WO2016069716A1 - Compositions and methods comprising tyrosyl-trna synthetases and resveratrol compounds - Google Patents

Abstract

Provided are combination therapies with tyrosyl-tRNA synthetase and resveratrol compounds, and related pharmaceutical compositions, for treating aging, physiological stress, and variety of disease indications, such as cardiovascular diseases, neurological diseases including neurodegenerative diseases, metabolic diseases, cancers, and obesity.

Description

COMPOSITIONS AND METHODS COMPRISING TYROSYL-TRNA SYNTHETASES AND RESVERATROL COMPOUNDS CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/072,561, filed on

October 30, 2014 all of which is incorporated by reference herein in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename:

ATY _122_01WO_SeqList_ST25.txt, date recorded: October 27, 2015, file size 34 kilobytes).

BACKGROUND

Technical Field

Embodiments of the present invention relate to combination therapies with tyrosyl-tRNA synthetase and resveratrol compounds, and related pharmaceutical compositions, for treating aging, physiological stress, and a variety of disease indications, such as cardiovascular diseases, neurological diseases, metabolic diseases, cancers, and obesity.

Description of the Related Art

Resveratrol (RSV) belongs to a class of polyphenolic compounds called stilbenes. Certain plants produce resveratrol and other stilbenes in response to stress, injury, fungal infection, or ultraviolet (UV) radiation. Resveratrol is reported to extend life span (see Howitz et al., Nature 425:191-196, 2003; and Viswanathan et al., Dev Cell 9:605-615, 2005), and provide cardio-neuro- protective (see Baur et al., Nature 444:337-342, 2006), anti-diabetic (Milne et al., Nature 450:712- 716, 2007), and anti-cancer effects (Jang et al., Science 275, 218-220, 1997)), for example, by initiating a stress response that induces survival genes.

Tyrosyl-tRNA synthetase (TyrRS) has been shown to possess a variety of non-canonical activities of therapeutic and diagnostic relevance. Examples of such activities include modulation of hematopoietic pathways such as thrombopoiesis, modulation of angiogenesis, and modulation of inflammatory pathways, among others. It also translocates to the nucleus under stress conditions (see Fu et al., JBC 287:9330-9334, 2012) and interacts with PARP-1 signaling pathways (see Sajish et al., Nature Chemical Biology 8:547-554, 2012). To best exploit these and other activities in therapeutic settings, there is a need in the art to identify interactions that can enhance beneficial stress response-associated pathways. The present invention provides these and other benefits.

BRIEF SUMMARY

Embodiments of the present invention relate to the discovery that the combination of tyrosyl-tRNA synthetase and resveratrol synergistically induces PARP-1- and NAD+-dependent signaling pathways, which results in the initiation of a cellular stress response that induces the expression of survival genes. Such could find therapeutic utility, for example, in treating or managing a variety of diseases, such as cardiovascular diseases, metabolic diseases, obesity, and neurological diseases, reducing physiological stress (e.g., oxidative stress) and the various symptoms and diseases that associate with the same, and reducing many of the harmful effects associated with aging, including combinations of the foregoing.

Embodiments of the present invention therefore include methods of increasing a stress response in a cell, for example, evoking therapeutic mild stress in the cell, comprising contacting the cell with (a) a tyrosyl-tRNA synthetase (TyrRS) polypeptide or a polynucleotide that encodes the TyrRS polypeptide, in combination with (b) at least one resveratrol compound.

In some embodiments, (a) is a TyrRS polypeptide that comprises any of SEQ ID NO:l-3 or a sequence having at least 90% identity to any of SEQ ID NOS:l-3, where the variant TyrRS polypeptide binds to the resveratrol compound and binds to Poly [ADP-ribose] polymerase 1 (PARP-1).

In certain embodiments, the resveratrol compound is a compound of Formula l-A or l-B, or a pharmaceutically acceptable salt thereof.

In certain embodiments, the combination of (a) and (b) synergistically increases the stress response in the cell relative to (a) alone or (b) alone.

In some embodiments, the stress response comprises evoking mild stress in the cell. In certain embodiments, the combination of (a) and (b) synergistically evokes mild stress in the cell relative to (a) alone or (b) alone, and optionally thereby induces or achieves protective effects in the cell.

In certain embodiments, the stress response comprises increased activation of Poly [ADP- ribose] polymerase 1 (PARP-1). In particular embodiments, the increased activation of PARP-1 comprises increased auto-PARylation of PARP-1. In certain embodiments, the increased activation of PARP-1 comprises increased expression of one or more PARP-1 target genes and optionally associated downstream signaling events. In some embodiments, the one or more PARP-1 target genes are selected from Hsp72, SIRT1, SIRT6, FOX03A, SESN2, NAMPT, PUMA, BRCA1, and pl4ARF, including combinations thereof.

In some embodiments, the cell is contacted with (a) and (b) simultaneously or sequentially. In some embodiments, the cell is in a subject, and the method comprises administering (a) and (b) to the subject. In certain embodiments, (a) and (b) are administered simultaneously or sequentially. In certain embodiments, the subject has or is at risk for having physiological stress, optionally selected from oxidative stress, viral stress, radiation-induced stress, drug-induced stress, light-induced stress, and combinations thereof. In some embodiments, the subject has a disease selected from a cardiovascular disease, a neurological disease, a metabolic disease, and cancer. In certain embodiments, the subject is elderly or middle-aged. In particular embodiments, the subject is at least 40, 50, 60, 70, 80, 90, or 100 years old. In certain embodiments, the subject has an aging- associated disease or condition. In some embodiments, the subject has atherosclerosis and/or hypertension.

In certain embodiments, administering (a) and (b) reduces oxidative stress and/or inflammation in the subject. In some embodiments, administering (a) and (b) increases the life expectancy of the subject.

Also included are methods of treating physiological stress in a subject, comprising administering to the subject (a) a tyrosyl-tRNA synthetase (TyrRS) polypeptide or a polynucleotide that encodes the TyrRS polypeptide, in combination with (b) at least one resveratrol compound.

In certain embodiments, the physiological stress is selected from oxidative stress, viral stress, radiation-induced stress, drug-induced stress, light-induced stress, and combinations thereof, for example, radiation-induced oxidative stress, drug-induced oxidative stress, or light-induced oxidative stress.

Certain embodiments relate to methods of treating a disease in a subject, where the disease is selected from a cardiovascular disease, a neurological disease, a metabolic disease, obesity, and a cancer, comprising administering to the subject (a) a tyrosyl-tRNA synthetase (TyrRS) polypeptide or a polynucleotide that encodes the TyrRS polypeptide, in combination with (b) at least one resveratrol compound.

In some embodiments, the cardiovascular disease is selected from coronary artery disease, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, cardiac dysrhythmias, inflammatory heart disease, valvular heart disease, cerebrovascular disease, peripheral arterial disease, congenital heart disease, and rheumatic heart disease.

In certain embodiments, the neurological disease is selected from neuro-inflammation, tumorigenesis of the brain, brain ischemia, neuropathy, and neurodegeneration associated with aging. In certain embodiments, the metabolic disease is impaired insulin sensitivity and/or glucose utilization.

In particular embodiments, the metabolic disease is selected from Type 1 diabetes, Type 2 diabetes, pre-diabetes, hyperglycemia, hyperinsulinaemia, and metabolic syndrome. In some embodiments, the subject is obese. In certain embodiments, the obese subject has one or more of a cardiovascular disease, a metabolic disease, obstructive sleep apnea, a cancer, or osteoarthritis, and where administering (a) and (b) reduces the symptoms or pathology of one or more of the foregoing. In certain embodiments, the obese subject has increased risk of developing one or more of a cardiovascular disease, a metabolic disease, obstructive sleep apnea, a cancer, or osteoarthritis, and where administering (a) and (b) reduces the risk of developing one or more of the foregoing. In specific embodiments, administering (a) and (b) increases the life expectancy of the obese subject.

In some embodiments, the cancer is selected from one or more of a breast cancer, cervical cancer, prostate cancer, pancreatic cancer, gastrointestinal cancer, lung cancer, ovarian cancer, testicular cancer, head and neck cancer, bladder cancer, kidney cancer, soft tissue sarcoma, squamous cell carcinoma, CNS or brain cancer, melanoma, non-melanoma cancer, thyroid cancer, endometrial cancer, an epithelial tumor, bone cancer, and hematopoietic cancer.

Also included are methods of treating aging in a subject, comprising administering to the subject (a) a tyrosyl-tRNA synthetase (TyrRS) polypeptide or a polynucleotide that encodes the TyrRS polypeptide, in combination with (b) at least one resveratrol compound.

In certain embodiments, the subject is elderly or middle-aged. In certain embodiments, the subject is at least 40, 50, 60, 70, 80, 90, or 100 years old. In certain embodiments, the subject has an aging-associated disease or condition. In certain embodiments, the subject has atherosclerosis and/or hypertension. In particular embodiments, the subject is obese. In some embodiments, administering (a) and (b) reduces oxidative stress and/or inflammation in the subject. In certain embodiments, administering (a) and (b) increases the life expectancy of the subject.

Also included are pharmaceutical compositions, comprising (a) a tyrosyl-tRNA synthetase (TyrRS) polypeptide or a polynucleotide that encodes the TyrRS polypeptide; (b) at least one resveratrol compound; and (c) a pharmaceutical-grade carrier.

In certain embodiments, the molar ratio of (a) to (b) is about 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In some embodiments, the molar ratio of (a) to (b) is about 1:2. Some pharmaceutical compositions or methods comprise or employ at least about 5 mg/ml of the TyrRS polypeptide. Particular pharmaceutical compositions or methods comprise or employ at least about 8 mg/ml, at least about 10 mg/ml, at least about 15 mg/mL, or at least about 20 mg/ml of the TyrRS polypeptide. Certain pharmaceutical compositions or methods comprise or employ about 0.001 mg to about 20,000 mg of the TyrRS polypeptide. Some pharmaceutical compositions or methods comprise or employ about 0.01 mg to about 5000 mg of the resveratrol compound. BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1C show that resveratrol binds at the active site of human TyrRS. Figure 1A provides an illustration of the domain organization of human TyrRS. Both domains are connected by a linker of about 20 amino acids in length. Figure IB shows that serum starvation and resveratrol treatment (5 μΜ) facilitated the nuclear translocation of TyrRS with a concomitant increase in the Poly(ADP-ribosyl)ation (PARylation) of PARP-1. Figure 1C shows the electron density of co-crystal X- ray structures (2.1 A") of TyrRS bound to cis-resveratrol and to L-tyrosine. At right, resveratrol induced a local conformational change relative to bound tyrosine at the active site.

Figures 2A-2E show that TyrRS facilitates the activation of PARP-1 in an active-site- dependent manner. I n Figure 2A (top), TyrRS activates PARP-1 in an in vitro assay. In Figure 2A (middle), resveratrol potentiates TyrRS mediated activation of PARP-1. In Figure 2A (bottom), Tyr-SA blocks the resveratrol-mediated activation of PARP-1. In Figure 2B (top), TyrRS-V5 overexpression activates PARP-1 in HeLa cells in a concentration-dependent manner. I n Figure 2B (middle), resveratrol treatment activates PARP-1 in HeLa cells and enhances TyrRS interaction with PARP-1. In Figure 2C (bottom), Tyr-SA blocks the resveratrol-mediated interaction of TyrRS and activation of PARP-1. Figure 2C shows an illustration of the C-domain disposition in TyrRS (left) and Y341ATyrRS (right). As shown in Figure 2D, Y341ATyrRS enhances its interaction and activates PARP-1 relative to WT. Figure 2E shows that resveratrol synergistically potentiates the recombinant TyrRS-dependent activation of PARP-1.

Figures 3A-3D show that resveratrol and serum starvation mimic similar downstream signaling events mediated by PARP-1 activation. Figure 3A illustrates the molecular basis and the integration of different signaling pathways to mediate a TyrRS/PARP-1 activated stress response evoked by either resveratrol or different stress conditions. Figure 3B shows that PARP-1 activating conditions enhance Tip60-mediated activation of ATM . Figures 3C and 3D shows a time course of poly-ADP-ribosylation status and associated signaling events as depicted in Figure 3A after serum starvation (3C) and 1 μΜ resveratrol treatment (3D).

Figures 4A-4E show that resveratrol treatment activates TyrRS-PARP-1 driven signaling events in mouse tissues. Figures 4A and 4B show that siRNA directed against PARP-1 (4A) or TyrRS (4B) abrogates resveratrol-mediated downstream signaling events. HeLa cells were treated with siRNA^{PARP 1} or siRNA^TvrRS for 60 hou rs to knockdown PARP-1 or TyrRS expression levels by ~70-80%. Knockdown efficiency was monitored by with a-PARP-1 or a-TyrRS. For siRNA^TyrRS, HeLa cells were treated with either serum starvation or resveratrol (5, 10 25 and 50 μΜ) for 45 minutes. The dashed line in Figure 4A represents the demarcation between siRNA^Con and siRNA^{PARP 1}; all the samples were run on the same gel. Figure 4C shows that NAMPT inhibition abrogates resveratrol-mediated downstream signaling events. HeLa cells were pre-treated with STF-118804 (100 nM), a NAMPT inhibitor, for 16 h and then treated with resveratrol (5 μΜ), with samples collected at intervals as indicated. Various signaling events, as depicted, were monitored using appropriate antibodies. Acetylation status was monitored using a-acetyl-lysine. The dashed line separates results without and with the inhibitor. Figure 4D shows that resveratrol-mediated activation of PARP-1 is blocked by Tyr-SA. Activation of Tip60 and AMPK were monitored by using a-AcK16-H4 and a-pSer36-H2B, respectively. Figure 4E shows that resveratrol-mediated interaction of TyrRS with PARP-1 and acetylation of p53 are blocked by Tyr-SA. Immunoprecipitation of PARP-1 and p53 from muscle tissue demonstrated RSV-mediated TyrRS-PARP-1 interaction and p53 acetylation.

Figure 5 shows the chemical structures of resveratrol and exemplary resveratrol analogs.

Figures 6A-6D show that resveratrol induces a distinct conformational change upon binding to active site of TyrRS. Figure 6A shows a comparison of the overall conformational change induced by resveratrol at the active site of TyrRS by structure based su perposition (yellow-tyrosine-bound structure and magenta-resveratrol-bound structure). Note the conformational change near the helix region (P331-P342) that connects the linker region with the C-domain. Figure 6B illustrates the interactions of resveratrol with the active site. In Figure 6C, trans-resveratrol docks (manual) into TyrRS active site without significant structural disturbances. In Figure 6D, generation of a new pocket through a RSV-induced conformational change in TyrRS accommodates the dihydroxy phenolic ring of RSV (otherwise exposed to the destabilizing aqueous environment in the trans form) and hence facilitates the trans to cis conversion of RSV.

Figures 7A-7C show that resveratrol inhibits the canonical enzymatic activation of TyrRS. In Figure 7A, the ATP-PPi exchange assay as described in Example 2 demonstrated the inhibitory effect of resveratrol on TyrRS. Figure 7B shows that resveratrol shifts the Km for tyrosine, and Figure 7C shows that resveratrol binds TyrRS better than tyrosine. The apparent Ki for resveratrol was deduced by varying the concentration of RSV and plotting the slope of (1/v vs l/[Tyr]) versus [RSV] as indicated. RSV strongly inhibited the aminoacylation enzymatic activity of TyrRS with a Ki-value of 22 μΜ.

Figures 8A-8H show that resveratrol facilitates the TyrRS/PARP-1 interaction in an active- site-dependent manner. In Figure 8A, both heat shock (42°C for 30 min) and tunicamycin-treatment (10 μg/ml, ER stress) facilitated the nuclear translocation of TyrRS and activation of PARP-1. I n Figure 8B, resveratrol or serum starvation facilitates TyrRS interaction with PARP-1 and Tyr-SA prevents this interaction. ZZ-PARP-1 was immuno-precipitated with IgG from HeLa cells treated with RSV or serum starvation alone or in combination with Tyr-SA. In Figure 8C, resveratrol or serum starvation mediated PARP-1 activation is blocked only by Tyr-SA and not by Gly-SA. In Figure 8D, TyrRS interacts directly with PARP-1. HeLa cell lysate after RSV treatment (5 μΜ, 30 min) was divided into three parts and treated with PARG and catalytically inactive PARG-MT. PARP-1 was immuno-precipitated and analyzed for TyrRS interaction. The model in Figure 8E illustrates the mechanism of RSV mediated TyrRS interaction with PARP-1 and subsequent release after auto-PARylation. In Figure 8F, Ni-NTA pull-down of N- and C-terminal fragments of PARP-1 overexpressed in E. coli demonstrated that TyrRS interacts with the C-terminal region of PARP-1. In Figure 8G, the full-length TyrRS (1-528) but none of TyrRS (mini-TyrRS (1-364), ΔΝ-TyrRS (228-528) or the C-domain (328-528)) interact with PARP-1. In Figure 8H, a Coomassie blue staining of a gel shows the total protein input in for the foregoing experiments.

Figures 9A-9E show that a Tyrosyl-AMP analogue (Tyr-SA) does not affect DNA-dependent auto-PARylation of PARP-1. Figure 9A is a silver stained SDS-PAGE gel showing the purity and input of PARP-1 and TyrRS in the in vitro PARylation experiments. Figure 9B shows a quantitation (Image J software) of the band intensity of PARylated PARP-1 from Figure 2A (top). Figure 9C shows that the Tyrosyl-AMP analogue (Tyr-SA) does not affect DNA-dependent auto-PARylation of PARP-1. In Figure 9D, overexpression of a nuclear translocation-weakened mutant of TyrRS (Fu et al., JBC. 287:9330- 34, 2012)) is less effective in activating PARP-1. In Figure 9E, Y314A-TyrRS is more sensitive to RSV than wild-type TyrRS in facilitating PARP-1 activation.

Figures 10A-10F show that resveratrol enhances the acetylation of Tip60 and modulates [NAD+] in a dose and time dependent manner. In Figure 10A, treatment of HeLa cells (1 h) with increasing concentration of resveratrol enhances the acetylation level of Tip60. Activation of Tip60 was monitored by histone acetylation status. In Figure 10B, total NAD+ content of serum starved cells or RSV treated samples were compared with untreated samples at 15 minutes using a commercially available BioVision NAD+/NADH quantitation colorimetric kit. In Figure IOC, total nicotinamide or ADP-ribose produced was deduced from the difference in the amount of NAD+ in each sample with respect to the untreated sample (consumption of one mole of NAD+ would give rise one mole of nicotinamide and one mole of ADP-ribose). In Figure 10D, total NAD+ content of the serum starved cells or RSV treated samples was compared with untreated samples at 1 hour.

Although the experiments were done in biological triplicates (all samples showing similar results), the error bars in the figure represent the deviations from the mean of the technical triplicates from one representative biological sample. Figures 10E-10F show a time course study of poly-ADP- ribosylation status and associated signaling events after (10E) serum starvation (extended time course data of the same image shown in figure 3c) and (10F) treatment with 5 μΜ RSV. Using the respective antibodies, activation of p53 was monitored by the induction of p21 and SIRT6. Activation of NRF2 was monitored by HO-1 induction.

Figure 11 shows that siRNA (siRNA^TyrRS or siRNA^{PARP 1}), with and without low RSV (5 μΜ), does not affect cell viability. HeLa cells (lx 10⁵) were reverse-transfected with siRNA targeted against TyrRS or PARP-1. An siRNA^Con (a scrambled sequence of siRNA^{PARP -1}) was used as a control. Viability was monitored using the RTCA iCELLigence System (ACEA Biosciences). Samples were treated with RSV (5 μΜ) at 60 hours and monitoring was continued for another 2 hours for siRNA^TvrRS (total 62 hours of monitoring) and for another 16 hours for siRNA^Con and siRNA^{PARP 1} (total 76 hours of monitoring).

Figure 12 shows that siRNA^SIRT1 did not affect downstream signaling events at low RSV (5 μΜ). HeLa cells were treated with siRNA^SIRT1 for 60 hours to knockdown SIRT1. HeLa cells were treated with RSV (5 μΜ) for another 4 h and samples were collected intervals as indicated. Samples were analyzed for downstream signaling markers using appropriate antibodies.

Figure 13 shows that siRNA-(siRNA^TyrRS or siRNA^{PARP _1}) treated cells did not up-regulate the levels of NAD+ in response to RSV (5 μΜ) after 1 hour. HeLa cells (lx 10⁶) were reverse-transfected, separately, with siRNA targeted against PARP-1 or TyrRS. A scrambled sequence of target siRNA was used as a control. Total NAD+ content of RSV (5 μlVI)-treated samples was compared with untreated samples at 1 hour using a commercially available BioVision NAD+/NADH quantitation colorimetric kit. Although the experiments were done in biological triplicates (all samples showing similar results), the error bars in the figure represent the deviations from the mean of the technical triplicates from one representative biological sample. The comparator (shown as a dashed bar) was taken from Figure 10D.

