effective to increase the TyrRS levels in the cell at least 5-fold.

13. The method of any one of claims 1 to 5, wherein the effective amount of disulfiram is an amount effective to increase the TyrRS levels in the cell at least 10-fold.

14. A method of treating a disorder in a subject associated with tyrosine-mediated transcription inhibition, the method comprising: administering an effective amount of disulfiram to the subject; where the effective amount is an amount sufficient to increase PARP1 -dependent neuronal DNA repair and / or to decrease CPT-mediated neurotoxicity.

15. The method of claim 14, further comprising measuring a serine ADP ribosylation level in the subject prior to administering disulfiram.

16. The method of claim 14 or 15, further comprising measuring a serine ADP ribosylation levels in the subject after administering disulfiram.

17. The method of any one of claims 14 to 16, further comprising measuring a TyrRS level in the subject prior to administering disulfiram.

18. The method of any one of claim 14 to 17, further comprising measuring TyrRS levels in the subject after administering disulfiram.

19. The method of any one of claims 14 to 18, wherein the effective amount of disulfiram is an amount effective to increase the TyrRS levels in the subject at least 2-fold after administration of disulfiram.

20. The method of any one of claims 14 to 18, wherein the effective amount of disulfiram is an amount effective to increase the TyrRS levels in the subject at least 5-fold after administration of disulfiram.

21. The method of any one of claims 14 to 18, wherein the effective amount of disulfiram is an amount effective to increase the TyrRS levels in the subject at least 10-fold after administration of disulfiram.

22. The method of any one of claims 14 to 18, wherein the effective amount of disulfiram sufficient to produce a disulfiram concentration of about 0.1 nM to about 10 nM in the subject’s blood, plasma, or tissues.

23. The method of any one of claims 14 to 18, wherein the effective amount of disulfiram sufficient to produce a disulfiram concentration of about 0.25 nM to about 5 nM in the subject’s blood, plasma, or tissues.

24. The method of any one of claims 14 to 18, wherein the effective amount of disulfiram sufficient to produce a disulfiram concentration of about 0.75 nM to about 4 nM in the subject’s blood, plasma, or tissues.

25. The method of claims 14 to 18, wherein the effective amount of disulfiram is a daily dose of about 100 mg to 1000 mg, or 250 mg, or 500 mg.

26. The method of any one of claims 1 to 13, additionally comprising contacting the cell with an effective amount of cis-RSV or a compound of Formula I, or salt thereof.

27. The method of any one of claims 14 to 25, additionally comprising administering an effective amount of cis-RSV or a compound of Formula I, or salt thereof to the subject.

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