US 20120028335A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0028335 A1 AGNEW (43) Pub. Date: Feb. 2, 2012

(54) ANTI-VIRAL AZIDE-CONTAINING Publication Classification COMPOUNDS (51) Int. Cl. CI2N 7/00 (2006.01) (75) Inventor: Brian AGNEW, Eugene, OR (US) (S2) usic... 435/235.1 (73) Assignee: LIFE TECHNOLOGIES (57) ABSTRACT CORPORATION, Carlsbad, CA Methods of using azide-modified biomolecules, such as fatty (US) acids, carbohydrates and lipids, to treat a plant or an animal infected with a virus or to inhibit infectivity of a virus, such as (21)21) Appl.App No.: 12/888,3719 the human immunodeficiencyy virus, are pprovided. Also prop vided are methods of labeling a human immunodeficiency (22) Filed: Sep. 22, 2010 virus with an azide-modified biomolecule, such as a fatty 9 acid, a carbohydrate, or an isoprenoid lipid. The azide-modi Related U.S. Application Data fied biomolecules may be combined with a pharmaceutically acceptable excipient to produce a pharmaceutical composi (60) Provisional application No. 61/368,558, filed on Jul. tion, optionally containing another anti-viral agent and/or a 28, 2010. delivery agent, such as a liposome. Patent Application Publication Feb. 2, 2012 Sheet 1 of 4 US 2012/0028335 A1

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ANTI-VIRAL AZDE-CONTAINING As a result, when the analogs are incorporated into a growing COMPOUNDS viral DNA chain, the incoming deoxynucleotide cannot form a phosphodiester bond with the analog that is needed to BACKGROUND extend the DNA chain. Thus, the analogs terminate viral 0001 Viral infections account for significant morbidity DNA replication. Another class of reverse transcriptase and mortality in humans and animals. In addition, viral infec inhibitors is the non-nucleoside reverse transcriptase inhibi tions also result in significant agricultural losses, with plant tors, such as Efavirenz, Nevirapine, Delavirdine, and Etravir viruses causing an estimated S60 billion in lost crop yields ine (El Safadi et al., Appl Microbiol Biotechnol, 2007, throughout the world each year. Although significant 75:723–37). They have a different mode of action than the resources have been dedicated to identifying compounds hav nucleoside and nucleotide inhibitors, binding to the reverse ing anti-viral properties, viral infections continue to present a transcriptase and interfering with its function. significant risk to human health and agriculture. 0005. The late stages of HIV replication involve process 0002. In addition, the usefulness of most existing anti ing of certain viral proteins prior to the final assembly of new viral treatments is limited by the development of multidrug virions. This late-stage processing is dependent, in part, on resistance, poor efficacy, and/or toxicity. In fact, many anti the activity of a viral protease. Thus, another area of focus in viral treatments are highly toxic and can cause serious side the development of antiretroviral drugs is protease inhibitors, effects, including heart damage, kidney failure and Such as saquinavir, ritonavir, indinavir, nelfinavir, osteoporosis. Other challenges include creating a drug that is amprenavir, lopinavir, and atazanavir (Erickson, J., 1990, broadly applicable in combating many different types of viral Science 249:527-533; Klei et al., J Virol, 81:9525-35). infections, which can be particularly important in the treat 0006. Other antiretroviral drugs target viral entry into the ment of immunocompromised individuals. cell, the earliest stage of HIV infection. For HIV to enter a 0003. One virus in particular, the human immunodefi cell, its surface gp120 protein binds to CD4, exposing a ciency virus (HIV), remains a global pandemic despite the conserved region of gp120 that binds to a CCR5 or a CXCR4 development of antiretroviral drugs targeting HIV. As of co-receptor. After gp120 binds to the co-receptor, a hydro 2007, it was estimated that more than 33 million people were phobic fusion peptide at the N-terminus of the gp41 envelope infected with HIV, and HIV associated diseases represent a protein is exposed and inserted into the membrane of the cell. major world health problem. HIV is a retrovirus that infects Entry inhibitors workby interfering with any stage of the viral CD4 cells of the immune system, destroying or impairing entry process. For example, recombinant soluble CD4, for their function. As the infection progresses, the immune sys example, has been shown to inhibit infection of CD-4T-cells tem becomes weaker, leaving the infected person more Sus by some HIV-1 strains (Smith, D. H. et al., 1987, Science ceptible to opportunistic infections and tumors, such as Kapo 238:1704-1707). Similarly, TNX-355 is a monoclonal anti si's sarcoma, cervical cancer, lymphoma, and neurological body that binds CD4 and inhibits binding to gp120 (Kuritzkes disorders. The most advanced stage of HIV infection is et al., J Infect Dis, 2004, 189:286-91). BMS-806 binds to the acquired immunodeficiency syndrome (AIDS). It can take viral envelope protein and inhibits binding to CD4 (Veazy et 10-15 years for an HIV-infected person to develop AIDS. al., Nature 2003, 438:99-102). Co-receptor binding can be Certain antiretroviral drugs can delay the process even fur inhibited by several CCR5 inhibitors, including SCH-C and ther. SCH-D, UK-427,857, maraviroc, Vicriviroc, and an anti 0004 Although much effort has been put forth into CCR5 antibody (PRO-140) (Emmelkamp et al., EurJ Med designing effective therapeutics against HIV, currently no Res, 2007, 12:409-17). Co-receptor binding can also be inhib curative anti-retroviral drugs against HIV exist. Several ited by the CXCR4 inhibitors AMD3100 and AMD070 (De stages of the HIV life cycle have been evaluated as targets for Clerq, Nature Reviews Drug Discovery 2003, 2:581-87). the development of therapeutic agents (Mitsuya, H. et al., Other compounds, such as enfuvirtide, bind to gp41 and 1991, FASEBJ5:2369-2381). One area of focus has been the interfere with its ability to mediate membrane fusion and HIV reverse transcriptase enzyme. Reverse transcriptase cop entry (La Bonte et al., Nature Reviews Drug Discovery 2003, ies the HIV, single stranded RNA genome into double 2:345-36). stranded viral DNA. The viral DNA is then integrated into the 0007 While beneficial, these antiretroviral drugs often host’s chromosomal DNA where the host’s cellular pro exhibit toxic side effects Such as bone marrow Suppression, cesses, like transcription and translation, are used to produce Vomiting, and liver function abnormalities. In addition, they viral proteins and ultimately new virus particles. Therefore, are not curative, probably due to the rapid appearance of drug interfering with reverse transcriptase inhibits HIV’s ability to resistant HIV mutants (Lander, B. et al., 1989, Science 243: replicate. One class of reverse transcriptase inhibitors is 1731-1734). Drug-resistant HIV strains develop due to the nucleoside analogs, such as Zidovudine (AZT), Didanosine very high genetic variability of HIV. This genetic variability (ddI), Zalcitabine (ddC), and Stavudine (d4T), Lamivudine results from several factors, including HIV’s fast replication (3TC), Abacavir (ABC), Emtricitabine (FTC), Entecavir cycle, with the generation of 10 to 10" virions per day, a high (INN), and Apricitabine (ATC) (Mitsuya, H. et al., 1991, mutation rate of approximately 3x10 per nucleotide base Science 249:1533-1544; El Kouni, Curr Pharm Des, 2002, per cycle of replication, and recombinogenic properties of 8:581-93; Sharma et al., Cur Top Med Chem, 2004, 4:895 reverse transcriptase. 919). Another class of reverse transcriptase inhibitors is 0008 To combat the development of drug resistant HIV nucleotide analogs, such as Tenofovir (tenofovir disoproxil strains, multiple drugs have been combined as part of highly fumarate) and Adefovir (bis-POM PMPA) (Palmer et al., active antiretroviral therapy (HAART) (El Safadi et al., Appl AIDS Res Hum Retroviruses, 2001, 17:1167–73). These Microbiol Biotechnol, 2007, 75:723-37; Sharma et al., Cur nucleoside and nucleotide compounds are analogs of the Top Med Chem, 2004, 4:895-919). Currently HAART typi naturally occurring deoxyribose nucleotides, however, the cally involves combining at least three drugs belonging to at analogs lack the 3'-hydroxyl group on the deoxyribose Sugar. least two classes of antiretroviral agents. As discussed above, US 2012/0028335 A1 Feb. 2, 2012

these classes include nucleoside or nucleotide analog reverse raacetylated N-azidoacetylgalactosamine (GalNaz), 15-azi transcriptase inhibitors, non-nucleoside reverse transcriptase dopentadecanoic acid (Palmitic), 12-azidododecanoic acid inhibitors, protease inhibitors, and entry inhibitors. (Myristic) and labeled with TAMRA. FIG. 2(B) shows a total 0009. Thus, although a great deal of effort is being protein stain using SYPROR Ruby protein stain (Sigma directed to the design and testing of anti-viral drugs, the Aldrich, St. Louis, Mo.). search for new and improved methods of treating viral infec (0018 FIG. 3 shows the results of a luciferase reporter tions, such as HIV, continues. assay (Applied Biosytems