Figures 14A-14H show that resveratrol treatment activates PARP-1 and associated signaling events in the mouse tissues. Figures 14A-14B show activation of PARP-1 in mouse muscle tissue treated with resveratrol as monitored by increased PARylation (14A) and increased acetylation status (14B). Activation of Tip60 and AMPK was monitored by using a-AcK16-H4 and -pSer36-H2B, respectively. Figures 14C-14D show activation of PARP-1 in mouse heart tissue treated with resveratrol as monitored by increased PARylation (14C) and increased acetylation status (14D). In Figure 14E, resveratrol treatment caused a transient activation of PARP-1. Immunoblotting of mouse muscle tissue samples after 24 hours of RSV treatment showed no significant difference in the level of PARP-1^PAR with respect to control. In Figure 14F, RSV treatment enhanced TyrRS interaction with and activation of PARP-1 in muscle tissue. In Figures 14G-14H, resveratrol-mediated activation of PARP-1 as monitored by PARylation status (14G) and acetylation status (14H) is blocked by Tyr-SA in mouse heart tissues. Figure 15 provides the data collection and refinement statistics obtained from co-crystals of resveratrol-TyrRS and L-Tyrosine-TyrRS (see Example 1). The numerical parameters for the co- crystals are listed and compared side-by-side in separate columns

DETAILED DESCRIPTION

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

By "about" is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

The term "amino acid" includes naturally occurring amino acids, non-naturally occurring amino acids, and amino acid analogs and mimetics. Naturally occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine, for example. Non- naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p- fluorophenylalanine, ethionine and the like, which are known to a person skilled in the art. Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivitization of the amino acid. Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics Arginine (Arg or R) would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid or of the amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

In certain aspects, the use of non-natural amino acids can be utilized to modify (e.g., increase) a selected non-canonical activity of an TyrRS polypeptide, or to alter the in vivo or in vitro half-life of the protein. Non-natural amino acids can also be used to facilitate (selective) chemical modifications (e.g., pegylation) of a TyrRS polypeptide. For instance, certain non-natural amino acids allow selective attachment of polymers such as PEG to a given protein, and thereby improve their pharmacokinetic properties.

Specific examples of amino acid analogs and mimetics can be found described in, for example, Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Eds. Gross and

Meinhofer, Vol. 5, p. 341, Academic Press, Inc., New York, N.Y. (1983), the entire volume of which is incorporated herein by reference. Other examples include peralkylated amino acids, particularly permethylated amino acids. See, for example, Combinatorial Chemistry, Eds. Wilson and Czarnik, Ch. 11, p. 235, John Wiley & Sons Inc., New York, N.Y. (1997), the entire book of which is incorporated herein by reference. Yet other examples include amino acids whose amide portion (and, therefore, the amide backbone of the resulting peptide) has been replaced, for example, by a sugar ring, steroid, benzodiazepine or carbo cycle. See, for instance, Burger's Medicinal Chemistry and Drug Discovery, Ed. Manfred E. Wolff, Ch. 15, pp. 619-620, John Wiley & Sons Inc., New York, N.Y. (1995), the entire book of which is incorporated herein by reference. Methods for synthesizing peptides, polypeptides, peptidomimetics and proteins are well known in the art (see, for example, U.S. Pat.

No. 5,420,109; M. Bodanzsky, Principles of Peptide Synthesis (1st ed. & 2d rev. ed.), Springer-Verlag, New York, N.Y. (1984 & 1993), see Chapter 7; Stewart and Young, Solid Phase Peptide Synthesis, (2d ed.), Pierce Chemical Co., Rockford, III. (1984), each of which is incorporated herein by reference). Accordingly, the TyrRS polypeptides described herein can be composed of naturally occurring amino acids, and non-naturally occurring amino acids, amino acid analogs and mimetics, and combinations thereof.

By "coding sequence" is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene. By contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene.

Throughout this specification, unless the context requires otherwise, the words "comprise,"

"comprises," and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of," Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

The term "endotoxin free" or "substantially endotoxin free" relates generally to

compositions, solvents, and/or vessels that contain at most trace amounts (e.g., amounts having no clinically adverse physiological effects to a subject) of endotoxin, and preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain bacteria, typically gram- negative bacteria, although endotoxins may be found in gram-positive bacteria, such as Listeria monocytogenes. The most prevalent endotoxins are lipopolysaccharides (LPS) or lipo-oligo- saccharides (LOS) found in the outer membrane of various Gram-negative bacteria, and which represent a central pathogenic feature in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans may produce fever, a lowering of the blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects.

Therefore, in pharmaceutical production of TyrRS polypeptides, it is often desirable to remove most or all traces of endotoxin from drug products and/or drug containers, because even small amounts may cause adverse effects in humans. A depyrogenation oven may be used for this purpose, as temperatures in excess of 300°C are typically required to break down most endotoxins. For instance, based on primary packaging material such as syringes or vials, the combination of a glass temperature of 250°C and a holding time of 30 minutes is often sufficient to achieve a 3 log reduction in endotoxin levels. Other methods of removing endotoxins are contemplated, including, for example, chromatography and filtration methods, as described herein and known in the art. Also included are methods of producing TyrRS polypeptides in and isolating them from eukaryotic cells such as mammalian cells to reduce, if not eliminate, the risk of endotoxins being present in a composition. Included are methods of producing TyrRS polypeptides in and isolating them from serum free cells.

Endotoxins can be detected using routine techniques known in the art. For example, the Limulus Amoebocyte Lysate assay, which utilizes blood from the horseshoe crab, is a very sensitive assay for detecting presence of endotoxin, and reagents, kits and instrumentation for the detection of endotoxin based on this assay are commercially available, for example from the Lonza Group. In this test, very low levels of LPS can cause detectable coagulation of the limulus lysate due a powerful enzymatic cascade that amplifies this reaction. Endotoxins can also be quantitated by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin free, endotoxin levels may be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU /mg of protein. Typically, 1 ng lipopolysaccharide (LPS) corresponds to about 1-10 EU.

In certain embodiments, the "purity" of a given agent (e.g., TyrRS polypeptide, resveratrol compound) in a composition may be specifically defined. For instance, certain compositions may comprise an agent that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between, as measured, for example and by no means limiting, by high performance liquid chromatography (HPLC), a well-known form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

As used herein, the terms "function" and "functional" and the like refer to a biological, enzymatic, or therapeutic function.

By "gene" is meant a unit of inheritance that may occupy a specific locus on a chromosome and consists of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e., introns, 5' and 3' untranslated sequences).

"Homology" refers to the percentage number of amino acids that are identical or constitute conservative substitutions. Homology may be determined using sequence comparison programs such as GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395), which is incorporated herein by reference. In this way sequences of a similar or substantially different length to those cited herein could be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated polynucleotide," as used herein, includes a polynucleotide that has been purified from the sequences that flank it in its naturally- occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment. Alternatively, an "isolated peptide" or an "isolated polypeptide" and the like, as used herein, includes the in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell; i.e., it is not significantly associated with in vivo substances.

The term "modulating" includes "increasing" or "enhancing" or "stimulating," as well as "decreasing" or "reducing," typically in a statistically significant or a physiologically significant amount as relative to a control. Accordingly a "modulator" may be an agonist, an antagonist, or any mixture thereof depending upon the conditions used. An "increased" or "enhanced" amount is typically a "statistically significant" amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the amount produced by no composition (the absence of an agent or compound) or a control composition. A "decreased" or "reduced" amount is typically a "statistically significant" amount, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in the amount produced by no composition (the absence of an agent or compound) or a control composition, including all integers in between. Other examples of "statistically significant" amounts are described herein.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic and naturally occurring analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers and naturally occurring chemical derivatives thereof. Such derivatives include, for example, post-translational modifications and degradation products including pyroglutamyl, iso-aspartyl, proteolytic, phosphorylated, glycosylated, oxidatized, isomerized, and deaminated variants of a TyrRS polypeptide.

The term "solubility" refers to the property of an agent provided herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as a concentration, either by mass of solute per unit volume of solvent (g of solute per kg of solvent, g per dL (100 mL), mg/ml, etc.), molarity, molality, mole fraction or other similar descriptions of concentration. The maximum equilibrium amount of solute that can dissolve per amount of solvent is the solubility of that solute in that solvent under the specified conditions, including temperature, pressure, pH, and the nature of the solvent. In certain embodiments, solubility is measured at physiological pH. In certain embodiments, solubility is measured in water or a physiological buffer such as PBS. In certain embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature can be about room temperature (e.g., about 20, 21, 22, 23, 24, 25°C) or about body temperature (37°C). In certain embodiments, an agent such as a TyrRS polypeptide has a solubility of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 mg/ml at room temperature or at 37°C.

By "statistically significant," it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.

A "subject," as used herein, includes any animal that exhibits a disease, condition, or symptom, or is at risk for exhibiting a disease, condition, or symptom, which can be treated with a method or composition described herein. Suitable subjects (patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Mammals, including non-human primates and, preferably, human patients, are included.

"Substantially" or "essentially" means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99% or greater of some given quantity.

"Treatment" or "therapy" or "treating," as used herein, includes any desirable effect on the symptoms or pathology of a disease or condition that can be affected by a method or composition described herein, and may include even minimal changes or improvements in one or more measurable markers of the disease or condition being treated. "Treatment" or "therapy" or "treating" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. The subject receiving this treatment is any subject in need thereof. Also included are "prophylactic" treatments or therapies, including those intended to reduce the risk of acquiring or worsening a disease, condition, or symptom. Exemplary markers of clinical improvement will be apparent to persons skilled in the art.

The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2000); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.);

Oligonucleotide Synthesis (N. Gait, ed., 1984); Oligonucleotide Synthesis: Methods and Applications (P. Herdewijn, ed., 2004); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Nucleic Acid Hybridization: Modern Applications (Buzdin and Lukyanov, eds., 2009); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Freshney, R.I. (2005) Culture of Animal Cells, a Manual of Basic Technique, 5th Ed. Hoboken NJ, John Wiley & Sons; B. Perbal, A Practical Guide to Molecular Cloning (3rd Edition 2010); Farrell, R., RNA Methodologies: A Laboratory Guide for Isolation and Characterization (3rd Edition 2005), Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) ( 1988, Cold Spring Harbor Laboratory Press, ISBN 0-87969-3, 4-2), 1855, Handbook of Drug Screening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, N.Y., Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Edited Jane Roskams and Linda Rodgers, (2002, Cold Spring Harbor Laboratory, ISBN 0-87969-630-3).

All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety to the extent they are consistent with the specification.

Tyrosyl-tRNA Synthetase Polypeptides and Polynucleotides

The methods and compositions described herein utilize or comprise one or more tyrosyl- tRNA synthetase (TyrRS or YRS) polypeptides, polynucleotides that encode the TyrRS polypeptide(s), and/or cells that comprise and express the foregoing. TyrRS belong to the class I tRNA synthetase family, which has two highly conserved sequence motifs at the active site, HIGH (SEQ ID NO: 8) and KMSKS (SEQ I D NO: 9). Class I tRNA synthetases aminoacylate at the 2'-OH of an adenosine nucleotide, and are usually monomeric or dimeric (one or two su bu nits, respectively).

Human TyrRS is a homodimer of a 528 amino acid polypeptide that is composed of three domains: 1) an amino-terminal Rossmann fold domain that is responsible for formation of the activated E-Tyr-AMP intermediate and is conserved among bacteria, archeae, and eukaryotes; 2) a tRNA anticodon recognition domain that has not been conserved between bacteria and eukaryotes; and 3) an appended eukaryote-specific C-terminal EMAP-II domain. It is believed that the carboxyl- terminal domain of human tyrosyl-tRNA synthetase evolved from gene duplication of the carboxyl- terminal domain of methionyl-tRNA synthetase and may direct tRNA to the active site of the enzyme.

Exemplary TyrRS polypeptide sequences are provided in Table 1 below.

EKWGGNKTYTAYVDLEKDFAAEVVHPGDLKNSVEVALNKLLDPIREKFNTPALKKL ASAAXPDPSKQKPMAKGPAKNSEPEEVI PSRLDIRVGKI ITVEKHPDADSLYVEKI DVGEAEPRTVVSGLVQFVPKEELQDRLVVVLCNLKPQKMRGVESQGMLLCASIEGI NRQVEPLDPPAGSAPGEHVFVKGYEKGQPDEELKPKKKVFEKLQADFKISEECIAQ WKQTNFMTKLGSISCKSLKGG IS

Where X is selected from alanine, glycine,

phenylalanine, valine, leucine, isoleucine, methionine, and proline

Human MGDAPSPEE LHLITRNLQEVLGEEKLKEILKERELKIYWGTATTG PHVAYFVPM Y341A SKIADFLKAGCEV ILFADLHAYLDNMKAPWELLELRVSYYEN IKAMLESIGVPL

EKLKFIKGTDYQLSKEYTLDVYRLSSVVTQHDSKKAGAEVVKQVEHPLLSGLLYPG LQALDEEYLKVDAQFGGIDQRKI F FAEKYLPALGYSKRVHLMNPMVPGLTGSKMS SSEEESKI DLLDRKEDVKKKLKKAFCEPGNVENNGVLSFIKHVLFPLKSEFVILRD EKWGG KTYTAYVDLEKDFAAEVVHPGDLKNSVEVALNKLLDPIREKFN PALKKL ASAAAPDPSKQKPMAKGPAKNSEPEEVI PSRLDIRVGKI ITVEKHPDADSLYVEKI DVGEAEPRTVVSGLVQFVPKEELQDRLVVVLCNLKPQKMRGVESQGMLLCASIEGI NRQVEPLDPPAGSAPGEHVFVKGYEKGQPDEELKPKKKVFEKLQADFKISEECIAQ WKQTNFMTKLGSISCKSLKGG IS

Bos taurus MGDSLSPEEK LSLITRNLQE VLGEEKLKEI LKERELKVYW GTATTGKPHV

(Bovine) AYFVPMSKIA DFLKAGCEVT ILFADLHAYL DNMKAPWDVL ELRTSYYENV

TyrRS IKAMLESIGV PLEKLRFIKG TDYQLSKEYT LDVYRLSSVV TQHDAKKAGA

EVVKQVEHPL LSGLLYPGLQ ALDEEYLKVD AQFGGVDQRK I FTFAEKYLP ALGYSKRIHL NPMVPGLTG SKMSSSEEES KIDLLDRKED VKKKLKKAFC EPGNVENNGV LAFIRHVLFP LKSEFVILRD EKWGGNKTYT AYLDLEKDFA DEVVHPGDLK NSVEVALNKL LDPIREKFNT PALKKLSSAA YPDPSKQKPA VKGPAKNSEP EEVIPSRLDI RVGKVISVDK HPDADSLYVE KIDVGEAEPR TVVSGLVQFV PKEELQDRLV VVLCNLKPQK RGVKSQGML LCASVEGVNR KVEPLDPPAG SAPGERVFVK GYEKGQPDEE LKPKKKVFEK LQADFKISDE YIAQWKQTNF MTKMGSVSCK SLKGGNIS

Rattus MGDAPSPEEK LHLITRNLQE VLGEEKLKEI LKERELKVYW GTATTGKPHV norvegicus AYFVPMSKIA DFLKAGCEVT ILFADLHAYL DNMKAPWELL ELRTSYYENV (Rat) IKAMLESIGV PLEKLKFTKG TDYQLSKEYT LDVYRLSSLV TQHDAKKAGA TyrRS EVVKQVEHPL LSGLLYPGLQ ALDEEYLKVD AQFGGIDQRK I FTFAEKYLP

TLGYSKRVHL MNPMVPGLTG SKMSSSEEES KIDLLDRKED VKKKLKKAFC EPGNVENNGV LSFVKHVLFP LKSEFVILRD EKWGGNKTYT IYQELEKDFA AEVVHPGDLK NSVEVALNKL LDPIREKFNT PALKKLASAA YPDPSKQKPT AKGPAKSSEP EEIIPSRLDI RVGKILSVEK HPDADSLYVE KIDVGEAEPR TVVSGLVQFV PKEELQDRLV VVLCNLKPQK MRGVDSQGML LCASVEGVSR QVEPLDPPAG SAPGERVFVQ GYEKGQPDEE LKPKKKVFEK LQADFKISDD CVAQWKQTNF MTKLGFVSCK SLKGGNIS

Mus MGDAPSPEEK LHLITRNLQE VLGEEKLKEI LKERELKVYW GTATTGKPHV musculus AYFVPMSKIA DFLKAGCEVT ILFADLHAYL DNMKAPWELL ELRTSYYENV (Mouse ) IKAMLESIGV PLEKLKFIKG TDYQLSKEYT LDVYRLSSVV TQHDAKKAGA TyrRS EVVKQVEHPL LSGLLYPGLQ ALDEEYLKVD AQFGGVDQRK I FTFAEKYLP

ALGYSKRVHL MNPMVPGLTG SKMSSSEEES KIDLLDRKED VKKKLKKAFC EPGNVENNGV LSFIKHVLFP LKSEFVILRD EKWGGNKTYT VYLELEKDFA AEVVHPGDLK NSVEVALNKL LDPIREKFNT PALKKLASAA YPDPSKQKPP AKGPAKNSEP EEVIPSRLDI RVGKILSVEK HPDADSLYVE KIDVGEAEPR TVVSGLVQFV PKEELQDRLV VVLCNLKPQK MRGVDSQGML LCASVEGVSR QVEPLDPPAG SAPGERVFVQ GYEKGQPDEE LKPKKKVFEK LQADFKISEE CIAQWKQTNF MTKLGFVSCK SLKGGNIS

Gallus METASGPQEK YQLITRNLQE VLGEDKLMAI LKEREVKIYW GTATTGKPHV gallus AYFVPMSKIA DFLKAGCEVT ILFADLHAYL DNMKAPWELL ELRTRYYEHV ( Chicken ) IKAMLESIGV PLEKLKFIRG TDYQLSKEYT LDVYRLSSVV TQHDAKKAGA TyrRS EVVKQVEHPL LSGLLYPGLQ ALDEEYLKVD AQFGGVDQRK I FTFAEKYLP

SLGYAKRIHL MNPMVPGLTG SKMSSSEEDS KIDLLDRKED VKKKLKKAFC EPGNIENNGV LSFIKHVLFP LKSEFVILRE EKWGGNKTYT AYETLEKDFA EQVVHPGDLK NSVEAALNKL LDPIREKFNS PELKKLTNAA YPNPSKAKPA EKGTKNSEPE TIVPSRLDIR VGKVVSVEKH PDADSLYVEK I DVGEPEPRT VVSGLVQFVP KEQLQDRLVV LLCNLKPQKM RGVESQGMVL CASSVGEPRQ VEPLDPPAEC CAGERVYVEG YEDGEPDDEL KPKKKVFEKL QADFRISEDC

IAQWKERNFL TKLGSISCKS L GGSIS

Moreover human TyrRS polypeptides include several hundred highly related polymorphic forms, and these are known in the art to be at least partially functionally interchangeable. It would thus be a routine matter to select a naturally occurring variant of TyrRS, including, for example, the single nucleotide polymorphic forms listed in Table 2 below, to create an AARS polypeptide containing one or more amino acid changes based on the sequence of any of the homologues, orthologs, and naturally-occurring isoforms of human TyrRS, as well as other species of TyrRS, including the various mammalian forms of TyrRS.

Table 2. SNPs of Human TyrRS

Table 2. SNPs of Human TyrRS

Table 2. SNPs of Human TyrRS

Gene Bank Nucleotide Gene Bank Nucleotide Change Accession Change Accession

Number Number

rs77572572 A/G rsl0914614 A/G

rs77564909 C/T rsl0914613 C/T

rs77508723 A/G rsl0914611 G/T

rs77395170 C/G rsl0798918 C/T

rs77372558 A/G rsl0753265 A/G

rs77240034 A/G rs9787263 A/C

rs77174196 A/T rs7548570 A/G

rs77116473 A/C rs7539432 C/G

rs77033253 -/AT rs7411174 A/G

rs76991974 A/C rs6686315 A/G

rs76862302 C/T rs35381223 C/G

rs76657858 A/C rs35287970 -/G

rs76611863 G/T rs35270871 -/T

rs76610248 C/G rs35204694 -/c

rs76497653 A/C rs34834736 -/T

rs76460696 A/C rs34800768 -/T

rs76435574 C/T rs34768899 -/T

rs76428484 A/C rs34704129 -/c

rs76313514 C/T rs34700143 A/G

rs76239948 C/T rs34619886 C/T

rs76213478 G/T rs34553868 G/T

rs76172167 C/T rs34533197 i C/T

rs76154286 A/G rs34506343 -/A

rs76066944 A/C rs34460415 C/T

rs75863818 A/T rs34458777 -/c

rs75658271 A/G rs34412390 -/T

rs75629042 A/C rs34366587 C/T

rs75556988 A/G rs34305494 -/T

rs75506593 G/T rs34297917 -/T

rs75499764 A/C rs34270752 -/T

rs75457356 C/T rs34240771 A/T

rs75420675 A/G rs34213904 -/T

rs75382318 A/T rs34204340 -/T

rs74852498 -/CAA rs34189200 -/T

rs74403587 A/C rs34134602 -/G

rs74319398 C/T rs34054038 -/c

rs74067514 C/T rs34046956 -/A

rs73618880 A/G rs28390180 A/G

rs72880571 C/T rsl7510718 C/T

rs72880567 A/G rsl6866009 C/T Table 2. SNPs of Human TyrRS

Gene Bank Nucleotide Gene Bank Nucleotide Change Accession Change Accession

Number Number

rs72880562 C/T rsl6835176 A/G

rs72880555 G/T rsl2760548 G/T

rs72880553 A/G rsl2759668 A/G

rs72880540 A/G rsl2729898 G/T

rs72874656 A/G rsl2724469 G/T

rs72654022 A/G rsl2564655 C/T

rs72654020 A/C rsl2406682 C/T

rs72654016 A/G rsl2136633 C/G

rs72510634 ~/CT rsl2133278 A/G

rs72418507 -/AA rs59754854 C/T

rs72391325 -/T rs59697437 A/T

rs72315501 -/AATA rs59686477 C/T

rs72147372 -/T rs59542013 -/A

rs72089012 -/AA rs59347601 C/T

rs72050208 -/T rs59076866 A/C

rs71910487 -/T rs58994767 -/T

rs71772495 -/T rs58957786 C/T

rs71665854 -/A rs58938776 C/T

rs71646400 C/T rs58905217 G/T

rs71646399 A/G rs58828752 C/T

rs71646397 C/G rs58621825 -/T

rs71516333 C/T rs58440351 A/T

rs71516332 C/G rs57928583 C/T

rs71516331 C/G rs57734982 A/T

rs71006371 -/A rs57578136 -/T

rs71006370 -/T rs57215825 C/G

rs68183151 A/C rs56697788 -/AA

rs68073354 -/T rs56064318 C/T

rs67839900 -/A rs56023272 A/G

rs67504155 -/A rs41265865 A/G

rs67210611 -/T rs41265863 G/T

rs67020459 -/TC rs41265861 A/G

rs66590952 -/CT rs41265859 A/G

rs61800867 A/G rs41265857 C/T

rs61800866 C/T rs36092637 A/T

rs61798853 C/G rs36061373 ~/C

rs61798852 A/G rs36056058 C/T

rs61798851 C/T

rs61798849 C/T

rs61798846 A/G Table 2. SNPs of Human TyrRS

Gene Bank Nucleotide Gene Bank Nucleotide Change

Accession Change Accession

Number Number

rs 617 9884 5 A/G

rs 61737 106 A/T

rs 61057 925 A/G

rs 60958 98 9 C/T

rs 60802 151 C/T

rs 607 04 395 ~ /T

rs 602 69206 A/G

rs 602 4 6059 C /G

Thus, TyrRS polypeptides may comprise, consist, or consist essentially of any of the sequences in Table 1, including variants and fragments thereof, for example, as illustrated by the SNP variants in Table 2. In certain embodiments, the TyrRS polypeptide is about, at least about, or no more than about, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 525, 528, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, or 750 amino acids in length (including all integers and ranges in between).

A TyrRS polypeptide can comprise all or a fragment of a reference sequence described herein, alone or in combination with other (e.g., heterologous) sequences. For instance, in certain embodiments, a TyrRS polypeptide comprises, consists, or consists essentially of 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 525, 528 contiguous or non-contiguous amino acids of a reference sequence (see, e.g., Table 1), including all integers and ranges in between.

Certain embodiments include "variants" of the TyrRS polypeptides described herein. Such variants can have one or more amino acid insertions, substitutions, additions, and/or deletions relative to a reference TyrRS polypeptide, and optionally retain one non-canonical activity, for example, the ability to interact with PARP-1. In some embodiments, the TyrRS polypeptide or variant binds to the resveratrol compound and binds to PARP-1. In particular embodiments, the TyrRS polypeptide or variant is able to stimulate PARylation (e.g., auto-PARylation) of PARP-1.

In certain embodiments, a variant TyrRS polypeptide comprises, consists, or consists essentially of an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or similarity to a TyrRS reference polypeptide sequence, as described herein, (see, e.g., Table 1). Also included are TyrRS polypeptides that differ from a reference polypeptide sequence by the addition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100 or more amino acids but which retain at least one property (e.g., biological activity such as interacting with/binding to PARP-1, binding to a resveratrol compound) of the reference polypeptide. In certain embodiments, the amino acid additions or deletions occur at the C-terminal end of the TyrRS polypeptide, the N-terminal end of the TyrRS polypeptide, or both.

In certain embodiments, a variant TyrRS polypeptide differs from a reference sequence by at least 1% but less than 20%, 15%, 10% or 5% of the residues. (If this comparison requires alignment, the sequences should be aligned for maximum similarity. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) Included are differences or changes at a non- essential residue or a conservative substitution. In certain embodiments, the molecular weight of a variant TyrRS polypeptide differs from that of the reference polypeptide by about, at least about, or no more than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, or 30%.

In some embodiments, a TyrRS polypeptide has a substitution of one more of residue at positions 46, 340, and/or 341 of SEQ ID NO:l (see, e.g., U.S. Patent No. 8,481,296, incorporated by reference). In particular embodiments, the substitution is a non-conservative substitution. In some embodiments, the one or more residues at positions 46, 340, and/or 341 of SEQ ID NO: l are substituted with an amino acid having an aliphatic side chain, for example, a non-polar aliphatic side chain. In specific embodiments, the amino acid having an aliphatic side chain is selected from one or more of alanine, glycine, phenylalanine, valine, leucine, isoleucine, methionine, and proline.

The terms "sequence identity" or, for example, comprising a "sequence 50% identical to," as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, H is, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the resu lt by 100 to yield the percentage of sequence identity.

Terms used to describe sequence relationships between two or more polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity" and "substantial identity." A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polypeptides may each comprise (1) a sequence (i.e., only a portion of the complete polypeptides sequence) that is similar between the two polypeptides, and (2) a sequence that is divergent between the two polypeptides, sequence comparisons between two (or more) polypeptides are typically performed by comparing sequences of the two polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wl, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel ef al., "Current Protocols in Molecular Biology," John Wiley & Sons Inc, 1994-1998, Chapter 15.

Calculations of sequence similarity or sequence identity between sequences (the terms are used interchangeably herein) can be performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In certain embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In some embodiments, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ( 1970, J. Mol. Biol. 48: 444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossu m 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A preferred set of parameters (and the one that should be used unless otherwise specified) includes a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (Cabios. 4: 11-17, 1989) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The sequences described herein can be used as a "query sequence" to perform a search against pu blic databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al., (1990, J. Mol. Biol. 215: 403-10). BLAST nucleotide searches can be performed with the N BLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to

polynucleotide sequences described herein. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to polypeptides described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

As noted above, TyrRS polypeptide may be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art, For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (PNAS USA. 82: 488-492, 1985), Kunkel et al. (Methods in Enzymol, 154: 367-382, 1987), U.S. Pat. No. 4,873,192, Watson, J. D. et al. ("Molecular Biology of the Gene", Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and the references cited therein. General guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).

Biologically active TyrRS polypeptides, including variants and fragments, may contain conservative amino acid substitutions at various locations along their sequence, as compared to a reference amino acid residue, and such additional substitutions may further enhance the activity or stability of the polypeptides. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub- classified as follows:

Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.

Basic: The residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof {e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having a basic side chain include arginine, lysine and histidine.

Charged: The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine).

Hydrophobic: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.

Neutral/polar: The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.

This description also characterizes certain amino acids as "small" since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity. With the exception of proline, "small" amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not. Amino acids having a small side chain include glycine, serine, alanine and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains. The structure of proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the a-amino group, as well as the -carbon. Several amino acid similarity matrices are known in the art (see e.g., PAM 120 matrix and PAM250 matrix as disclosed for example by Dayhoff et al., 1978, A model of evolutionary change in proteins). Matrices for determining distance relationships In M . 0. Dayhoff, (ed.), Atlas of protein sequence and structure, Vol. 5, pp. 345-358, National Biomedical Research Foundation, Washington DC; and by Gonnet et al., [Science, 256: 14430-1445, 1992), however, include proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a "small" amino acid.

The degree of attraction or repulsion required for classification as polar or nonpolar is arbitrary and, therefore, amino acids specifically contemplated by the invention have been classified as one or the other. Most amino acids not specifically named can be classified on the basis of known behavior.

Amino acid residues can be further su b-classified as cyclic or non-cyclic, and aromatic or non-aromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always non-aromatic. Dependent on their structu ral properties, amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, su b-classification according to this scheme is presented in Table A.

Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional truncated and/or variant TyrRS polypeptide can readily be determined by assaying its non-canonical activity, as described herein. Conservative substitutions are shown in Table B under the heading of exemplary substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, (c) the bulk of the side chain, or (d) the biological function. After the substitutions are introduced, the variants are screened for biological activity.

Alternatively, similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains. The first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochemistry, third edition, Wm. C. Brown Publishers ( 1993).

In conjunction with the primary amino acid sequences, the detailed physical descriptions of the protein (e.g., the crystal structure) provide precise insights into the roles played by specific amino acids within the protein. Persons skilled in the art can thus use this information to identify structurally-conserved domains, linking regions, secondary structures such as alpha-helices, surface or solvent-exposed amino acids, non-exposed or internal regions, catalytic sites, and ligand- interacting surfaces, among other structural features. Such persons can then use that and other information to readily engineer Tyr S variants that retain or improve the activity of interest, for instance, by conserving or altering the characteristics of the amino acid residues within or adjacent to these and other structural features, such as by conserving or altering the polarity, hydropathy index, charge, size, and/or positioning {i.e., inward, outward) of selected amino acid side chain(s) relative to wild-type residues (see, e.g., Zaiwara et al., Mol Biotechnol. 51:67-102, 2012; Perona and Hadd, Biochemistry. 51:8705-29, 2012; Morin et al., Trends Biotechoi. 29:159-66, 2011; Collins et al., Annu. Rev. Biophys. 40:81-98, 2011; and U.S. Application No. 61/674,639).

Thus, in some embodiments, a predicted non-essential amino acid residue in a TyrRS polypeptide can be replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of TyrRS sequence, such as by satu ration mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide to identify mutants which retain that activity. Following mutagenesis of the coding sequences, the encoded peptide can be expressed recombinantly and the activity of the peptide can be determined. A "non-essential" amino acid residue is a residue that can be altered from the reference sequence of an embodiment polypeptide without abolishing or substantially altering one or more of its non-canonical activities. Suitably, the alteration does not substantially abolish one of these activities, for example, the activity is at least 20%, 40%, 60%, 70% or 80% 100%, 500%, 1000% or more of the reference sequence. An "essential" amino acid residue is a residue that, when altered from the reference sequence of a TyrRS polypeptide, results in abolition of an activity of the parent molecule such that less than 20% of the reference activity is present.

In general, polypeptides and fusion polypeptides (as well as their encoding polynucleotides) are isolated. An "isolated" polypeptide or polynucleotide is one that is removed from its original environment. For example, a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are at least about 90% pu re, at least about 95% pure, at least about 98% pure, or at least about 99% pure. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.

The TyrRS polypeptides provided herein can be modified in a variety of ways. For instance, in certain embodiments, a TyrRS is fused to a heterologous polypeptide. Examples of heterologous polypeptides include purification tags, epitope tags, targeting sequences, signal peptides, membrane translocating sequences, and pharmacokinetic property modifiers ("PK modifiers") such as immunoglobulin Fc domains, human albumin (Osborn et al., Eur. J. Pharmacol. 456:149-158, 2002), poly Glu or poly Asp sequences, and transferrin. Additionally, fusion to conformationally disordered polypeptide sequences composed of the amino acids Pro, Ala, and Ser (PASylation) or hydroxyethyl starch (sold under the trademark HESYLATION^®) provides one way to increase the hydrodynamic volume of the TyrRS polypeptide. This additional extension adopts a bulky random structure, which significantly increases the size of the resulting fusion protein. By this means the typically rapid clearance of smaller TyrRS polypeptides via kidney filtration is retarded by several orders of magnitude. Additionally use of Ig G fusion proteins has also been shown to enable some fusion protein proteins to penetrate the blood brain barrier (Fu et al., Brain Res. 1352:208-13, 2010).

Examples of fusion proteins that improve penetration across cellular membranes include fusions to membrane translocating sequences. In this context, the term "membrane translocating sequences" refers to naturally occurring and synthetic amino acid sequences that are capable of membrane translocation across a cellular membrane. Representative membrane translocating sequences include those based on the naturally occurring membrane translocating sequences derived from the Tat protein, and homeotic transcription protein Antennapedia, as well as synthetic membrane translocating sequences based in whole or part on poly Arginine and Lysine resides. Representative membrane translocating sequences include for example those disclosed in the following patents, US5,652,122; US 5,670,617; US5,674,980; US5,747,641; US5,804,604;

US6,316,003; US7,585,834; US7,312,244; US7,279,502; US7,229,961; US7,169,814; US7,453,011; US7,235,695; US6,982,351; US6,605,115; US7,306,784; US7,306,783; US6,589,503; US6,348,185; US6,881,825; US7,431,915; WO0074701A2; WO2007111993A2; WO2007106554A2;

WO02069930A1; WO03049772A2; WO03106491A2; and WO2008063113A1.

It will be appreciated that a flexible molecular linker (or spacer) optionally may be interposed between, and covalently join, the TyrRS polypeptide and any of the fusion proteins disclosed herein.

Additionally in some embodiments, the TyrRS polypeptide can include synthetic, or naturally occurring secretion signal sequences, derived from other well characterized secreted proteins. In some embodiments such proteins, may be processed by proteolytic cleavage to form the TyrRS polypeptide in situ. Such fusions proteins include for example fusions of TyrRS polypeptide to ubiquitin to provide a new N-terminal amino acid, or the use of a secretion signal to mediate high level secretion of the TyrRS polypeptide into the extracellular medium, or N, or C-terminal epitope tags to improve purification or detection.

Also included as modifications are chemical and/or enzymatic derivatizations at one or more constituent amino acid(s) of a TyrRS polypeptide, including side chain modifications, backbone modifications, and N- and C-terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid moieties, cofactors, and the like.

Exemplary modifications also include pegylation of a TyrRS polypeptide (see, e.g., Veronese and Harris, Advanced Drug Delivery Reviews 54: 453-456, 2002; and Pasut et al., Expert Opinion. Ther. Patents 14:859-894, 2004, both herein incorporated by reference). PEG is a well-known polymer having the properties of solubility in water and in many organic solvents, lack of toxicity, and lack of immunogenicity. It is also clear, colorless, odorless, and chemically stable. For these reasons and others, PEG has been selected as the preferred polymer for attachment, but it has been employed solely for purposes of illustration and not limitation. Similar products may be obtained with other water-soluble polymers, including without limitation; polyvinyl alcohol, other poly(alkylene oxides) such as poly(propylene glycol) and the like, poly(oxyethylated polyols) such as poly(oxyethylated glycerol) and the like, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl purrolidone, poly-1,3- dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride, and polyaminoacids. One skilled in the art will be able to select the desired polymer based on the desired dosage, circulation time, resistance to proteolysis, and other considerations.

A wide variety of PEG derivatives are both available and suitable for use in the preparation of PEG-conjugates. For example, NOF Corp.'s PEG reagents sold under the trademark SUNBRIGHT^® Series provides numerous PEG derivatives, including methoxypolyethylene glycols and activated PEG derivatives such as methoxy-PEG amines, maleimides, N-hydroxysuccinimide esters, and carboxylic acids, for coupling by various methods to the N-terminal, C-terminal or any internal amino acid of the TyrRS polypeptide. Nektar Therapeutics' Advanced PEGylation technology also offers diverse PEG-coupling technologies to potentially improve the safety and efficacy of a TyrRS polypeptide based therapeutic. See also US Pat. Nos. 6,436,386; 5,932,462; 5,900,461; 5,824,784; and 4,904,584, which are incorporated by reference.

In certain aspects, chemoselective ligation technology may be utilized to modify a TyrRS polypeptides, such as by attaching polymers in a site-specific and controlled manner. Such technology typically relies on the incorporation of chemoselective anchors into the protein backbone by either chemical or recombinant means, and subsequent modification with a polymer carrying a complementary linker. As a result, the assembly process and the covalent structure of the resulting protein-polymer conjugate may be controlled, enabling the rational optimization of drug properties, such as efficacy and pharmacokinetic properties (see, e.g., Kochendoerfer, Current Opinion in Chemical Biology 9:555-560, 2005).

The TyrRS polypeptides described herein may be prepared by any suitable procedure known to those of skill in the art, such as by recombinant techniques. Thus, in some embodiments, the TyrRS polypeptide is a recombinant polypeptide. In addition to recombinant production methods, polypeptides of the invention may be produced by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Alternatively, various fragments may be chemically synthesized separately and combined using chemical methods to produce the desired molecule. In certain embodiments, the TyrRS polypeptide is a synthetic polypeptide.

Also included are TyrRS polynucleotides, or polynucleotides that encode a TyrRS polypeptide described herein. The term "polynucleotide" or "nucleic acid" as used herein designates mRNA RNA, cRNA, cDNA or DNA. The term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

The terms "DNA" and "polynucleotide" and "nucleic acid" refer to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, an isolated DNA segment encoding a polypeptide refers to a DNA segment that contains one or more coding sequences yet is substantially isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the terms "DNA segment" and "polynucleotide" are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.

Additional coding or non-coding sequences may, but need not, be present within a TyrRS polynucleotide, and a polynucleotide may, but need not, be linked to other molecules and/or support materials. Hence, the TyrRS polynucleotides, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably.

Polynucleotides and fusions thereof may be prepared, manipulated and/or expressed using any of a variety of well-established techniques known in the art. For example, polynucleotide sequences which encode TyrRS polypeptides, or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of a TyrRS polypeptide in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence. Such polynucleotides are commonly referred to as "codon-optimized." Any of the polynucleotides described herein may be utilized in a codon-optimized form. In certain embodiments, a polynucleotide can be codon optimized for use in specific bacteria such as E. coli or yeast such as S. cerevisiae (see, e.g., Burgess-Brown et al., Protein Expr Purif. 59:94-102, 2008; Ermolaeva, Curr. Iss. Mol. Biol. 3:91-7, 2001; Welch et al., PLoS ONE 4(9): e7007

doi:10.1371/journal.pone.0007002).

Moreover, the polynucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, expression and/or activity of the gene product.

In some embodiments, polynucleotides encoding polypeptides of the invention may be delivered to a subject in vivo, e.g., using gene therapy techniques. Gene therapy refers generally to the transfer of heterologous nucleic acids to the certain cells, target cells, of a mammal, particularly a human, with a disorder or conditions for which such therapy is sought. The nucleic acid is introduced into the selected target cells in a manner such that the heterologous DNA is expressed and a therapeutic product encoded thereby is produced.

Various viral vectors that can be utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, adeno-associated virus (AAV), or, preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus, or is a lentiviral vector. The preferred retroviral vector is a lentiviral vector. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting a zinc finger derived- DNA binding polypeptide sequence of interest into the viral vector, along with another gene that encodes the ligand for a receptor on a specific target cell, for example, the vector may be made target specific. Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a protein (dimer). Illustrative targeting may be accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the zinc finger- nucleotide binding protein polynucleotide.

Since recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsulation. Helper cell lines which have deletions of the packaging signal include but are not limited to PSI.2, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced. The vector virions produced by this method can then be used to infect a tissue cell line, such as NIH 3T3 cells, to produce large quantities of chimeric retroviral virions.

"Non-viral" delivery techniques for gene therapy can also be used including, for example, DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaP04 precipitation, gene gun techniques, electroporation, liposomes, lipofection, and the like. Any of these methods are widely available to one skilled in the art and would be suitable for use in the present invention. Other suitable methods are available to one skilled in the art, and it is to be understood that the present invention can be accomplished using any of the available methods of transfection. Lipofection can be accomplished by encapsulating an isolated DNA molecule within a liposomal particle and contacting the liposomal particle with the cell membrane of the target cell. Liposomes are self-assembling, colloidal particles in which a lipid bilayer, composed of amphiphilic molecules such as phosphatidyl serine or phosphatidyl choline, encapsulates a portion of the surrounding media such that the lipid bilayer surrounds a hydrophilic interior. Unilammellar or multilammellar liposomes can be constructed such that the interior contains a desired chemical, drug, or, as in the instant invention, an isolated DNA molecule. In some aspects, polynucleotides encoding TyrRS polypeptides may be used to express and delivery a TyrRS polypeptide via cell therapy. Some aspects thus include cell therapy for treating a disease or disorder, comprising administering a host cell expressing, or capable of expressing, a TyrRS polypeptide.

Cell therapy involves the administration of cells which have been selected, multiplied and pharmacologically treated or altered (i.e. genetically modified) outside of the body (Bordignon et al, Cell Therapy: Achievements and Perspectives, Haematologica, 84, pp.1110-1149, 1999). Such host cells include for example, primary cells, including macrophages, and stem cells which have been genetically modified to express a TyrRS polypeptide. Transplanted cells may thus function by releasing bioactive compounds such as TyrRS polypeptide(s).

Accordingly, the methods and compositions described herein can employ any one or more of the foregoing TyrRS polypeptides, polynucleotides, or host cells, including combinations and mixtures thereof. Resveratrol Compounds

The methods and compositions described herein utilize or comprise one or more resveratrol compounds. Examples of "resveratrol compounds" include resveratrol and its enantiomers, stereoisomers, diastereomers, and other stereoisomeric forms, racemates, tautomers, metabolites, analogs, and prodrugs thereof. Also included are pharmaceutically acceptable salts of the foregoing, including acid and base addition salts.