luciferase reagent) to measure the infectivity of unlabeled HIV (CONTROL) or HIV labeled SUMMARY with 15-azidopentadecanoic acid (PALM), 12-azidodode canoic acid (MYR), tetraacetylated N-azidoacetyl-D-man 0010. The present disclosure provides methods of using nosamine (MAN), or tetraacetylated N-azidoacetylgalac azide-modified biomolecules, such as azide-modified fatty tosamine (GAL). acids, azide-modified carbohydrates, or azide-modified iso 0019 FIG. 4 shows the results of a luciferase reporter prenoid lipids, for treating viral infections, such as HIV infec assay (Promega luciferase reagent) to measure the infectivity tions, or for labeling a protein of a virus, such as HIV, as well of unlabeled HIV (CONTROL) or HIV labeled with 15-azi as pharmaceutical compositions containing an azide-modi dopentadecanoic acid (PALM), 12-azidododecanoic acid fied biomolecule. (MYR), tetraacetylated N-azidoacetyl-D-mannosamine 0011. One aspect of the disclosure is directed to a method (MAN), or tetraacetylated N-azidoacetylgalactosamine of treating a plant or an animal infected with a virus, the method comprising administering to the plant or animal an (GAL). effective amount of an azide-modified fatty acid, an azide modified carbohydrate, oran azide-modified isoprenoid lipid. DEFINITIONS 0012 Another aspect of the disclosure is directed a 0020. Unless defined otherwise, all technical and scien method of inhibiting the infectivity of a virus, the method tific terms used herein have the same meaning as commonly comprising contacting a cell infected with the virus with an understood by one of ordinary skill in the art to which this azide-modified fatty acid, an azide-modified carbohydrate, or invention is related. In order that the present invention may be an azide-modified isoprenoid lipid in an amount effective to more readily understood, certainterms are first defined. Addi inhibit the infectivity of the virus. tional definitions are set forth throughout the detailed descrip 0013 A third aspect of the disclosure is directed to a tion. In case of conflict, the present specification, including method of producing a human immunodeficiency virus definitions, will control. labeled with an azide-modified fatty acid, an azide-modified 0021. As used herein, “azide-modified fatty acid refers to carbohydrate, or an azide-modified isoprenoid lipid, the a fatty acid that comprises an azido group and has the follow method comprising contacting a cell infected with the human ing formula, R N where R comprises a hydrocarbon chain immunodeficiency virus with the azide-modified fatty acid, with at least one carboxylic acid functional group, which is the azide-modified carbohydrate, or the azide-modified iso usually, although not necessarily, at a terminal position. prenoid lipid so that the azide-modified fatty acid, the azide 0022. As used herein, “azide-modified carbohydrate' modified carbohydrate, or the azide-modified isoprenoid refers to a carbohydrate that comprises an azido group and has lipid enters the cell and is incorporated into a protein of the the following formula, R N where R is a carbohydrate. virus, thereby producing the labeled virus. 0023. As used herein, “azide-modified isoprenoid lipid 0014. Yet another aspect of the disclosure is directed to a refers to an isoprene-containing lipid, or derivative thereof. pharmaceutical composition comprising an azide-modified The azide-modified isoprenoid comprises an azido group and fatty acid, an azide-modified carbohydrate, or an azide-modi has the following formula, R N where R is an isoprene fied isoprenoid lipid and a pharmaceutically acceptable containing lipid, such as the Cs farnesyl isoprenoid lipid or excipient. the Co geranylgeranyl isoprenoid lipid, or a derivative thereof, including, but not limited to, an azido farnesyl BRIEF DESCRIPTION OF THE DRAWINGS diphosphate, an azido farnesyl alcohol, an azido geranylgera 0.015 The accompanying drawings, which are incorpo nyl diphosphate, or an azido geranylgeranyl alcohol. rated in and constitute a part of this specification, illustrate 0024. As used herein, “animal virus' refers to a virus that certain embodiments of the invention, and together with the infects a non-human, animal cell. In certain instances, a virus written description, serve to explain certain principles of the that infects non-human animal cells is also capable of infect invention. ing human cells. 