In some embodiments, the resveratrol compound has a tyrosine-like phenolic ring, for example, that fits into the active pocket of a TyrRS polypeptide, as illustrated in Figures 1C-1D and Figures 6A-6D. In some embodiments, the resveratrol compound potentiates the translocation of a TyrRS polypeptide into the nucleus of a cell. In particular embodiments, the resveratrol compound potentiates (e.g., synergistically potentiates) an activity of a TyrRS polypeptide, for instance, the activation of Poly [ADP-ribose] polymerase 1 (PARP-1), optionally including the increased PARylation (e.g., auto-PARylation) of PARP-1 and/or the expression of one or more downstream PARP-1 target genes, as described herein and known in the art,

As noted above, resveratrol (3,4',5-trihydroxystilbene) belongs to a class of polyphenolic compounds called stilbenes. It exists as two geometric isomers: cis- (Z) and trans- (E). Resveratrol is also referred to as 3,5,4'-trihydroxy-trans-stilbene; trans-3,5,4'-Trihydroxystilbene; 3,4',5- Stilbenetriol; trans-Resveratrol; (E)-5-(p-Hydroxystyryl)resorcinol; and (E)-5-(4- hydroxystyryl)benzene-l,3-diol. In certain embodiments, a resveratrol compound has the following formula of trans- resveratrol (l-A):

(l-A)

In certain embodiments, a resveratrol compound has the following formula of cis-resveratrol

(l-B):

(l-B)

"Resveratrol compounds" also include analogs of resveratrol. Exemplary resveratrol analogs are described, for example, in U.S. Patent No. 7,026,518; U.S. Application No. 2010/0185006; which are incorporated by reference.

Also included are hexahydroxystilbene, 3,4,5,4'-tetrahydroxystilbene, and other analogs described, for example, in Szekeres et al. (Pharm Res. 27:1042-48, 2010, incorporated by reference), Lu et al. (Carcinogenesis 22:321-328, 2001, incorporated by reference), and Calil et al (Letters in Drug Design & Discovery 9:8-11(4), 2012, incorporated by reference). Specific embodiments include 4- acetoxy-resveratrol (or 4'-acetoxy-resveratrol) as described, for example, in U.S. Application No. 2013/0123357, which is incorporated by reference. Some embodiments include Aza-resveratrol analogs as described, for example, in Siddiqui et al. (Bioorg Med Chem Lett. 23:635-40, 2013 incorporated by reference), or imine-resveratrol analogs as described, for example, in Li et al. (PLoS ONE 9(7): el01455, 2014, incorporated by reference). Additional examples of resveratrol compounds are shown in Figure 5.

"Pharmaceutically acceptable salt" includes both acid and base addition salts.

"Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-l,2-disulfonic acid, ethanesulfonic acid, 2- hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid,

glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-l,5-disulfonic acid, naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

"Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2 dimethylaminoethanol, 2 diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

Often crystallizations produce a solvate of the resveratrol compound. As used herein, the term "solvate" refers to an aggregate that comprises one or more molecules of a resveratrol compound with one or more molecules of solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the resveratrol compounds may exist as a hydrate, including a monohydrate, dihydrate, hemihydrate,

sesquihydrate, trihydrate, tetrahydrate and the like, as well as the corresponding solvated forms. In some instances the resveratrol compounds may be true solvates, while in other instances the compounds may merely retain adventitious water or be a mixture of water plus some adventitious solvent.

The resveratrol compounds, or their pharmaceutically acceptable salts may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomer^'^ forms that may be defined, in terms of absolute stereochemistry, as (R) or (S). The present invention is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and ( ), (R) and (S) isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centres of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms of resveratrol are also intended to be included.

"Prodrug" is meant to indicate a resveratrol compound that may be converted under physiological conditions or by solvolysis to a biologically active compound. Thus, the term "prodrug" refers to a metabolic precursor of resveratrol that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subject in need thereof, but is converted in vivo to an active compound. Prodrugs are typically rapidly transformed in vivo to yield the parent compound, for example, by hydrolysis in blood. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7 9, 21 24 (Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi, T., et al., A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

The term "prodrug" is also meant to include any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a mammalian subject. Prodrugs of resveratrol may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Prodrugs include resveratrol compounds wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug is administered to a mammalian subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol or amide derivatives of amine functional groups in resveratrol and the like. Also included are in vivo metabolic products of resveratrol compounds. Such products may result from, for example, the oxidation, reduction, hydrolysis, amidation, esterification, and the like of the administered compound, primarily due to enzymatic processes. Accordingly, certain embodiments include resveratrol compounds produced by a process comprising administering a compound of this invention to a mammal for a period of time sufficient to yield a metabolic product thereof. Such products are typically identified by administering a radiolabeled compound in a detectable dose to an animal, such as rat, mouse, guinea pig, monkey, or to human, allowing sufficient time for metabolism to occur, and isolating its conversion products from the urine, blood or other biological samples.

The "purity" of any given resveratrol compound or mixture of resveratrol compounds in a composition may be specifically defined. For instance, certain compositions may comprise a resveratrol compound that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between, as measured, for example, by high pressure liquid

chromatography (HPLC).

"Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. A "stereoisomer" refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof and includes "enantiomers", which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.

A "tautomer" refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present invention includes tautomers of any said compounds.

Accordingly, the methods and compositions described herein can employ any one or more of the foregoing resveratrol compounds, including combinations and mixtures thereof.

Methods of Use and Compositions

Certain embodiments include methods of using the TyrRS polypeptides and resveratrol compounds, alone or in combination, including compositions comprising the same, in methods of modulating a stress response in a cell. Such methods can be practiced in vitro or in vivo, for example, in a subject, to protect or enhance recovery from a broad array of stressful stimuli. Certain embodiments therefore include therapeutic methods, for example, methods of treating or managing a variety of diseases and conditions associated with aging; diseases and conditions associated with or triggered by oxidative stress, viral infections, radiation exposure, drugs, and/or light exposure; and chronic diseases such as cardiovascular diseases, among others.

Some embodiments include methods of increasing a stress response in a cell, comprising contacting the cell with (a) a tyrosyl-tRNA synthetase (TyrRS) polypeptide or a polynucleotide that encodes the TyrRS polypeptide, in combination with (b) at least one resveratrol compound. In some embodiments, the stress response includes evoking or inducing mild stress in the cell, for example, therapeutic mild stress that likewise induces or achieves protective effects in the cell.

In specific embodiments, the combination of (a) and (b) synergistically increases the stress response relative to (a) alone and (b) alone. In particular embodiments, the combination of (a) and (b) synergistically evokes mild stress in the cell relative to (a) alone and (b) alone, and thereby induces or achieves protective effects in the cell. In certain embodiments, the stress response comprises increased activation of Poly [ADP-ribose] polymerase 1 (PARP-1).

In particular aspects, the increased activation of PARP-1 comprises increased PARylation (e.g., auto-PARylation) of PARP-1. For example, in some instances PARylation of PARP-1 is increased by about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10-fold or more, relative to a control (for instance, where the control is (a) alone, (b) alone, or both). In some instances, the combination of (a) and (b) synergistically increases PARylation of PARP-1 relative to (a) alone and (b) alone.

In certain aspects, the increased activation of PARP-1 comprises increased expression of one or more PARP-1 target genes. Particular examples of PARP-1 target genes include, without limitation, Hsp72, SIRT1, SIRT6, FOX03A, SESN2, NAMPT, PUMA, BRCA1, and pl4ARF, including combinations thereof (see also Figures 3A-3D, Figures 4A-4E, Figures 10A-10F, and Figure 12 for relevant target genes). In some instances, the expression of one or more PARP-1 target genes is increased by about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10-fold or more, relative to a control (for instance, where the control is (a) alone, (b) alone, or both). In some instances, the combination of (a) and (b) synergistically increases expression of one or more PARP-1 target genes relative to (a) alone and (b) alone.

In some embodiments, the cell is contacted with (a) and (b) simultaneously or concurrently (i.e., at the same or substantially the same time). In some embodiments, (a) and (b) are part of the same composition, for example, a pharmaceutical or therapeutic composition. In some

embodiments, (a) and (b) are in separate compositions, which are contacted separately but at the same (or substantially the same) time.

In certain embodiments, the cell is contacted with (a) and (b) sequentially (i.e., at separate times). In these embodiments, (a) and (b) are typically in separate compositions.

In some embodiments, the cell is contacted with a defined molar ratio of (a):(b), for example, a molar ratio of about, at least about, or no more than about 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, or a molar ratio of about, at least about, or no more than about 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.5:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1. 0.4:1, 0.3:1, 0.2:1, or 0.1:1.

In certain embodiments, the cell being contacted is a cancer or cancerous cell. In specific embodiments, the cancer cell is a metastatic or invasive cancer cell. Examples of cancer cells include breast cancer cell, a cervical cancer cell, a prostate cancer cell, a pancreatic cancer cell, a gastrointestinal cancer cell, a lung cancer cell, an ovarian cancer cell, a testicular cancer cell, a head and neck cancer cell, a bladder cancer cell, a kidney cancer cell (e.g., renal cell carcinoma), a squamous cell carcinoma, a CNS or brain cancer cell, a melanoma cell, a non-melanoma cancer cell, a thyroid cancer cell, a endometrial cancer cell, an epithelial tumor cell, a bone cancer cell, or a hematopoietic cancer cell.

Examples or primary bone cancer cells include osteosarcomas, chondrosarcomas, and cells of the Ewing Sarcoma Family of Tumors (ESFTs). Examples of gastrointestinal cancer cells include esophageal cancer cells, stomach (gastric) cancer cell, pancreatic cancer cells, liver cancer cells, gallbladder (biliary) cancer cells, small intestinal cancer cells, colorectal cancer cells, anal or rectal cancer cells, and gastrointestinal carcinoid or stromal tumors.

Examples of lung cancer cells include adenocarcinomas, squamous-cell lung carcinomas, small-cell lung carcinomas, and large-cell lung carcinomas.

Particular examples of CNS or brain cancer cells include gliomas, meningiomas, pituitary adenomas, vestibular schwannomas, primary CNS lymphomas, neuroblastomas, and primitive neuroectodermal tumors (medulloblastomas). In some embodiments, the glioma is an astrocytoma, oligodendroglioma, ependymoma, or a choroid plexus papilloma. In some aspects, the brain cancer cell is a glioblastoma multiforme. In some embodiments, the glioblastoma multiforme is a giant cell gliobastoma or a gliosarcoma. In particular embodiments, the cancer cell is a metastatic cancer of the CNS, for instance, a cancer cell that has metastasized to the brain. Examples of such cancer cells include, without limitation, metastatic breast cancer cells, metastatic lung cancer cells, metastatic genitourinary tract cancer cells, metastatic gastrointestinal tract cancer cells (e.g., colorectal cancer cells, pancreatic carcinomas), osteosarcomas, melanomas, metastatic head and neck cancer cells, metastatic prostate cancer cells (e.g., prostatic adenocarcinomas), and metastatic lymphomas.

Examples of melanoma cells include those derived from lentigo maligna, lentigo maligna melanomas, superficial spreading melanomas, acral lentiginous melanomas, mucosal melanomas, nodular melanomas, polypoid melanomas, desmoplastic melanomas, amelanotic melanomas, soft- tissue melanomas, and uveal melanomas. Examples of hematopoietic cancer cells include lymphoma cells, leukemia cells, and multiple myeloma cells. In some instances, the lymphoma cell is a T-cell lymphoma, B-cell lymphoma, small lymphocytic lymphoma, mangle cell lymphoma, anaplastic large cell lymphoma (ALCL), follicular lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma. In particular instances, the leukemia cell is chronic lymphocytic leukemia (CLL), hairy cell leukemia, acute lymphoblastic leukemia, myelocytic leukemia, acute myeloid or myelogenous leukemia, or chronic myelogenous leukemia.

In come embodiments, the cell is in vitro, for example, in tissue culture. Methods of culturing cells including cancer or transformed cells are well-known in the art (see, for example, Animal Cell Culture ( . Freshney, ed., 1986); Freshney, R.I. (2005) Culture of Animal Cells, a Manual of Basic Technique, 5th Ed. Hoboken NJ, John Wiley & Sons).

In certain embodiments, the cell is in a subject, and the method comprises administering (a) the TyrRS polypeptide or TyrRS polynucleotide and (b) the resveratrol compound to the subject. Thus, certain embodiments include methods of treating a subject in need thereof.

In some embodiments, the subject has a disease or condition associated with or triggered by stress, for example, physiological stress or biological stress. Examples of stress include oxidative stress, viral stress (e.g., mediated by viral infection), radiation-induced stress (e.g., mediated by radiation exposure, recovery from exposure to high radiation), drug-induced stress (e.g., mediated by toxic drug exposure, drug overdoses), light-induced stress (e.g., mediated by ultraviolet (UV) light exposure, sunlight exposure), or any combination thereof (e.g., drug- or light-induced oxidative stress).

Oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen species and a biological system's ability to detoxify the reactive intermediates or repair the resulting damage. Disturbances in the normal redox state of cells lead to the production of toxic peroxides and free radicals. Further, some reactive oxidative species act as cellular messengers in redox signaling. Oxidative stress can cause disruptions in normal mechanisms of cellular signaling. In some embodiments, the disease or condition associated with or triggered by oxidative stress is selected from one or more of cancer, Parkinson's disease, Alzheimer's disease, atherosclerosis, heart failure, myocardial infarction, fragile X syndrome, Sickle Cell Disease, lichen planus, vitiligo, autism, infection, and chronic fatigue syndrome.

Radiation exposure can occur, for example, from exposure to nuclear medicine or radiotherapy (e.g., cancer treatment), X-rays, particle accelerators, nuclear weapons, nuclear power plants including nuclear fuel reprocessing plants, cosmic radiation (e.g., high-altitude flight or space travel), mining (e.g., uranium mining), radionuclides (e.g., phosphorus-32 , sulfur-35, americium-241, cesium-137, cobalt-60, iodine-129 and -131, plutonium, radium, radon, strontium-90, technetium- 99, tritium, thorium, uranium), and various other forms of ionizing radiation. Thus, in certain embodiments the subject has radiation-induced stress, for instance, from exposure to one or more of the foregoing, and the methods include the treatment of said radiation exposure.

In some embodiments, the subject has or is at risk for having drug-induced stress, including drug-induced oxidative stress (see, e.g., Deavall et al., Journal of Toxicology. Volume 2012 (2012), Article ID 645460). Non-limiting examples of drugs that induce oxidative stress include many cancer drugs (e.g., cisplatin and others described herein and known in the art) and anti-viral agents (e.g., anti-retrovirals such as AZT and HAART therapies).

In some embodiments, the subject has or is at risk for having light-induced stress, including light-induced oxidative stress. Examples include exposure to sunlight (solar) and/or UV radiation (see, e.g., Bickers and Athar, Journal of Investigative Derm. 126:2565-2575, 2006). Certain subjects have one or more skin diseases/conditions associated with light-induced stress, such as a skin cancer, an inflammatory skin disease (e.g., vitiligo), allergic reactions in the skin, cutaneous inflammation, varicose ulcers, drug-induced photosensitization, or combinations thereof.

In some embodiments, the subject has or is at risk for having a disease or condition such as a cardiovascular disease, a neurological disease, a metabolic disease, obesity, or a cancer.

Certain embodiments include the treatment of one or more cardiovascular diseases.

Particular embodiments include the prophylactic treatment or management of one or more cardiovascular diseases. Cardiovascular disease (also called heart disease) includes a broad class of diseases that involve the heart and/or the blood vessels (e.g., arteries, capillaries, veins). Included are cardiac diseases, vascular diseases of the brain and kidney, and peripheral arterial disease.

Particular examples of cardiovascular diseases include, without limitation, coronary artery disease (i.e., coronary heart disease, ischemic heart disease), cardiomyopathy, hypertensive heart disease (diseases of the heart secondary to high blood pressure), heart failure, pulmonary heart disease (e.g., failure at the right side of the heart with respiratory system involvement), cardiac dysrhythmias, inflammatory heart diseases such as endocarditis, inflammatory cardiomegaly, and myocarditis, valvular heart disease, cerebrovascular disease, peripheral arterial disease, congenital heart disease, and rheumatic heart disease.

Cardiovascular diseases are often associated with atherosclerosis and/or hypertension. Thus, in certain embodiments, a subject has a cardiovascular disease, or is at risk for having a

cardiovascular disease, for instance, because they have atherosclerosis and/or hypertension. The methods and compositions described herein can therefore be used in the treatment of any of the foregoing cardiovascular diseases. Some embodiments include the treatment of one or more neurological or neuronal diseases or conditions. Particular embodiments include the prophylactic treatment or management of one or more neurological or neuronal diseases or conditions. Examples include, without limitation, neuro- inflammation, tumorigenesis of the brain, brain ischemia and repair, neuropathies such as peripheral neuropathy, and neurodegeneration or neurodegenerative diseases. Specific examples of neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Friedreich's ataxia, Motor neuron diseases (MND),

Spinocerebellar ataxia (SCA), Lewy body disease, spinal muscular atrophy, and neurodegeneration associated with aging.

Thus, in certain embodiments, a subject has a neurological disease or condition, or is at risk for having a neurological disease or condition. The methods and compositions described herein can therefore be used in the treatment of any of the foregoing neurological diseases/conditions.

Some embodiments include the treatment of one or more metabolic diseases, including congenital and acquired metabolic diseases. Particular embodiments include the prophylactic treatment or management of one or more metabolic diseases. In particular embodiments, the metabolic disease is a disease of glucose metabolism, for example, a disease resulting from impaired insulin sensitivity and/or glucose utilization. Particular examples of metabolic diseases include diabetes (e.g., Type 1 diabetes, Type 2 diabetes), pre-diabetes, hyperglycemia, hyperinsulinaemia, and metabolic syndrome (i.e., metabolic syndrome X, cardiometabolic syndrome, syndrome X, insulin resistance syndrome, eaven's syndrome).

Thus, in certain embodiments, a subject has a metabolic disease, or is at risk for having a metabolic disease. The methods and compositions described herein can therefore be used in the treatment of any of the foregoing metabolic diseases.

Some embodiments include the treatment of obesity. Particular embodiments include the prophylactic treatment or management of obesity. Obesity is a condition in which excess body fat accumulates to the extent that it may have a negative impact on health, leading to reduced life expectancy and/or increased health problems. In some embodiments, a subject is obese if their body mass index (BMI = m/h²) is about or greater than about 25 to 30 kg/m² or more.

Obesity is associated with an increased risk of many diseases, for example, heart disease, type 2 diabetes, obstructive sleep apnea, certain types of cancer, osteoarthritis, and many of the other cardiovascular and metabolic diseases described herein. Thus, certain obese subjects have or are at risk for having one or more of such disease, or any of the diseases described herein. In some embodiments, the methods and compositions described herein reduce the symptoms or pathology of one or more diseases associated with obesity. In certain embodiments, the methods and compositions reduce the risk of developing one or more diseases associated with obesity.

Obesity is also associated with reduced life expectancy. In some instances, the methods and compositions described herein can be used to increase the life expectancy of an obese subject (or a population of obese subjects), for instance, by about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years or more.

Certain methods include the treatment of cancer. "Cancer" relates generally to a class of diseases or conditions in which a group of cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., intrusion on and destruction of adjacent tissues), and/or metastasis (i.e., spread to other locations in the body via lymph or blood). These malignant properties of cancers differentiate them from benign cancers, which are self-limited, and typically do not invade or metastasize. Also included are myelodysplastic syndromes.

In some embodiments, the subject has a cancer selected from one or more of breast cancer, cervical cancer, prostate cancer, pancreatic cancer, gastrointestinal cancer, lung cancer, ovarian cancer, testicular cancer, head and neck cancer, bladder cancer, kidney cancer (e.g., renal cell carcinoma), soft tissue sarcoma, squamous cell carcinoma, CNS or brain cancer, melanoma, non- melanoma cancer, thyroid cancer, endometrial cancer, an epithelial tumor, bone cancer, or a hematopoietic cancer.

Examples of lung cancers include adenocarcinomas, squamous-cell lung carcinomas, small- cell lung carcinomas, and large-cell lung carcinomas.

Examples or primary bone cancers include osteosarcoma, chondrosarcoma, and the Ewing Sarcoma Family of Tumors (ESFTs).

Examples of gastrointestinal cancers include esophageal cancer, stomach (gastric) cancer, pancreatic cancer, liver cancer, gallbladder (biliary) cancer, small intestinal cancer, colorectal cancer, anal or rectal cancer, and gastrointestinal carcinoid or stromal tumors.

Examples of CNS or brain cancers include primary brain cancers and metastatic brain cancers. Particular examples of brain cancers include gliomas, meningiomas, pituitary adenomas, vestibular schwannomas, primary CNS lymphomas, neuroblastomas, and primitive neuroectodermal tumors (medulloblastomas). In some embodiments, the glioma is an astrocytoma,

oligodendroglioma, ependymoma, or a choroid plexus papilloma. In some aspects, the subject has a glioblastoma multiforme. In specific aspects, the glioblastoma multiforme is a giant cell gliobastoma or a gliosarcoma. In particular embodiments, the cancer is a metastatic cancer of the CNS, for instance, a cancer that has metastasized to the brain. Examples of such cancers include, without limitation, breast cancers, lung cancers, genitourinary tract cancers, gastrointestinal tract cancers (e.g., colorectal cancers, pancreatic carcinomas), osteosarcomas, melanomas, head and neck cancers, prostate cancers (e.g., prostatic adenocarcinomas), and lymphomas.

Examples of melanomas include lentigo maligna, lentigo maligna melanoma, superficial spreading melanoma, acral lentiginous melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft-tissue melanoma, and uveal melanoma.

Examples of hematopoietic cancers include lymphomas, leukemias, and multiple myelomas. In some instances, the lymphoma is a T-cell lymphoma, B-cell lymphoma, small lymphocytic lymphoma, mangle cell lymphoma, anaplastic large cell lymphoma (ALCL), follicular lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma. In particular instances, the leukemia is chronic lymphocytic leukemia (CLL), hairy cell leukemia, acute lymphoblastic leukemia, myelocytic leukemia, acute myeloid or myelogenous leukemia, or chronic myelogenous leukemia.

The use of TyrRS and resveratrol compounds for treating cancers can be combined with other therapeutic modalities. For example, the combination of TyrRS and one or more resveratrol compounds can be administered to a subject before, during, or after other therapeutic

interventions, including symptomatic care, radiotherapy, surgery, transplantation, hormone therapy, immunotherapy, photodynamic therapy, antibiotic therapy, and administration of anti-cancer agents, including any combination thereof. Symptomatic care includes administration of corticosteroids, to reduce cerebral edema, headaches, cognitive dysfunction, and emesis, and administration of anti-convulsants, to reduce seizures. Radiotherapy includes whole-brain irradiation, fractionated radiotherapy, and radiosurgery, such as stereotactic radiosurgery, which can be further combined with traditional surgery.

Thus, in certain embodiments, a subject has a cancer, or is at risk for having a cancer. The methods and compositions described herein can therefore be used in the treatment of any of the foregoing cancers, alone or in combination with other therapeutic modalities.

In some embodiments, the subject is aging, for example, a middle-aged or elderly subject. In certain embodiments, the subject has a disease or condition associated with aging, for example, an aging-associated disease. In some embodiments, the methods include slowing or reducing aging, reducing the effects of aging, or reducing age-related tissue degeneration, for example, age-related neurodegeneration. In some embodiments, the methods include treating an aging-associated disease, such as cardiovascular diseases such as atherosclerosis and/or hypertension, cancer, arthritis, dementia, cataracts, osteoporosis, osteoarthritis, diabetes such as type 2 diabetes, hearing loss, and neurodegenerative diseases such as Alzheimer's disease, among other diseases described herein. In some embodiments, the subject is about or at least about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 years old. In some instances, the methods and compositions described herein can be used to increase the life expectancy of a subject (or a population of subjects), for instance, by about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years or more. In some embodiments, the administration of (a) and (b) reduces oxidative stress and/or inflammation in the subject.

In some embodiments, (a) the TyrRS polypeptide or TyrRS polynucleotide and (b) the resveratrol compound are administered simultaneously or concurrently (i.e., at the same or substantially the same time). In some embodiments, (a) and (b) are administered as part of the same composition, for example, a pharmaceutical or therapeutic composition. In some embodiments, (a) and (b) are administered in separate compositions, which are administered separately but at the same (or substantially the same) time.

In certain embodiments, (a) and (b) are administered sequentially (i.e., at separate times). In these embodiments, (a) and (b) are typically administered in separate compositions.

In some embodiments, the (a) and (b) are administered at a defined molar ratio of (a):(b), for example, a molar ratio of about, at least about, or no more than about 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, or a molar ratio of about, at least about, or no more than about 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.5:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1. 0.4:1, 0.3:1, 0.2:1, or 0.1:1.

For the purposes of administration, the TyrRS polypeptides and resveratrol compounds may be administered to a patient or subject as a raw chemical (for resveratrol compounds) or may be formulated as pharmaceutical compositions. Pharmaceutical compositions generally comprise a TyrRS polypeptide and/or a resveratrol compound, and a pharmaceutical grade (i.e., USP grade) or pharmaceutically acceptable carrier, diluent, or excipient. The TyrRS polypeptide and/or the resveratrol compound is typically present in the composition in an amount which is effective to treat or manage a particular disease or condition of interest, as described herein, and preferably with acceptable toxicity to the subject. The activity of compound(s) can be determined by one skilled in the art, for example, as described in the Examples below.

Embodiments of the present invention therefore include compositions comprising TyrRS polypeptides and/or resveratrol compounds formulated in pharmaceutically-acceptable or physiologically-acceptable solutions for administration to a cell, subject, or an animal, either alone, or in combination with one or more other modalities of therapy. It will also be understood that, if desired, the compositions may be administered in combination with other agents as well, for example, other proteins or polypeptides or various pharmaceutically-active agents. There is virtually no limit to other components that may also be included in the compositions, provided that the additional agents do not adversely affect the modulatory or other effects desired to be achieved.

For pharmaceutical production, pharmaceutical or therapeutic compositions will typically be substantially endotoxin free. Endotoxins are toxins associated with certain bacteria, typically gram- negative bacteria, although endotoxins may be found in gram-positive bacteria, such as Listeria monocytogenes. The most prevalent endotoxins are lipopolysaccharides (LPS) or lipo-oligo- saccharides (LOS) found in the outer membrane of various Gram-negative bacteria, and which represent a central pathogenic feature in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans may produce fever, a lowering of the blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects.

Endotoxins can be detected using routine techniques known in the art. For example, the Limulus Amoebocyte Lysate assay, which utilizes blood from the horseshoe crab, is a very sensitive assay for detecting presence of endotoxin. In this test, very low levels of LPS can cause detectable coagulation of the limulus lysate due a powerful enzymatic cascade that amplifies this reaction. Endotoxins can also be quantitated by enzyme-linked immunosorbent assay (ELISA).

To be substantially endotoxin free, endotoxin levels may be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU/mg of protein. Typically, 1 ng lipopolysaccharide (LPS) corresponds to about 1-10 EU.

In certain embodiments, as noted herein, the compositions have an endotoxin content of less than about 10 EU / mg of polypeptide, or less than about 5 EU / mg of polypeptide, less than about 3 EU / mg of polypeptide, or less than about 1 EU / mg of polypeptide or less than about 0.1 EU/ mg of polypeptide, or less than about 0.01EU / mg of polypeptide. In certain embodiments, as noted above, the pharmaceutical compositions are about 95% endotoxin free, preferably about 99% endotoxin free, and more preferably about 99.99% endotoxin free on wt/wt protein basis.

In one aspect such compositions may comprises polypeptides that are substantially monodisperse, meaning that the TyrRS polypeptide exists primarily (i.e., at least about 90%, or greater) in one apparent molecular weight form when assessed for example, by size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation.

In some aspects, compositions have a purity (e.g., on a protein basis) of at least about 90%, or in some aspects at least about 95% purity, or in some embodiments, at least 98% purity. Purity may be determined via any routine analytical method as known in the art.

In some aspects, compositions have a high molecular weight aggregate content of less than about 10%, compared to the total amount of protein present, or in some embodiments such compositions have a high molecular weight aggregate content of less than about 5%, or in some aspects such compositions have a high molecular weight aggregate content of less than about 3%, or in some embodiments a high molecular weight aggregate content of less than about 1%. High molecular weight aggregate content may be determined via a variety of analytical techniques including for example, by size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation.

Pharmaceutical compositions may include pharmaceutically acceptable salts of a TyrRS polypeptide and/or a resveratrol compound. For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002). Suitable base salts are formed from bases which form non-toxic salts. Representative examples include the aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts.

Hemisalts of acids and bases may also be formed, e.g., hemisulphate and hemicalcium salts.

Compositions to be used in the invention suitable for parenteral administration may comprise sterile aqueous solutions and / or suspensions of the pharmaceutically active ingredients preferably made isotonic with the blood of the recipient, generally using sodium chloride, glycerin, glucose, mannitol, sorbitol, and the like. Organic acids suitable for forming pharmaceutically acceptable acid addition salts include, by way of example and not limitation, acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, oxalic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, alkylsulfonic acids (e.g., methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid), arylsulfonic acids (e.g., benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2- naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid), 4-methylbicyclo(2.2.2)-oct- 2-ene-l-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like.

In particular embodiments, the carrier may include water. In some embodiments, the carrier may be an aqueous solution of saline, for example, water containing physiological concentrations of sodium, potassium, calcium, magnesium, and chloride at a physiological pH. In some embodiments, the carrier may be water and the formulation may further include NaCI. In some embodiments, the formulation may be isotonic. In some embodiments, the formulation may be hypotonic. In some embodiments, the formulation may be hypertonic. In some embodiments, the formulation may be isomostic. In some embodiments, the formulation is substantially free of polymers (e.g., gel-forming polymers, polymeric viscosity-enhancing agents). In some embodiments, the formulation is substantially free of viscosity-increasing agents (e.g., carboxymethylcellulose, polyanionic polymers). In some embodiments, the formulation is substantially free of gel-forming polymers. In some embodiments, the viscosity of the formulation is about the same as the viscosity of a saline solution containing the same concentration of agent(s) (or a pharmaceutically acceptable salt thereof).

Formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation.

In certain embodiments, the TyrRS polypeptide and/or the resveratrol compounds have a solubility that is desirable for the particular mode of administration, such intravenous

administration. Examples of desirable solubility's include at least about 1 mg/ml, at least about 10 mg/ml, at least about 25 mg/ml, and at least about 50 mg/ml.

In certain applications, the pharmaceutical compositions disclosed herein may be delivered via oral administration to a subject. As such, these compositions may be formulated with an inert diluent or with an edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.

Pharmaceutical compositions suitable for the delivery of polypeptides and small molecules and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).

Administration of a composition may be by any suitable method known in the medicinal arts, including for example, oral, intranasal, parenteral administration include intravitreal, subconjuctival, sub-tenon, retrobulbar, suprachoroidal intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, intraocular, topical and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques.

Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates, and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. The preparation of parenteral formulations under sterile conditions, for instance, by lyophilization, may readily be accomplished using standard pharmaceutical techniques well-known to those skilled in the art. Formulations for parenteral administration may be formulated to be immediate and / or sustained release. Sustained release compositions include delayed, modified, pulsed, controlled, targeted and programmed release. Thus a composition may be formulated as a suspension or as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing sustained release of agent(s). Examples of such formulations include without limitation, drug-coated stents and semi-solids and suspensions comprising drug-loaded poly(DL-lactic-co-glycolic)acid (PGLA), poly(DL-lactide-co-glycolide) (PLG) or poly(lactide) (PLA) lamellar vesicles or microparticles, hydrogels (Hoffman AS: Ann. N.Y. Acad. Sci. 944: 62-73 (2001)), poly-amino acid nanoparticles systems, such as the Medusa system developed by Flamel Technologies Inc., non-aqueous gel systems such as Atrigel developed by Atrix, Inc., and SABER (Sucrose Acetate Isobutyrate Extended Release) developed by Durect Corporation, and lipid-based systems such as DepoFoam developed by SkyePharma.

Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, incorporated by reference in its entirety). In all cases the form should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the

maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion (see, e.g., Remington's

Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologies standards.

Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent with the various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The compositions disclosed herein may be formulated in a neutral or salt form.

Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.

As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.

Compositions and agents can also be administered topically, (intra)dermally, or transdermally to the skin, mucosa, or surface of the eye, either alone or in combination with one or more antihistamines, one or more antibiotics, one or more antifungal agents, one or more beta blockers, one or more anti-inflammatory agents, one or more antineoplastic agents, one or more immunosuppressive agents, one or more antiviral agents, one or more antioxidant agents, or other active agents. Formulations for topical and ocular administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release.

Typical formulations for this purpose include gels, hydrogels, lotions, solutions, eye drops, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, bandages, and microemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol, and propylene glycol. Penetration enhancers may be incorporated (see, e.g., Finnin and Morgan: J. Pharm. Sci. 88:955-958, 1999). Other means of topical administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis, and microneedle or needle-free injection (e.g., the systems sold under the trademarks POWDEFUECT™, BIOJECT™).

Examples of antihistamines include, but are not limited to, loradatine, hydroxyzine, diphenhydramine, chlorpheniramine, brompheniramine, cyproheptadine, terfenadine, clemastine, triprolidine, carbinoxamine, diphenylpyraline, phenindamine, azatadine, tripelennamine, dexchlorpheniramine, dexbrompheniramine, methdilazine, and trimprazine doxylamine, pheniramine, pyrilamine, chiorcyclizine, thonzylamine, and derivatives thereof.

Examples of antibiotics include, but are not limited to, aminoglycosides (e.g., amikacin, apramycin, arbekacin, bambermycins, butirosin, dibekacin, dihydrostreptomycin, fortimicin(s), gentamicin, isepamicin, kanamycin, micronomicin, neomycin, neomycin undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin, trospectomycin), amphenicols (e.g., azidamfenicol, chloramphenicol, florfenicol, thiamphenicol), ansamycins (e.g., rifamide, rifampin, rifamycin sv, rifapentine, rifaximin), lactams (e.g., carbacephems (e.g., loracarbef), carbapenems (e.g., biapenem, imipenem, meropenem, panipenem), cephalosporins (e.g., cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefcapene pivoxil, cefclidin, cefdinir, cefditoren, cefepime, cefetamet, cefixime, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cefpimizole, cefpiramide, cefpirome, cefpodoxime proxetil, cefprozil, cefroxadine, cefsulodin, ceftazidime, cefteram, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephacetrile sodium, cephalexin, cephaloglycin, cephaloridine, cephalosporin, cephalothin, cephapirin sodium, cephradine, pivcefalexin), cephamycins (e.g., cefbuperazone, cefmetazole, cefminox, cefotetan, cefoxitin), monobactams (e.g., aztreonam, carumonam, tigemonam), oxacephems, flomoxef, moxalactam), penicillins (e.g., amdinocillin, amdinocillin pivoxil, amoxicillin, ampicillin, apalcillin, aspoxicillin, azidocillin, azlocillin, bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium, carbenicillin, carindacillin,

clometocillin, cloxacillin, cyclacillin, dicloxacillin, epicillin, fenbenicillin, floxacillin, hetacillin, lenampicillin, metampicillin, methicillin sodium, mezlocillin, nafcillin sodium, oxacillin, penamecillin, penethamate hydriodide, penicillin g benethamine, penicillin g benzathine, penicillin g

benzhydrylamine, penicillin g calcium, penicillin g hydrabamine, penicillin g potassium, penicillin g procaine, penicillin n, penicillin o, penicillin v, penicillin v benzathine, penicillin v hydrabamine, penimepicycline, phenethicillin potassium, piperacillin, pivampicillin, propicillin, quinacillin, sulbenicillin, sultamicillin, talampicillin, temocillin, ticarcillin), other (e.g., ritipenem), lincosamides (e.g., clindamycin, lincomycin), macrolides (e.g., azithromycin, carbomycin, clarithromycin, dirithromycin, erythromycin, erythromycin acistrate, erythromycin estolate, erythromycin glucoheptonate, erythromycin lactobionate, erythromycin propionate, erythromycin stearate, josamycin, leucomycins, midecamycins, miokamycin, oleandomycin, primycin, rokitamycin, rosaramicin, roxithromycin, spiramycin, troleandomycin), polypeptides (e.g., amphomycin, bacitracin, capreomycin, colistin, enduracidin, enviomycin, fusafungine, gramicidin s, gramicidin(s), mikamycin, polymyxin, pristinamycin, ristocetin, teicoplanin, thiostrepton, tuberactinomycin, tyrocidine, tyrothricin, vancomycin, viomycin, virginiamycin, zinc bacitracin), tetracyclines (e.g., apicycline, chlortetracycline, clomocycline, demeclocycline, doxycycline, guamecycline, lymecycline, meclocycline, methacycline, minocycline, oxytetracycline, penimepicycline, pipacycline,

rolitetracycline, sancycline, tetracycline), and others (e.g., cycloserine, mupirocin, tuberin). 2.4- Diaminopyrimidines (e.g., brodimoprim, tetroxoprim, trimethoprim), nitrofurans (e.g., furaltadone, furazolium chloride, nifuradene, nifuratel, nifurfoline, nifurpirinol, nifurprazine, nifurtoinol, nitrofurantoin), quinolones and analogs (e.g., cinoxacin, ciprofloxacin, clinafloxacin, difloxacin, enoxacin, fleroxacin, flumequine, grepafloxacin, lomefioxacin, miloxacin, nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pazufloxacin, pefloxacin, pipemidic acid, piromidic acid, rosoxacin, rufloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin), sulfonamides (e.g., acetyl sulfamethoxypyrazine, benzylsulfamide, chloramine-b, chloramine-t, dichloramine t, n2- formylsulfisomidine, mafenide, 4'-(methylsulfamoyl)sulfanilanilide, noprylsulfamide,

phthalylsulfacetamide, phthalylsulfathiazole, salazosulfadimidine, succinylsulfathiazole, sulfabenzamide, sulfacetamide, sulfachlorpyridazine, sulfachrysoidine, sulfacytine, sulfadiazine, sulfadicramide, sulfadimethoxine, sulfadoxine, sulfaethidole, sulfaguanidine, sulfaguanol, sulfalene, sulfaloxic acid, sulfamerazine, sulfameter, sulfamethazine, sulfamethizole, sulfamethomidine, sulfamethoxazole, sulfamethoxypyridazine, sulfametrole, sulfamidochrysoidine, sulfamoxole, sulfanilamide, 4-sulfanilamidosalicylic acid, n4-sulfanilylsulfanilamide, sulfanilylurea, n-sulfanilyl-3,4- xylamide, sulfanitran, sulfaperine, sulfaphenazole, sulfaproxyline, sulfapyrazine, sulfapyridine, sulfasomizole, sulfasymazine, sulfathiazole, sulfathiourea, sulfatolamide, sulfisomidine, sulfisoxazole) sulfones (e.g., acedapsone, acediasulfone, acetosulfone sodium, dapsone, diathymosulfone, glucosulfone sodium, solasulfone, succisulfone, sulfanilic acid, p- sulfanilylbenzylamine, sulfoxone sodium, thiazolsulfone), and others (e.g., clofoctol, hexedine, methenamine, methenamine anhydromethylene-citrate, methenamine hippurate, methenamine mandelate, methenamine subsalicylate, nitroxoline, taurolidine, xibornol).

Examples of antifungal agents include, but are not limited to Polyenes (e.g., amphotericin b, candicidin, dermostatin, filipin, fungichromin, hachimycin, hamycin, lucensomycin, mepartricin, natamycin, nystatin, pecilocin, perimycin), others (e.g., azaserine, griseofulvin, oligomycins, neomycin undecylenate, pyrrolnitrin, siccanin, tubercidin, viridin), Allylamines (e.g., butenafine, naftifine, terbinafine), imidazoles (e.g., bifonazole, butoconazole, chlordantoin, chlormidazole, cloconazole, clotrimazole, econazole, enilconazole, fenticonazole, flutrimazole, isoconazole, ketoconazole, lanoconazole, miconazole, omoconazole, oxiconazole nitrate, sertaconazole, sulconazole, tioconazole), thiocarbamates (e.g., tolciclate, tolindate, tolnaftate), triazoles (e.g., fluconazole, itraconazole, saperconazole, terconazole) others (e.g., acrisorcin, amorolfine, biphenamine, bromosalicylchloranilide, buclosamide, calcium propionate, chlorphenesin, ciclopirox, cloxyquin, coparaffinate, diamthazole dihydrochloride, exalamide, flucytosine, halethazole, hexetidine, loflucarban, nifuratel, potassium iodide, propionic acid, pyrithione, salicylanilide, sodium propionate, sulbentine, tenonitrozole, triacetin, ujothion, undecylenic acid, zinc propionate).

Examples of beta blockers include but are not limited to acebutolol, atenolol, labetalol, metoprolol, propranolol, timolol, and derivatives thereof.

Examples of antineoplastic agents include, but are not limited to antibiotics and analogs (e.g., aclacinomycins, actinomycin fl, anthramycin, azaserine, bleomycins, cactinomycin, carubicin, carzinophilin, chromomycins, dactinomycin, daunorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, idarubicin, menogaril, mitomycins, mycophenolic acid, nogalamycin, olivomycines, peplomycin, pirarubicin, plicamycin, porfiromycin, puromycin, streptonigrin, streptozocin, tubercidin, zinostatin, zorubicin), antimetabolites (e.g. folic acid analogs (e.g., denopterin, edatrexate, methotrexate, piritrexim, pteropterin, Tomudex^®, trimetrexate), purine analogs (e.g., cladribine, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine), pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, doxifluridine, emitefur, enocitabine, floxuridine, fluorouracil, gemcitabine, tagafur).

Examples of anti-inflammatory agents include but are not limited to steroidal antiinflammatory agents and non-steroidal anti-inflammatory agents. Exemplary steroidal antiinflammatory agents include acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide,

desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate,

paramethasone, prednicarbate, prednisolone, prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, and triamcinolone hexacetonide.

Exemplary non-steroidal anti-inflammatory agents include aminoarylcarboxylic acid derivatives (e.g., enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, mefenamic acid, niflumic acid, talniflumate, terofenamate, tolfenamic acid), arylacetic acid derivatives (e.g., aceclofenac, acemetacin, alclofenac, amfenac, amtolmetin guacil, bromfenac, bufexamac, cinmetacin, clopirac, diclofenac sodium, etodolac, felbinac, fenclozic acid, fentiazac, glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac, metiazinic acid, mofezolac, oxametacine, pirazolac, proglumetacin, sulindac, tiaramide, tolmetin, tropesin, zomepirac), arylbutyric acid derivatives (e.g., bumadizon, butibufen, fenbufen, xenbucin), arylcarboxylic acids (e.g., clidanac, ketorolac, tinoridine), arylpropionic acid derivatives (e.g., alminoprofen, benoxaprofen, bermoprofen, bucloxic acid, carprofen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, loxoprofen, naproxen, oxaprozin, piketoprolen, pirprofen, pranoprofen, protizinic acid, suprofen, tiaprofenic acid, ximoprofen, zaltoprofen), pyrazoles (e.g., difenamizole, epirizole), pyrazolones (e.g., apazone, benzpiperylon, feprazone, mofebutazone, morazone, oxyphenbutazone, phenylbutazone, pipebuzone, propyphenazone, ramifenazone, suxibuzone, thiazolinobutazone), salicylic acid derivatives (e.g., acetaminosalol, aspirin, benorylate, bromosaligenin, calcium acetylsalicylate, diflunisal, etersalate, fendosal, gentisic acid, glycol salicylate, imidazole salicylate, lysine acetylsalicylate, mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide, salicylamide o-acetic acid, salicylsulfuric acid, salsalate, sulfasalazine), thiazinecarboxamides (e.g., ampiroxicam, droxicam, isoxicam, lornoxicam, piroxicam, tenoxicam), ε-acetamidocaproic acid, s- adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,, bucolome, difenpiramide, ditazol, emorfazone, fepradinol, guaiazulene, nabumetone, nimesulide, oxaceprol, paranyline, perisoxal, proquazone, superoxide dismutase, tenidap, and zileuton.

Examples of antiviral agents include interferon gamma, zidovudine, amantadine hydrochloride, ribavirin, acyclovir, valciclovir, dideoxycytidine, phosphonoformic acid, ganciclovir, and derivatives thereof

Examples of antioxidant agents include ascorbate, alpha-tocopherol, mannitol, reduced glutathione, various carotenoids, cysteine, uric acid, taurine, tyrosine, superoxide dismutase, lutein, zeaxanthin, cryotpxanthin, astazanthin, lycopene, N-acetyl-cysteine, carnosine, gamma- glutamylcysteine, quercitin, lactoferrin, dihydrolipoic acid, citrate, Ginkgo Biloba extract, tea catechins, bilberry extract, vitamins E or esters of vitamin E, retinyl palmitate, and derivatives thereof. Other therapeutic agents include squalamine, carbonic anhydrase inhibitors, alpha-2 adrenergic receptor agonists, antiparasitics, antifungals, and derivatives thereof.

The exact dose of each component administered will, of course, differ depending on the specific components prescribed, on the subject being treated, on the severity of the disease, for example, severity of the inflammatory reaction, on the manner of administration and on the judgment of the prescribing physician. Thus, because of patient-to-patient variability, the dosages provided herein are a guideline and the physician may adjust doses of the compounds to achieve the treatment that the physician considers appropriate.

In particular embodiments, the dosage of a resveratrol compound or the total amount (by weight) of a resveratrol compound in a composition (e.g., therapeutic or pharmaceutical composition) ranges from about 0.01 mg to about 10,000 mg, or about 1 mg to 5000 mg, or about 1 mg to about 1000 mg, or about 1 mg to about 500 mg, including all integers and ranges in between, for example, about 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 mg.

In some embodiments, the dosage of a TyrRS polypeptide ranges from about 0.1 μg/ g to about 0.1 mg/kg to about 50 mg/kg to about 100 mg/kg of the subject's body weight, including all ranges and integers in between, for example, about, at least about, or no more than about 0.0001, 0.001, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 mg/kg of the subject's body weight. In some embodiments, the dosage of a TyrRS polypeptide or the total amount (by weight) of a TyrRS polypeptide in a composition (e.g., therapeutic composition, pharmaceutical composition, solid, liquid) is about or at least about 0.001 mg to about 20,000 mg, including all integers and ranges in between, for example, about or at least about 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, or 20,000 mg. In particular embodiments, the concentration of a TyrRS polypeptide in a composition (e.g., a liquid therapeutic or pharmaceutical composition) is about or at least about 1 mg/ml to about 100 mg/ml, including all integers and ranges in between, for example, about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 mg/ml.

Suitable aqueous saline solutions will be understood by those of skill in the art and may include, for example, solutions at a pH of from about pH 4.5 to about pH 8.0. In further variations of aqueous solutions (where water is included in the carrier), the pH of the formulation is between any of about 6 and about 8.0; between about 6 and about 7.5; between about 6 and about 7.0; between about 6.2 and about 8; between about 6.2 and about 7.5; between about 7 and about 8; between about 6.2 and about 7.2; between about 5.0 and about 8.0; between about 5 and about 7.5;

between about 5.5 and about 8.0; between about 6.1 and about 7.7; between about 6.2 and about 7.6; between about 7.3 and about 7.4; about 6.0; about 7.1; about 6.2; about 7.3; about 6.4; about 6.5; about 6.6; about 6.7; about 6.8; about 6.9; about 7.0; about 7.1; about 7.2; about 7.3; about 7.4; about 7.5; about 7.6; or about 8.0. In some variations, the formulation has a pH of about 6.0 to about 7.0. In some variations, the formulation has a pH of about 7.4. In particular variations, the formulation has a pH of about 6.2 to about 7.5.

In certain embodiments the concentration of the salt (e.g., NaCI) will be, for example, from about 0% to about 0.9% (w/v). For example, the concentration of salt may be from about 0.01 to about 0.9%, from about 0.02% to about 0.9%, from about 0.03% to about 9%, from about 0.05% to about 0.9% from about 0.07% to about 0.9%, from about 0.09% to about 0.9%, from about 0.1% to about 0.9% from about 0.2% to about 0.9%, from about 0.3% to about 0.9%, from about 0.4% to about 0.9% from about 0.5% to about 0.9%, from about 0.6% to about 0.9%, from about 0.7% to about 0.9%, from about 0.8% to about 0.9%, about 0.9%, about 0%, about 0.05%, about 0.01%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, or about 0.8%. In certain embodiments, the aqueous saline solution will be isotonic (e.g., NaCI concentration of about 0.9% NaCI (w/v)). In certain embodiments, the aqueous solution will contain a NaCI concentration of about 0.5%, about 0.7%, about 0.8%, about 0.85, or about 0.75%. As will be appreciated the skilled artisan, depending on the concentrations of other components, for example where the agent(s) are present as salts of, the concentration of NaCI or other salt needed to achieve an formulation suitable for administration may vary.

In some embodiments, where the formulation is substantially free of viscosity-increasing agents, the formulation may be substantially free of viscosity-increasing agents such as, but not limited to polyanionic polymers, water soluble cellulose derivatives (e.g., hypromellose (also known as HPMC, hydroxypropylmethyl cellulose, and hydroxypropylcellulose), hydroxyethylcellulose, carboxmethylcellulose, etc.), polyvinyl alcohol, polyvinyl pyrrolidone, chondroitin sulfate, hyaluronic acid, soluble starches, etc. In some variations, the formulation does not incorporate a hydrogel or other retention agent (e.g., such as those disclosed in U.S. Pat. Pub. No. 2005/0255144

(incorporated by reference herein in its entirety)), e.g., where the hydrogel may include hydrogels incorporating homopolymers; copolymers (e.g., tetrapolymers of hydroxymethylmethacrylate, ethylene glycol, dimethylmethacrylate, and methacrylic acid), copolymers of trimethylene carbonate and polyglycolicacid, polyglactin 910, glyconate, poly-p-dioxanone, polyglycolic acid, polyglycolic acid felt, poly-4-hydroxybutyrate, a combination of poly(L-lactide) and poly(L-lactide-co-glycolide), glycol methacrylate, poly-DL-lactide, or Primacryl); composites of oxidized regenerated cellulose, polypropylene, and polydioxanone or a composite of polypropylene and poligelcaprone; etc. In some variations, the formulations do not include one or more of polyvinyl alcohol, hydroxypropyl methylcellulose, polyethylene glycol 400 castor oil emulsion, carboxymethylcellulose sodium, propylene glycol, hydroxypropyl guar, carboxymethylcelluose sodium, white petrolatum, mineral oil, dextran 70, glycerin, hypromellose, flaxseed oil, fish oils, omega 3 and omega 6 fatty acids, lutein, or primrose oil. In some variations, the formulations do not include one or more of the carriers described in U.S. Pat. No. 4,888,354 (incorporated by reference herein in its entirety), e.g., such as one or more of oleic acid, ethanol, isopropanol, glycerol monooleate, glycerol diooleate, methyl laurate, propylene glycol, propanol or dimethyl sulfoxide. In some variations, the formulations are substantially free of glycerol diooleate and isopropanol.

In particular embodiments, the gel-forming polymer may be, for example, a polysaccharide. In certain embodiments, the polysaccharide is gellan gum. Gellan gum refers to a

heteropolysaccharide elaborated by the bacterium Pseudomonas elodea, though the name "gellan gum" is more commonly used in the field. Gellan gum, in particular the formulation GELRITE^® is described in detail in U.S. Pat. No. 4,861,760 (hereby incorporated by reference in its entirety), in particular in its use in formulation of timolol. GELRITE^®, a low acetyl clarified grade of gellan gum, is commercially available from Merck & Co (Rahway, N.J.) and gellan gum can be commercially obtained from, among others CPKelco (Atlanta, Ga.). The preparation of polysaccharides such as gellan gum is described in, for example, U.S. Pat. Nos. 4,326,053 and 4,326,052, which are hereby incorporated by reference in their entirety.

In certain embodiments, the gel-forming polymer is present at a concentration of from about 0.03% to about 2% (w/v). In some embodiments, the gel-forming polymer is present at a concentration from about 0.03% to about 1.75%; from about 0.03% to about 1.5%, from about 0.03% to about 1.25%, from about 0.03% to about 1%, from about 0.03% to about 0.9%, from about 0.03% to about 0.8%, from about 0.03% to about 0.7%, from about 0.03% to about 0.6%, from about 0.03% to about 0.5%, from about 0.05% to about 2%, from about 0.05% to about 1.75%; from about 0.05% to about 1.5%, from about 0.05% to about 1.25%, from about 0.05% to about 1%, from about 0.05% to about 0.9%, from about 0.05% to about 0.8%, from about 0.05% to about 0.7%, from about 0.05% to about 0.6%, from about 0.05% to about 0.5%, from about 0.1% to about 2%, from about 0.1% to about 1.75%; from about 0.1% to about 1.5%, from about 0.1% to about 1.25%, from about 0.1% to about 1%, from about 0.1% to about 0.9%, from about 0.1% to about 0.8%, from about 0.1% to about 0.7%, from about 0.1% to about 0.6%, from about 0.1% to about 0.5%, from about 0.2% to about 2%, from about 0.2% to about 1.75%; from about 0.2% to about 1.5%, from about 0.2% to about 1.25%, from about 0.2% to about 1%, from about 0.2% to about 0.9%, from about 0.2% to about 0.8%, from about 0.2% to about 0.7%, from about 0.2% to, about 0.6%, from about 0.2% to about 0.5%, or from about 0.5% to about 1.5%. In some embodiments, the concentration of gel- forming polymer is about 0.1%, about 0.2%, about 0.4%, about 0.6%, about 0.8%, about 1%.

In particular embodiments, the gel-forming polymer is gellan gum at a concentration of from about 0.05% to about 2% (w/v), from about 0.1% to about 2% (w/v), from about 0.1% to about 1% (w/v), from about 0.05% to about 1% (w/v) or from about 0.1% to about 0.6% (w/v). In some embodiments, the concentration of gellan gum is about 0.1%, about 0.2%, about 0.4%, about 0.6%, about 0.8%, about 1%.

In some embodiments of the formulations, the formulation may include additional components such as one or more preservatives, one or more surfactants, or one or more pharmaceutical agents. In particular embodiments, the formulation may include additional components such as one or more preservatives, one or more surfactants, one or more tonicity agents, one or more buffering agents, one or more chelating agents, one or more viscosity- increasing agents, one or more salts, or one or more pharmaceutical agents. In certain

embodiments, the formulation may include one or more preservatives, one or more buffering agents (e.g., one, two, three, etc.), one or more chelating agents, and one or more salts. In some embodiments, the formulation may include one or more preservatives, one or more tonicity agents, one or more buffering agents, one or more chelating agents, and one or more viscosity-increasing agents.

In some embodiments, the viscosity of the formulation is about the same as the viscosity of a saline solution containing the same concentration of an agent (or a pharmaceutically acceptable salt thereof). In some embodiments, the formulation is substantially free of gel-forming polymers. In certain embodiments, where the carrier is water, the formulation may additionally include one or more chelating agents (e.g., EDTA disodium (EDTA), one or more preservatives (e.g., benzalkonium chloride, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, phenylethyl alcohol, propylparaben, thimerosal, phenylmercuric nitrate, phenylmercuric borate, phenylmercuric acetate, or combinations of two or more of the foregoing), salt (e.g., NaCI) and one or more buffering agents (e.g., one or more phosphate buffers (e.g., dibasic sodium phosphate, monobasic sodium phosphate, combinations thereof, etc.), citrate buffers, maleate buffers, borate buffers, and combination of two or more of the foregoing.).

In particular embodiments, the chelating agent is EDTA disodium, the preservative is benzalkonium chloride, the salt is NaCI, and the buffering agents are dibasic sodium phosphate and monobasic sodium phosphate. In certain of these embodiments, the formulation is substantially free of polymer. In some embodiments, the formulation is substantially free of substantially viscosity- increasing agent(s) (e.g., carboxymethylcellulose, polyanionic polymers, etc.). In some embodiments, the viscosity of the formulation is about the same as the viscosity of a saline solution containing the same concentration of an agent (or a pharmaceutically acceptable salt thereof). In some of these embodiments, the concentration of an agent (or a pharmaceutically acceptable salt thereof) if from about 0.02% to about 3%, from about 0.02% to about 2%, from about 0.02% to about 1% (w/v). In certain embodiments, the concentration of an agent (or a pharmaceutically acceptable salt thereof), is about 0.01%, about 0.02%, about 0.03%, about 0.05%, about 0.07%, about 0.1%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.8% or about 1% (w/v).

In certain embodiments, where the carrier includes water, a viscosity-increasing agent may also be included in the formulation. The skilled artisan will be familiar with viscosity-increasing agents that are suitable (e.g., water-soluble cellulose derivatives (e.g., hypromellose (also known as HPMC, hydroxypropylmethyl cellulose, and hydroxypropylcellulose), hydroxyethylcellulose, carboxmethylcellulose, etc.), polyvinyl alcohol, polyvinyl pyrrolidone, chondroitin sulfate, hyaluronic acid, and soluble starches. It is intended that when viscosity-increasing agents are used, they are not included in high enough concentrations such that the formulation would form a gel prior to or after administration (e.g., wherein the concentration of the viscosity-increasing agent is not sufficient to induce gel formation). While exact concentrations of viscosity-increasing agents will depend upon the selection and concentration of other components in the formulation as well as the particular viscosity-increasing agent(s) selected, in general, viscosity-increasing agents may be present in a concentration such that the viscosity of the resulting solution is less than about 1000 centipoise. In certain embodiments, the viscosity of the formulation is less than about 900, less than about 800, less than about 700, less than about 600, less than about 500, less than about 400, less than about 300, less than about 200, less than about 150, less than about 100, less than about 50 centipoise. In some embodiments, the viscosity of the formulation is about 200, about 150, about 100, about 50 centipoise. In particular embodiments, the viscosity is less than about 200 centipoise. In some, less than about 120 centipoise or less than about 100 centipoise. In some embodiments, the viscosity is about 100 centipoise. In certain aspects, about 50 centipoise. In some embodiments the viscosity is about 200 centipoise. Methods for measuring viscosity are well known to the skilled artisan. For example, as described in United States Pharmacopoeia 29 (Chapter 911) Viscosity, page 2785 (which is herein incorporated by reference in its entirety). As is well known to the skilled artisan, formulations commonly considered "gels" will have viscosity significantly greater than 1000 centipoise, for example, greater than about 2000 centipoise, greater than about 5000 centipoise.

In some embodiments, including (but not limited to) where the use of salts is

contraindicated as described above, the formulation may further include one or more tonicity agents. As used herein, the term "tonicity agent" and its cognates refers to agents that adjust the tonicity of the formulation, but are not salts (e.g., not NaCI), which, as will be appreciated by the skill artisan in view of the teaching provided herein, are contraindicated for some formulations due to the presence of certain of the gel-forming polymers or viscosity-increasing agents. These agents may be used to prepare formulations that are isotonic or near isotonic (e.g., somewhat hyper- or hypo- isotonic; e.g., within about ±20%, about ±15%, about ±10%, about ±5% of being isotonic). Tonicity agent(s) may also be used in formulations where the use of salts is not contraindicated.

Tonicity agents that may be used to adjust the tonicity of formulation the formulations described herein and are known to the skilled artisan and can be selected based on the teaching provided herein. For example, tonicity agents include polyols (e.g., sugar alcohols (e.g., mannitol, etc.), trihydroxy alcohols (e.g., glycerin, etc.), propylene glycol or polyethylene glycol, etc.), or combinations of two or more polyols. Likewise, the concentration of the tonicity agent(s) will depend upon the identity and concentrations of the other components in the formulation and can be readily determined by the skilled artisan in view of the teaching provided herein.

In certain embodiments, the tonicity agent is glycerin or mannitol. In some embodiments, the tonicity agent is glycerin. In some embodiments it is mannitol. In certain embodiments a combination of mannitoi and glycerin may be used. Exemplary concentrations of tonicity agents include, for example from about 0.001 to about 3%. In some embodiments, the concentration of the tonicity agent (e.g., mannitoi or glycerin) is, for example, about 0.001% to about 2.7%, about 0.001% to about 2.5%, about 0.001% to about 2%, about 0.001% to about 1.5%, about 0.001% to about 1%, about 0.01% to about 3%, about 0.01% to about 2.7%, about 0.01% to about 2.5%, about 0.01% to about 2%, about 0.01% to about 1.5%, about 0.01% to about 1%, about 0.1% to about 3%, about 0.1% to about 2.7%, about 0.1% to about 2.5%, about 0.1% to about 2%, about 0.1% to about 1.5%, about 0.1% to about 1%, about 0.01% about 1% to about 3%; about 1% to about 2.5%; about 1% to about 2%; about 1% to about 1.8%; about 1% to about 1.5%; or about 0.001%, about 0.01%, about 0.05%, about 0.08%, about 0.1%, about 0.2%, about 0.5%, about 0.8%, about 1%, about 1.5%, about 1.8%, about 2%, about 2.2%, about 2.5%, about 2.8%, or about 3% (w/v). In certain embodiments, the tonicity agent is mannitoi. In some of these embodiments, the carrier includes a gel-forming agent (e.g., gellan gum).

In some embodiments, the tonicity agent is mannitoi. In certain of these embodiments, the carrier includes a viscosity-increasing agent (e.g., water soluble cellulose derivatives (e.g., hypromellose), polyvinyl alcohol, polyvinyl pyrrolidone, chondroitin sulfate, hyaluronic acid, or soluble starches).

In some embodiments, the formulation may additionally include a preservative (e.g., benzalkonium chloride, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, Phenylethyl alcohol, propylparaben, thimerosal, phenylmercuric nitrate, phenylmercuric borate, or phenylmercuric acetate, peroxides), or a combination of two or more of the foregoing preservatives. In certain embodiments, the preservative is benzalkonium chloride.

As will be appreciated by the skilled artisan, preservatives may be present in concentrations of from about 0.001% to about 0.7% (w/v). In particular embodiments, the preservative(s) may be present in a concentration of from about 0.001% to about 0.5% (w/v); from about 0.001% to about 0.05% (w/v), from about 0.001% to about 0.02% (w/v), from about 0.001% to about 0.015% (w/v), from about 0.001% to about 0.005% (w/v), from about 0.01% to about 0.02%, from about 0.002% to about 0.01%, from about 0.015% to about 0.05%, less than about <0.5%, from about 0.005% to about 0.01%, from about 0.001% to about 0.15%, from about 0.002% to about 0.004%, from about 0.001% to about 0.002%. In some embodiments the concentration of the preservative may be, for example, about 0.001%, about 0.005%, about 0.01%, about 0.02%, about 0.03%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, or about 0.7% (w/v). Typical concentrations (w/v) for various commonly used preservatives are listed in Table C below. Table C

Preservative Approximate Concentration

Range (w/v)

Benzalkoniu m chloride 0.01-0.02%

Benzethonium chloride 0.01-0.02%

Chlorhexidine 0.002-0.01%

Chlorobutanol <0.5%

Methylparaben 0.015-0.05%

Phenylethyl alcohol <0.5%

Propylparaben 0.005-0.01%

Thimerosal 0.001-0.15%

Phenylmercuric nitrate 0.002-0.004%

Phenylmercuric borate 0.002-0.004

Phenylmercuric acetate 0.001-0.002

In certain embodiments, the formulation may additionally include a surfactant, or combinations of two or more surfactants. In particular embodiments, the formulation is substantially free of surfactant. As used herein, the term "substantially free" is intended to refer to levels of a particular component that are undetectable using routine detection methods and protocols known to the skilled artisan. For example, HPLC (including chiral HPLC, chiral HPLC/MS, LC/MS/MS etc.), thin layer chromatography, mass spectrometry, polarimetry measu rements, gas-chromatography-mass spectrometry, or others.

In particular embodiments, the formulation may further include a chelating agent (e.g., EDTA disodium (EDTA) (e.g., EDTA disodium (dihydrate), etc.) citrates, etc.). In some embodiments, a combination of chelating agents may be present. As will be appreciated by those of skill in the field, chelating agents can be used to hinder degradation of the formulation components and thereby increase the shelf life of formulations. As will be appreciated by the skilled artisan, use of EDTA in combination with gellan gum formulation may be contraindicated as the EDTA can cause gel formation prior to administration of the gellan gum formulation.

Typical concentrations for chelating agents are from about 0.005% to 0.1% (w/v). For example, from about 0.005% to about 0.09%, from about 0.005% to about 0.08%, from about 0.005% to about 07%, from about 0.005%, to about 0.06%, from about 0.005% to about 0.05%, from about 0.005 to about 0.04%, from about 0.005% to about 0.03%, from about 0.01% to about 0.1%, from about 0.01% to about 0.09%, from about 0.01% to about 0.08%, from about 0.01% to about 0.07%, from about 0.01% to about 0.06%, from about 0.01% to about 0.05%, from about 0.01% to about 0.04%, etc. In certain embodiments, the concentration of chelating agent(s) is about 0.005%, about 0.01%, about 0.02%, about 0.03%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1%. In particular embodiments, the chelating agent is EDTA disodium. In certain embodiments, the chelating agent is EDTA disodium (dihydrate). In some of these embodiments, the EDTA disodium dihydrate is present at a concentration of about 0.01% (w/v).

In some embodiments, the formulation may additionally include one or more buffering agents (e.g., phosphate buffer(s) (e.g., sodium phosphate buffers (e.g., dibasic sodium phosphate, monobasic sodium phosphate, etc.), citrate buffers, maleate buffers, borate buffers, etc.). As will be appreciated by the skilled artisan, the one or more buffering agent(s) should be selected in combination with the other components of a given formulation to achieve a pH suitable for use (e.g., pH of about 4.5 to about 8).

In certain embodiments, the buffering agent is a phosphate buffer or combination of two or more phosphate buffers. In certain embodiments, the buffering agents are dibasic sodium phosphate and monobasic sodium phosphate.

Typical concentrations for buffering agent(s) for example, phosphate buffering agent(s) may be from about 0.005 molar to 0.1 molar. In some embodiments, the buffering agent(s) may be at a concentration of about 0.01 to about 0.1, from about 0.01 to about 0.08, from about 0.01 to about 0.05, from about 0.01 to about 0.04, from about 0.02 to about 0.1, from about 0.02 to about 0.08, from about 0.02 to about 0.06, from about 0.02 to about 0.05, from about 0.02 to about 0.04 molar, etc. In particular embodiments, there are two buffering agents. Exemplary buffering agents include a combination of dibasic sodium phosphate (e.g., dibasic sodium phosphate.7H20) and monobasic sodium phosphate (e.g., monobasic sodium phosphate anhydrous). In some embodiments, the concentration of the buffering agent(s) is about 0.005 molar, about 0.01 molar, about 0.02 molar, about 0.03 molar, about 0.04 molar, about 0.05 molar, about 0.06 molar, about 0.07 molar, or about 0.1 molar.

An additional aspect of the invention includes use of the formulations as described herein in the manufacture of a medicament. Particularly, the manufacture of a medicament for use in the treatment and/or prevention of conditions as described herein. Further, the formulations, variously described herein, are also intended for use in the manufacture of a medicament for use in treatment and/or prevention of the conditions and, in accordance with the methods, described herein, unless otherwise noted.

Methods of formulation are well known in the art and are disclosed, for example, in

Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th Edition (1995). The compositions and agents provided herein may be administered according to the methods of the present invention in any therapeutically effective dosing regimen. The dosage amount and frequency are selected to create an effective level of the agent without harmful effects. The effective amount of a compound of the present invention will depend on the route of administration, the type of warm-blooded animal being treated, and the physical characteristics of the specific warm-blooded animal under consideration. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical arts. This amount and the method of administration can be tailored to achieve optimal efficacy but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.

In certain embodiments, the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering genes, polynucleotides, and peptide compositions directly to the lungs via nasal aerosol sprays have been described e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871, specifically incorporated herein by reference in its entirety) are also well-known in the

pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).

In certain embodiments, the delivery may occur by use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, for the introduction of the compositions of the present invention into suitable host cells. In particular, the compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, a nanoparticle or the like. The formulation and use of such delivery vehicles can be carried out using known and conventional techniques.

In certain embodiments, the agents provided herein may be attached to a pharmaceutically acceptable solid substrate, including biocompatible and biodegradable substrates such as polymers and matrices. Examples of such solid substrates include, without limitation, polyesters, hydrogels (for example, poly(2 hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and y-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as poly(lactic-co~glycolic acid) (PLGA) and the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), poly-D-(-)-3-hydroxybutyric acid, collagen, metal, hydroxyapatite, bioglass, aluminate, bioceramic materials, and purified proteins.

In some embodiments, the solid substrate comprises Atrigel™ (QLT, Inc., Vancouver, B.C.). The Atrigel^® drug delivery system consists of biodegradable polymers dissolved in biocompatible carriers. Pharmaceuticals may be blended into this liquid delivery system at the time of manufacturing or, depending upon the product, may be added later by the physician at the time of use. When the liquid product is injected into the subcutaneous space through a small gauge needle or placed into accessible tissue sites through a cannula, water in the tissue fluids causes the polymer to precipitate and trap the drug in a solid implant. The drug encapsulated within the implant is then released in a controlled manner as the polymer matrix biodegrades with time.

For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. The progress of these and other therapies (e.g., ex vivo therapies) can be readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art.

It will be further appreciated that for sustained delivery devices and compositions the total dose of agent(s) contained in such delivery system will be correspondingly larger depending upon the release profile of the sustained release system. Thus, a sustained release composition or device that is intended to deliver an agent (e.g., TyrRS polypeptide, resveratrol compound) over a period of 5 days will typically comprise at least about 5 to 10 times the daily dose of the agent; a sustained release composition or device that is intended to deliver an agent over a period of 365 days will typically comprise at least about 400 to 800 times the daily dose of the agent (depending upon the stability and bioavailability of the agent(s) when administered using the sustained release system).

In certain embodiments, a composition or agent is administered intravenously, e.g., by infusion over a period of time of about, e.g., 10 minutes to 90 minutes. In some embodiments, a composition or agent is administered by continuous infusion, e.g., at a dosage of between about 0.1 to about 10 mg/kg/hr over a time period. While the time period can vary, in certain embodiments the time period may be between about 10 minutes to about 24 hours or between about 10 minutes to about three days.

In some embodiments, an effective amount or therapeutically effective amount is an amount sufficient to maintain a concentration of the agent(s) in the blood plasma of a subject above about 300 M, above about 1 nM, above about 10 nM, above about 100 nM, or above about 1000 nM.

In certain embodiments, an IV or SC dosage is an amount sufficient to achieve a blood plasma concentration (Cmax) of between about 1,000 nM to about 5,000 nM or between about 200 nM to about 1,000 nM, or about 20 nM to about 200 nM.

Certain embodiments provide kits comprising one or more containers filled with one or more of the TyrRS, polypeptides, TyrRS polynucleotides, and/or resveratrol compounds, compositions thereof, etc., of the invention, as described herein. The kits can include written instructions on how to use such compositions (e.g., to modulate cellular signaling, treat disease, etc.).

The kits herein may also include a one or more additional therapeutic agents or other components suitable or desired for the indication being treated, or for the desired diagnostic application. An additional therapeutic agent may be contained in a second container, if desired.

Examples of additional therapeutic agents include, but are not limited to anti-neoplastic agents, antiinflammatory agents, antibacterial agents, antiviral agents, angiogenic agents, etc.

The kits herein can also include one or more syringes or other components necessary or desired to facilitate an intended mode of delivery (e.g., stents, implantable depots, etc.).

EXAMPLES

Example 1

Resveratrol Binds to the Active Site of TyrRS

Crystal structures were prepared to assess the potential physical interactions between human TyrRS and resveratrol, which possesses a tyrosine-like phenolic ring.

Materials and Methods:

Co-crystal preparation. Briefly, mini-TyrRS was mixed with either 1 mM RSV or 2 mM Tyrosine and incubated at 4°C for 16h. Before setting up the crystallization trials, the protein samples were subjected to high-speed centrifugation (13000 rpm for 15 min) to remove precipitants and the clear soluble fraction was transferred to a new tube. The crystallization trials were performed using 2.1 M (NH4)2S04, 0.1 M NaH2P04/K2HP04 (pH 6-8), and 2% acetone at room temperature. Co-crystals with L-tyrosine were grown in 3 days (data collected at Stanford

Synchrotron Radiation Lightsource), and crystals with RSV were grown in about 3-4 months at around pH 7.

Data collection and analysis. X-ray data were collected at 2.1 A using an in-house x-ray diffraction facility (The Scripps Research Institute, La Jolla). Data were integrated and scaled using HKL2000. The electron density was refined by molecular replacement (CNS and CCP4/REFMAC suite and Coot) using the known structure of mini-TyrRS and structures were deposited to PDB (ID code 4Q93 and 4QBT).

Results and Discussion:

Crystallization of mini-TyrRS with RSV and, separately, with tyrosine yielded co-crystal structures (at 2.1 A) (see Figure 1C, Figures 6A-6B, and Figure 15). While the phenolic ring of RSV and of the tyrosine have the same disposition in the respective co-crystals, accommodation of the cis-conformation of the dihydroxy ring of RSV forces a local structural change near the linker to the C-domain (See Figure 1C, Figure 6A). An RSV-promoted conformational change in TyrRS may drive the predominant trans-RSV (in solution) into a cis conformation (see Figure 6C-6D).

Example 2

Functional Synergies Between Resveratrol and TyrRS

Experiments were then performed to evaluate the effects of resveratrol on the nuclear translocation and downstream activities of human TyrRS, particularly its effects on PARP-1 and related signaling pathways.

Materials and Methods:

Cell culture, transfection and resveratrol treatment. HeLa cells (from ATCC, mycoplasma free) were cultured in a humidified incubator with 5% C02 in DMEM medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Invitrogen) and lx penicillin/streptomycin. The cells were transfected with pcDNA6-TyrRS-V5 (wild type), or pcDNA3.1-ZZ-PARP-l using Lipofectamine LTX (Invitrogen). Resveratrol (SantaCruz Biotechnologies), at a series of concentrations from 0 to 50 μΜ, was used to treat HeLa cells 0-24 hour in experiments as described.

Because the effects of RSV are strongly influenced by the background level of PARP-1 activity, the experiments were done only after checking to verify that background PARP-1 activation was low. The criterion was to observe PARP-1 activation upon serum starvation, along with Hsp72 induction. When serum starvation induces PARP-1 activation with Hsp72 induction within 30-45 min (from the same batch of a split of HeLa cells), PARP-1 activation was consistently observed at lower concentrations of RSV (1 μΜ).

Cell culture medium was supplemented with Tyr-SA or Gly-SA (RNA-TEC, Coralville, IA) where mentioned. Silencer^® Select Pre-designed siRNAs against PARP-1 (siRNA ID#: sl099, 5'- GCAGCUUCAUAACCGAAGAtt-3' (SEQ ID NO: 14)), SIRT1 (siRNA ID#: s23770, 5'- GGCUUGAUGGUAAUCAGUAtt-3' (SEQ ID NO: 15)) and TyrRS (siRNA ID#: s443, 5'-

GGACUUUGCUGCUGAGGUUtt-3' (SEQ ID NO: 16)) were purchased from Invitrogen, Carlsbad, CA and transfected into HeLa cells using Lipofectamine RNAiMAX (Invitrogen). NAMPT, the rate-limiting enzyme in the NAD+ salvage pathway, was inhibited using STF-11880453 according to the manufacturer's protocol (SelleckChem). The effect of a concomitant increase in nicotinamide production with a reduction of NAD+ concentration (leading to SIRT1 inhibition) was monitored by immunoblotting with ct-AcK382-p53, an acetylation site known to be specifically targeted by SIRT11. ct-pSerl5-p53 monitoring indicated if enhanced acetylation preceded the phosphorylation event54. Total p53 was blotted with a-p53. Transactivation of p53 was determined by monitoring its known targets, such as p21, PUMA, 14-3-3, FOX03A, SESN2 and SIRT6. Activation of NRF2 was further confirmed by following HO-1 expression, using the cognate antibodies. Activation of AM PK (Thrl72- phosphorylation) mediated through AMP and Ca2+ influx25 was monitored using ot-pThrl72-AM PK. Activation of AMPK on its targets was further determined by both ot-pSer36-H2B and by the expression levels of NAMPT.

Cell viability assay. HeLa (1x106) cells were reverse-transfected with siRNAs and viability was monitored using RTCA iCELLigence System (ACEA Biosciences). HeLa cells were cultured in a humidified incubator with 5% C02 in DMEM mediu m (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Invitrogen) and lx penicillin/streptomycin..

Preparation of cell fractions. The protocol for cell fractionation was followed as described previously (Sajish et al., Nature Chemical Biology 8:547-554, 2012). Briefly, for whole cell lysate preparation, HeLa cells were dissolved in lxSDS-PAGE loading buffer containing 300 mM NaCl. For cytoplasmic fraction preparation, HeLa cells were suspended in 0.1 ml of swelling buffer for 6 min on ice, and then incubated with 0.1 ml of plasma membrane lysis buffer for 5 min on ice. The cells were immediately passed through a 21-G needle 10 times and centrifuged for 10 min at 3220g, 4°C. The supernatant was harvested, while the pellets were used for the preparation of nuclear fractions. For this purpose, the pellet (nuclei) was incubated for 30 min at 4°C with 0.2 ml nuclear extraction buffer. Whenever required, PARP-1 (AG14361) and PARG inhibitors (ADP-HPD, Millipore) were added to the cell lysis buffer to ensure unwanted PARylation and removal of PAR chains with PARG.

Antibodies. All antibodies were obtained from Cell Signaling Technology (Danvers, MA) unless and otherwise mentioned. a-ATM was from Abeam (Cambridge, MA) and GeneTex (San

Antonio, TX). a-PARP-1 and cx-poly(ADP-ribose) were from BD Biosciences (Franklin Lakes, NJ). S-p84 was from Sigma (St. Louis, MO). cx-pSer36-H2B from ECM Biosciences. Rabbit α-human TyrRS polyclonal antibodies were homemade. ct-Tip60 was from Millipore (Billerica, MA). ot-AcK16-H4 was from Active Motif (Carlsbad, CA) and ct-Hsp72 (cat # SPA-812) was from assay designs (Ann Arbor, Ml).

Immuno-precipitation and Ni-NTA pull-down assays. Protocol for immuno-precipitation was followed as described previously (Sajish et al., supra). Briefly, the supernatants were pre-cleared by incubation with protein G beads. The pre-cleared cell lysates were incubated at 4°C for 1 h with either a-PARP-1, a-ATM, a-DNA-PKcs, a-TyrRS, a-Tip60 or non-immune IgG, at a concentration of 5 g/vn\ followed by incubation with 30 μΙ Protein G-beads (pretreated with 10 mg/ml BSA) at 4°C for lh with rotation. PARP-1 (AG14361) and PARG inhibitors (ADP-HPD, Millipore) were added to the cell lysis buffer to ensure unwanted PARylation and removal of PAR chains with PARG. Immuno- precipitates were washed three times, subjected to SDS-PAGE and immunoblotted with specific antibodies. Whenever mentioned, the ZZ domain allowed immuno-precipitation of ectopically expressed ZZ-PARP-1, using anti-lgG. Whenever Ni-NTA pull-down was performed, proteins with a 6X-His tag were overexpressed in E. coli. Cells were lysed and the supernatant fractions containing the soluble proteins were mixed with HeLa cell lysates. For Ni-IMTA pull-downs, normal procedures for immunoprecipitation were followed with 15-20 mM imidazole in the washing buffer.

Animal experiments. All experiments were approved by the Scripps Research Institute,

IACUC (protocol number 13-0002) and conducted by the mice facility at The Scripps Research Institute. BALB/cByJ mice were purchased from Jackson Laboratories. Six week old male mice were kept with a 12 h light-dark cycle with free access to food and water for 3 days before conducting the experiment.

For all studies, mice were dosed once and collected sample after either at 30 minutes or at

24 hours. Briefly, activation of PARP-1 in mouse tissues treated with resveratrol was performed by the intravenous (IV) injection of a 100 μΙ sample of resveratrol (10 μΜ) in PBS into 8 mouse tails (0.012 mg/kg). A 100 μΙ PBS injection was used as a control in 6 mice. Tissue samples were collected from 4 resveratrol treated mice and 3 control mice after 30 min treatment. Tissue samples from the remaining mice were collected after 24h.

In a different experiment, blocking of resveratrol mediated activation of PARP-1 by Tyr-SA was analyzed with 5 groups of two mice each, a 100 μΙ sample of resveratrol (10 μΜ ) in PBS alone and, separately, with Tyr-SA (5 μΜ), Gly-SA (5 μΜ), cycloheximide (5 μΜ) or the AG14361 PARP-1 inhibitor (10 μΜ) was IV-injected. A 100 μΙ PBS injection with Tyr-SA was used in a control group (2 mice) as indicated. Tissue samples were collected after 30 min treatment. Muscle or heart tissues samples were homogenized and analyzed by immunoblotting for the status of poly-ADP-ribosylation and associated signaling events.

Recombinant protein purification. Human TyrRS and PARP-1 and their variants were purified as previously described (see Yang et al., PNAS USA. 99:15369-15374, 2002; and Sajish et al., supra). Briefly, DNA encoding either full-length human TyrRS (aa 1-528), mini-TyrRS (aa 1-341), dN- TyrRS (aa 237-528), or CT-TyrRS (aa 324-528) was cloned into Nde-l/Hind-lll sites of pET-20b vector (Novagen, Gibstown, NJ). The expressed proteins included a 6-His tag from the vector sequence.

The full-length PARP-1 and its variants (N-terminal domain (NTD) and C-terminal domain (CTD)) were cloned into the pET 20b vector. All proteins having a C-terminal His-tag were expressed in E. coli strain BL21 (DE3) by induction for 4h with 1 mM isopropyl β-D-thiogalactopyranoside. Proteins were purified from the supernatants of lysed cells using Ni-NTA agarose (Qiagen,

Chatsworth, CA) column chromatography according to the manufacturer's instruction. We also included an additional high salt 2-column wash (1-1.5 M NaCI), to remove the endogenous DNA/RNA associated with the purified protein. The NaCI concentration was increased to 1.5M in increments and returned to final elution buffer having 250 m NaCI in decrements. All the proteins were subjected to a gel-filtration (S-200) chromatography and collected the protein peak corresponding to homogenous protein. As endotoxin associated with purified protein interfered with the inhibitory/activating effect of RSV on TyrRS/PARP-1, the complete protein purification included an additional endotoxin removal step. The purified protein was further passed through a column containing Detoxi-GelTM (Pierce-Thermo Scientific, IL, USA) and followed the manufactures instructions. The final endotoxin level (0.5 EU/mg/ml) was measured using The EndosafeR-PTS kit (Charles River Laboratories, Charleston, SC). The quality of each protein purification was validated by SDS-PAGE analysis.

ATP-PPi exchange assay. Tyrosyl adenylate synthesis was measured by using the tyrosine- dependent ATP-pyrophosphate (PPi) exchange assay. A mixture containing 100 mM Hepes (pH 7.5), 20 mM KCI, 2 mM ATP, 1 mM NaPPi, 2 mM DTT, 250-500 μΜ L-tyrosine, 10 mM MgCI2, and =0.01 mCi/ml Na[³²P]PPi was added to 50-100 nM purified TyrRS (endotoxin free, 0.5EU/mg/ml), pre- incubated with 0-lmM RSV at 4°C for 30 min. The ATP-PPi exchange reaction was incubated at room temperature, and aliquots were removed at specified time intervals and quenched in a mixture containing 40 mM NaPPi, 1.4% HCI04, 0.4% HCI, and 8% (wt/vol) of activated charcoal. After thoroughly mixing, the charcoal was filtered and washed with a solution of 7% HCI04 and 200 mM NaPPi using Spin-X Centrifuge Filters (Corning, Corning, NY) containing 0.45-μηι pore-size cellulose acetate filters. After drying, the charcoal was punched into scintillation vials and the radioactivity of the ATP bound to the charcoal mixture was measured by scintillation counting.

In vitro PARylation assay. The protocol followed as described previously (see Sajish et al., supra). Briefly, 5 μΙ recombinant PARP-1 (2 μg ) was mixed with 5 μΙ recombinant TyrRS (0-50 μΜ) and depending on the experiment either RSV, TyrSA, DNA or AG14361 were also added (5 μΙ) and adjusted the volume to 25 μΙ. This mixture was incubated at 4°C for 15 min, then incubated with 25 μΙ 2x PAR assay cocktail (50 mM HEPES, pH 7.5, 100 mM KCI, 10 mM MgCI2, 0.2 mM EGTA, 0.1 mM EDTA, 40 nM NAD and 1 μϋ [³²P]-NAD (Perkin-Elmer, Boston, MA)) at 30°C for 30 min. The RSV concentrations used in Figure 2A (middle) was 0, 5, 10, 20, 30, 40, 60 and 80 nM and in Figure 2A (bottom) was 40 nM. The reaction was stopped by addition of 50μΙ 2x SDS sample buffer (5% SDS,) and heated at 95"C for 1 min. The [³²P]-PARylated PARP-1 was separated from free [³²P]-NAD on SDS- PAGE, vacuum-dried and the poly-ADP-ribosylation activity of PARP-1 was analyzed using visualized by a phosphorimager (TyphoonTM FLA 7000, GE Healthcare).

Construction of a working model. Because both RSV and serum starvation activate PARP-1 leading to the metabolic break-down of NAD+ into nicotinamide and ADP-ribose, downstream signaling markers associated with the NAD+ metabolic flux were investigated. Targets were selected if they were activated and/or inhibited under conditions of RSV-treatment, stress or NAD+ metabolic byproduct-treatment (nicotinamide and ADP-ribose). For example, nicotinamide activates Tip60 through SIRT1 inhibition and Tip60 activates ATM, the p53 kinase. p53 is a master regulator of NRF2 through p21 induction and inducible expression of survival genes like SIRT6, SIRT1, Sestrins, and FOX03A. (SIRT6 promotes DNA repair under stress by activating PARP-1 and SIRT1 down-regulates the over-activation of PARP-1). Similarly, ADP-ribose (metabolic byproduct of NAD+ and an inhibitor of SIRT1) is a potent Ca2+ channel opener and a substrate for AMP production and hence an indispensable component for AMPK activation by resveratrol and the stress response by phosphorylating H2B. AMPK is known to up-regulate NAMPT expression, the major regulator of NAD+ levels in the cell. NAD+ is major substrate for sirtuins (deacetylation or mono-ADP- ribosylation) and PARPs ((mono/poly)-ADP-ribosylation). Although not depicted, p38 and NF-kB (major transducers of stress signaling) are also regulated by TAK1 in a PARP-l/ATM dependent manner.

Results and Discussion:

In these studies, a distinct TyrRS-PARP-1 interaction was observed. PARP-1 is a major modulator of NAD+ metabolism and its related signaling (Luo et al., Genes & development 26:417- 432, 2012). Because RSV acts through NAD+-dependent proteins (Sinclair et al., Annual review of pharmacology and toxicology 54:363-380, 2014), the TyrRS-PARP-1 interaction was further studied. Given that RSV treatment elicits a stress response (Viswanathan et al., Dev Cell 9:605-615, 2005), serum starvation (SS) was used to mimic a general 'stand-alone' stress condition so that common signaling pathways, if any, between RSV treatment and a general stress condition, could be compared ex-vivo. Both serum starvation and RSV treatment promoted nuclear translocation of endogenous TyrRS in HeLa cells (see Figure IB). Translocation was observed under different stress conditions (heat shock and ER stress, Figure 8A), suggesting that TyrRS is a general stress transducer. Nuclear translocation of endogenous TyrRS was concomitant with strong auto-PARylation of PARP-1 (PARP-1PAR) (see Figure IB and Figure 8A).

Ex-vivo RSV also strongly promoted association of TyrRS with PARP-1, and robust auto-poly- ADP-ribosylation of PARP-1 (Figure 8B). Effects of RSV were blocked by a Tyr-AMP analog (Tyr-SA (5'- 0-[N-(9L-tyrosyl) sulfamoyl] adenosine)), but not by Gly-SA (a control targeting GlyRS) (Figure 8B- 8C). Similar, but less pronounced, PARylation was seen with serum starvation. Enhanced PARylation correlated with increased amounts of TyrRS in the nucleus, which occurred upon serum starvation. Thus, both serum starvation and RSV promoted nuclear translocation of TyrRS and activation of PARP-1. Cell lysates treated with the PARG hydrolyase and its hydrolase-inactive mutant su pported that TyrRS preferentially bound to non-PARylated PARP-1 (Figure 8D-8E). TyrRS interacted specifically with the C-domain of PARP-1 (CT-PARP-1) (Figure 8F). In pull-down assays, PARP-1 bound to full-length TyrRS but not mini-TyrRS or the TyrRS C-domain (Figure 8G-8H). In the absence of RSV, concentration-dependent activation of PARP-1 by TyrRS was observed in vitro (See Figure 2A (top) and Figures 9A-9B). RSV enhanced in-vitro auto-PARylation with the half-maximal effect at roughly 10 nM (Figure 2A, middle), well below the Ki (about 22 μΜ). Thus, PARP-1 may alter the apparent affinity of RSV for TyrRS. Also, concentration-dependent quenching of PARylation of PARP-1 by Tyr- SA was evident (Figure 2A, bottom). Lastly, while broken DNA normally activates PARP-113, Tyr-SA did not interfere with this DNA-dependent-pathway of PARP-1 activation in vitro (Figure 9C).

Therefore, the TyrRS-RSV activation of PARP-1 is distinct from the DNA-dependent pathway.

Ectopically expressed TyrRS in HeLa cells for 0-24 hou rs caused a progressive increase in cellular concentrations of the synthetase (Figure 2B, top) and a correlated progressive increase in PARP-1PAR (Figure 2B, top). A TyrRS mutant (TyrRS-dNLS), with reduced nuclear localizations, reduced activation of PARP-1 (Figure 9D). Effects of RSV ex-vivo at various concentrations (Figure 2B, middle) and of Tyr-SA (Figure 2B, bottom) mirrored those seen in vitro (Figure 2A, middle and bottom).

RSV and L-tyrosine produce different bound conformations of TyrRS, seen locally in the high- resolution structures of the two co-crystals (Figure 1C and Figure 6A). These differences could affect the disposition of the C-domain needed for the interaction of TyrRS with PARP-1. This domain is tethered to the mini-TyrRS catalytic unit by a flexible linker (Figure 2C, left). By breaking the Y341 OH--H-bond to a backbone carbonyl oxygen, the Y341A mutation releases tight tethering of the C- domain to the catalytic domainl5 and its reorientationl6 (Figure 2C, right).

In the absence of RSV, Y341A-TyrRS showed robust interaction with and activation of PARP-1 in vitro (Figure 2D). If Y341A is shifted more towards a conformation needed to bind to PARP-1, then it would be more sensitive than WT TyrRS to low concentrations of RSV. This expectation was fulfilled (Figure 9E). TyrRS-Tyr may thus have a conformation that prevents interaction with PARP-1, while TyrRS-RSV has a distinct conformation that binds PARP-1. In the absence of either ligand, a dynamic equilibrium populates both forms (Figure 2C).

A working model of downstream signaling markers was developed as detailed above (Figure

3A). Effects from serum starvation and RSV administration involve cascades through protein acetylation and phosphorylation effects of serum starvation and of RSV, which have not been linked to TyrRS nor activation of PARP-1. Under conditions of serum starvation or RSV administration, the response in HeLa cells of PARylated and acetylated proteins, together with the aforementioned cell- signaling proteins, was monitored for up to 1 hour. Either RSV or serum-starvation promoted production of AcK-Tip60 and AcK-ATM (Figure 3B). In a temporal response from 0 to 8 hours, either or both of serum starvation or 1 μΜ RSV promoted increased levels of whole cell acetylated proteins, Hsp72, AcK382-p53, p-AMPK, pSer36-H2B, p21, FOX03A, 14-3-3, HO-1, NAMPT, SIRT1 and SIRT6 (Figures 3C-3D). Also, RSV activated acetylation of Tip60 ex-vivo in a concentration-dependent manner (Figure 10A). As expected from previous reports that acetylation of p53 is indispensable for its activation, a transient increase in acetylation of p53 (AcK382) was evident (Figures 3C-3D), possibly through a transient inhibition of SIRT1 by the nicotinamide being produced. Consistently, at 15 minutes, a RSV concentration-dependent reduction in total NAD+ with concomitant production of nicotinamide and ADP-ribose was observed (Figures lOB-lOC). Thus, serum starvation and RSV rapidly increased all monitored proteins in a way that is dependent on having active PARylation. This rapid increase is consistent with work showing rapid up-regulation of PARP-1 target genes, possibly due to a broader effect of PARP-1 on transcription and associated stress signaling.

At longer times, and continuing at low concentrations (< 10 μΜ) of RSV, similarity between responses to RSV and serum starvation was ostensibly seen. By 1 hour, low concentrations of RSV elevated the NAD+ levels similar to those of serum-starvation conditions (Figure 10D). Responses of key stress-signaling markers in a more extended period of 8-24 hours at 5 μΜ RSV were mostly similar to those of serum starvation (Figures 10E-10F). At 5 μΜ RSV, levels of PARP-1PAR were sustained longer when compared with what was observed at 1 μΜ RSV (compare the 2 and 8 hour time points in Figure 3D with Figure 10F) and thus further studies were continued at 5 μΜ RSV.

An siRNA directed against PARP-1 (siRNA^{PARP _1}) effectively abrogated the RSV-stimulated expression of Hsp72, p-AMPK, SIRT1, FOX03A, SESN2, NAMPT, PUMA and SIRT6 (Figure 4A). Also, 5 μΜ RSV promoted induction of BRAC1 and pl4ARF (whose genes are directly regulated by PARP-1). Consistently, the RSV-stimulated induction of BRCA1 and pl4ARF were prevented by siRNA^PARP"1 (Figure 4A), further showing involvement of PARP-1 in RSV-mediated induction of these proteins.

In Figure 4A, RSV treatment led to enhanced activation of p53. However, in the absence of RSV, knockdown of PARP-1 resulted in a background increase in the products of these p53-regulated genes. Possibly p53 was activated by the knockdown of PARP-1, because removal of PARP-1 would enhance background levels of DNA damage. Indeed, activation of p53, as seen by enhanced acetylation and phosphorylation of p53, was observed (Figure 4A).

siRNA^TyrRS-knockdown at 5 μΜ RSV eliminated induction of, and dramatically reduced amounts of, PARylated PARP-1, of whole acetylated proteins, and of AcK382-p53, AcK16-H4 (Tip60 activation), pAMPK, and pSer36-H2B (AMPK activation) (Figure 4B). (Separately, RSV (5 μΜ) addition did not affect the viability of HeLa cells expressing either siRNA^TyrRS or siRNA^{PARP 1} (Figure 11). In contrast, siRNA -knockdown did not affect RSV (5 μM)-mediated production of whole acetylated proteins or, among other downstream markers, activation of PARP-1 and induction of SIRT6, FOX03A, NAMPT, AcK16-H4, p-AMPK24, and pSer36-H2B (Figure 12). Collectively, Figures 4A-4B showed that ex-vivo TyrRS and PARP-1 collaborate to activate RSV- and serum-starvation responses.

Although the RSV-TyrRS-PARP-1 axis initially consumes NAD+, at low RSV (5 μΜ), NAD+ levels are transiently raised after 1 hour due to NAMPT activation (Figure 10D). At 1 hour at low RSV, induction of NAD+ levels was abolished with either siRNA^TyrRS or siRNA^{PARP 1} (Figure 13). NAD+ depletion by NAMPT inhibition abolished RSV-mediated induction of BRCA1, FOX03A, NAMPT, SESN2 and SIRT6 (Figure 4C). These results are consistent with those using inhibitors against PLC-yl and the ryanodine receptor, which inhibit PARP-1 activation and ADP-ribose-mediated calcium signaling, and which prevented RSV-mediated AMPK activation. Similarly, a PARP-1 inhibitor prevented LKB1 activation by RSV.

In the animal studies, mice were injected through tail vein-IV with 100 μΙ of 10 μΜ RSV and, after 30 min, were sacrificed. Compared to the PBS IV-injected control, PARylated and acetylated proteins, along with AcK16-H4 and pSer36-H2B, were significantly increased in skeletal muscle

(Figures 14A-14B). Results in cardiac tissue were similar (Figures 14C-14D). In addition, PARP-1 activation diminished back to normal by 24 hours (Figure 14E). Consistent with ex-vivo assays, tissue samples from RSV-injected mice showed higher levels of TyrRS-PARP-1 interaction together with increased auto-PARylation (Figure 14F).

Tyr-SA (5 μΜ) was added to the IV-injection with RSV. No interaction of TyrRS with PARP-1 could be detected in skeletal or cardiac muscle and levels of PARylated and acetylated proteins did not increase. In contrast, co-injection of Gly-SA or cycloheximide (CHX, protein synthesis inhibitor) did not block RSV/TyrRS-mediated activation of PARP-1 and its downstream signals (Figure 4D

(muscle), Figures 14G-14H (cardiac)). Consistent with ex-vivo assays, immuno-precipitation of PARP- 1 (from harvested muscle tissue) pulled-down TyrRS, and immuno-precipitation of p53 showed its increased acetylation. Pull-down of TyrRS and p53 acetylation were blocked in mice that had co- injections of Tyr-SA or the AG14361 (SelleckChem) PARP-1 inhibitor, but not of Gly-SA or cycloheximide (Figure 4E). These responses in vivo parallel ex-vivo assays, and support that TyrRS is a major effector target for RSV that acts through PARP-1 as described in Figure 3A.

The mechanism of action of RSV and of the stress response are both linked here to the activation of PARP-1 through TyrRS and NAD+. The interaction of RSV with TyrRS could be viewed as an example of xenohormesis through interactions of a natural ligand with a protein target. In this instance, the natural ligand blocks the active site to create a tRNA synthetase catalytic null with a new, orthogonal function. This kind of catalytic null is in contradistinction to those created by alternative splicing events that specifically remove the aminoacylation active site. The RSV-TyrRS activation of PARP-1 is readily observable in a functional in vitro assay even at sub-micromolar concentrations (e.g., 10-20 nM; see Figure 2A, middle). Thus, TyrRS-RSV-induced PARP-1 activation appears at significantly lower RSV concentrations than seen with RSV functional binding to other targets. As a consequence, the direct effects of RSV binding to these targets of RSV are layered over a pre-existing foundation that comes from the TyrRS-RSV-PARP-l-NAD+ connection.

These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

1. A method of increasing a stress response in a cell, comprising contacting the cell with (a) a tyrosyl-tRNA synthetase (TyrRS) polypeptide or a polynucleotide that encodes the TyrRS polypeptide, in combination with (b) at least one resveratrol compound.

2. The method of claim 1, where (a) is a TyrRS polypeptide that comprises any of SEQ ID NO:l-3 or a sequence having at least 90% identity to any of SEQ ID NOS:l-3, where the variant TyrRS polypeptide binds to the resveratrol compound and binds to Poly [ADP-ribose] polymerase 1 (PARP-1).

3. The method of claim 1 or 2, where the resveratrol compound is a compound of Formula l-A or l-B, or a pharmaceutically acceptable salt thereof.

4. The method of any of the preceding claims, where the combination of (a) and (b) synergistically increases the stress response in the cell relative to (a) alone or (b) alone.

5. The method of any of the preceding claims, where increasing the stress response comprises evoking mild stress in the cell.

6. The method of claim 5, where the combination of (a) and (b) synergistically evokes mild stress in the cell relative to (a) alone or (b) alone

7. The method of any of the preceding claims, where the stress response comprises increased activation of Poly [ADP-ribose] polymerase 1 (PARP-1).

8. The method of claim 7, where increased activation of PARP-1 comprises increased auto-PARylation of PARP-1.

9. The method of claim 7, where increased activation of PARP-1 comprises increased expression of one or more PARP-1 target genes and optionally associated downstream signaling events.

10. The method of claim 8, where the one or more PARP-1 target genes are selected from Hsp72, SIRT1, SIRT6, FOX03A, SESN2, NAMPT, PUMA, BRCA1, and pl4ARF, including combinations thereof.

11. The method of any of the preceding claims, where the cell is contacted with (a) and (b) simultaneously or sequentially.

12. The method of any of the preceding claims, where the cell is in a subject, and the method comprises administering (a) and (b) to the subject.

13. The method of claim 11, where (a) and (b) are administered simultaneously or sequentially.

14. The method of claim 12 or 13, where the subject has or is at risk for having physiological stress, optionally selected from oxidative stress, viral stress, radiation-induced stress, drug-induced stress, light-induced stress, and combinations thereof.

15. The method of any one of claims 12-14, where the subject has a disease selected from a cardiovascular disease, a neurological disease, a metabolic disease, and cancer.

16. The method of any one of claims 12-15, where the subject is elderly or middle-aged.

17. The method of any one of claims 12-16, where the subject is at least 40, 50, 60, 70, 80, 90, or 100 years old.

18. The method of any one of claims 12-17, where the subject has an aging-associated disease or condition.

19. The method of any one of claims 12-18, where the subject has atherosclerosis and/or hypertension.

20. The method of any one of claims 12-19, where administering (a) and (b) reduces oxidative stress and/or inflammation in the subject.

21. The method of any one of claims 12-20, where administering (a) and (b) increases the life expectancy of the subject.

22. A method of treating physiological stress in a subject, comprising administering to the subject (a) a tyrosyl-t NA synthetase (TyrRS) polypeptide or a polynucleotide that encodes the TyrRS polypeptide, in combination with (b) at least one resveratrol compound.

23. The method of claim 22, where physiological stress is selected from oxidative stress, viral stress, radiation-induced stress, drug-induced stress, light-induced stress, and combinations thereof.

24. A method of treating a disease in a subject, where the disease is selected from a cardiovascular disease, a neurological disease, a metabolic disease, obesity, and a cancer, comprising administering to the subject (a) a tyrosyl-tRNA synthetase (TyrRS) polypeptide or a polynucleotide that encodes the TyrRS polypeptide, in combination with (b) at least one resveratrol compound.

25. The method of claim 24, where the cardiovascular disease is selected from coronary artery disease, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, cardiac dysrhythmias, inflammatory heart disease, valvular heart disease, cerebrovascular disease, peripheral arterial disease, congenital heart disease, and rheumatic heart disease.

26. The method of claim 24, where the neurological disease is selected from neuro- inflammation, tumorigenesis of the brain, brain ischemia, neuropathy, and neurodegeneration associated with aging, and a neurodegenerative disease.

27. The method of claim 24, where the metabolic disease is impaired insulin sensitivity and/or glucose utilization.

28. The method of claim 24, where the metabolic disease is selected from Type 1 diabetes, Type 2 diabetes, pre-diabetes, hyperglycemia, hyperinsulinaemia, and metabolic syndrome.

29. The method of claim 24, where the subject is obese.

30. The method of claim 29, where the obese subject has one or more of a

cardiovascular disease, a metabolic disease, obstructive sleep apnea, a cancer, or osteoarthritis, and where administering (a) and (b) reduces the symptoms or pathology of one or more of the foregoing.

31. The method of claim 29, where the obese subject has increased risk of developing one or more of a cardiovascular disease, a metabolic disease, obstructive sleep apnea, a cancer, or osteoarthritis, and where administering (a) and (b) reduces the risk of developing one or more of the foregoing.

32. The method of claim 29, where administering (a) and (b) increases the life expectancy of the obese subject.

33. The method of claim 24, where the cancer is selected from one or more of a breast cancer, cervical cancer, prostate cancer, pancreatic cancer, gastrointestinal cancer, lung cancer, ovarian cancer, testicular cancer, head and neck cancer, bladder cancer, kidney cancer, soft tissue sarcoma, squamous cell carcinoma, CNS or brain cancer, melanoma, non-melanoma cancer, thyroid cancer, endometrial cancer, an epithelial tumor, bone cancer, and hematopoietic cancer.

34. A method of treating aging in a subject, comprising administering to the subject (a) a tyrosyl-tRNA synthetase (TyrRS) polypeptide or a polynucleotide that encodes the TyrRS polypeptide, in combination with (b) at least one resveratrol compound.

35. The method of any one of claims 22-34, where the subject is elderly or middle-aged.

36. The method of any one of claims 22-34, where the subject is at least 40, 50, 60, 70, 80, 90, or 100 years old.

37. The method of any of one of claims 22-36, where the subject has an aging- associated disease or condition.

38. The method of any one of claims 22-37, where the subject has atherosclerosis and/or hypertension.

39. The method of any one of claims 22-38, where the subject is obese.

40. The method of any one of claims 22-39, where administering (a) and (b) reduces oxidative stress and/or inflammation in the subject.

41. The method of any one of claims 22-40, where administering (a) and (b) increases the life expectancy of the subject.

42. A pharmaceutical composition, comprising (a) a tyrosyl-tRNA synthetase (TyrRS) polypeptide or a polynucleotide that encodes the TyrRS polypeptide; (b) at least one resveratrol compound; and (c) a pharmaceutical-grade carrier.

43. The pharmaceutical composition of claim 42, where the molar ratio of (a) to (b) is about 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5.

44. The pharmaceutical composition of claim 43, where the molar ratio of (a) to (b) is about 1:2.

45. The pharmaceutical composition of any one of claims 42-44, comprising at least about 5 mg/ml of the TyrRS polypeptide.

46. The pharmaceutical composition of claim 45, comprising at least about 8 mg/ml, at least about 10 mg/ml, at least about 15 mg/mL, or at least about 20 mg/ml of the TyrRS polypeptide.

47. The pharmaceutical composition of any of claims 42-44, comprising about 0.001 mg to about 20,000 mg of the TyrRS polypeptide.

48. The pharmaceutical composition of any one of claims 42-47, comprising about 0.01 mg to about 5000 mg of the resveratrol compound.