0016 FIG. 1 shows a time course of azide-modified pro 0025. As used herein, “biomolecule.” refers to proteins, teins in HIV-infected CEMX174 cells. CEMX174 infected peptides, amino acids, glycoproteins, nucleic acids, nucle cells were labeled with either (A) 15-azidopentadecanoic otides, nucleosides, oligonucleotides, Sugars, oligosaccha acid, (B) 12-azidododecanoic acid, (C) tetraacetylated N-azi rides, lipids, hormones, proteoglycans, carbohydrates, doacetylgalactosamine, or (D) tetraacetylated N-azidoacetyl polypeptides, polynucleotides, polysaccharides, which hav D-mannosamine and were harvested at 12, 24, 72 hours, and ing characteristics typical of molecules found in living organ 14 days post-infection. FIG. 1 (E) shows a representative gel isms and may be naturally occurring or may be artificial (not that was post-stained with the total protein stain: SYPROR) found in nature and not identical to a molecule found in Ruby protein stain (Sigma-Aldrich, St. Louis, Mo.). nature). 0017 FIG. 2 shows gel electrophoresis of azide-modified 0026. As used herein, "click chemistry.” refers to the cop proteins from HIV produced from chronically infected per(I)-catalyzed variant of the Huisgen cycloaddition or the CEMx 174 cells. FIG. 2(A) shows viral proteins tagged with 1,3-dipolar cycloaddition between an azide and a terminal tetraacetylated N-azidoacetyl-D-mannosamine (Man), tet alkyne to form a 1,2,4-triazole. Such chemical reactions can US 2012/0028335 A1 Feb. 2, 2012

use, but are not limited to, simple heteroatomic organic reac 0037. As used herein, “RNA virus' refers to a virus that tants and are reliable, selective, Stereospecific, and exother has ribonucleic acid (RNA) as its genetic material. RNA mic. viruses are usually single Stranded but may also be double 0027. As used herein, "cycloaddition” refers to a chemical Stranded. reaction in which two or more t (pi)-electron systems (e.g., 0038. As used herein, the term “subject' is intended to unsaturated molecules or unsaturated parts of the same mol include human and non-human animals. Subjects may ecule) combine to form a cyclic product in which there is a net include a human patient having a viral infection, including, reduction of the bond multiplicity. In a cycloaddition, the It but not limited to, an HIV infection. The term “non-human (pi) electrons are used to form new it (pi) bonds. The product animals' of the invention includes all vertebrates, such as of a cycloaddition is called an “adduct” or “cycloadduct’. non-human primates, sheep, dogs, cows, chickens, amphib Different types of cycloadditions are known in the art includ ians, reptiles, etc. ing, but not limited to. 3+2cycloadditions and Diels-Alder 0039. As used herein, “treatment” or “treating” refers to a reactions. 3+2cycloadditions, which are also called 1.3- therapeutic or preventative measure. The treatment may be dipolar cycloadditions, occur between a 1,3-dipole and a administered to a Subject having a medical disorder or who dipolarophile and are typically used for the construction of ultimately may acquire the disorder, in order to prevent, cure, five-membered heterocyclic rings. The term "3+2cycload delay, reduce the severity of and/or ameliorate one or more dition” also encompasses "copperless' 3+2cycloadditions symptoms of a disorder or recurring disorder, or in order to between azides and cyclooctynes and difluorocyclooctynes prolong the survival of a subject beyond that expected in the described by Bertozzi et al. J. Am. Chem. Soc., 2004, 126: absence of Such treatment. 15046-15047. 0040. As used herein, a “therapeutically effective amount 0028. As used herein, “DNA virus' refers to a virus that or “effective amount’ means the amount of a compound that, has deoxyribonucleic acid (DNA) as its genetic material. when administered to a mammal or other Subject for treating DNA viruses are usually double stranded but may also be a disease, is sufficient to effect such treatment for the disease. single stranded. The “effective amount” will vary depending on the com pound, the disease and its severity and the age, weight, etc., of 0029. As used herein, “glycoprotein’ refers to a protein the subject to be treated. that has been glycosylated and those that have been enzymati 0041. It must be noted that, as used in this specification cally modified, in vivo or in vitro, to comprise a carbohydrate and the appended claims, the singular form “a”, “an and group. “the include plural referents unless the context dictates oth 0030. As used herein, “HIV” and “human immunodefi erwise. Thus, for example, reference to “a virus' includes a ciency virus' refer to human immunodeficiency virus 1 and 2 plurality of viruses unless the context dictates otherwise. (HIV-1 and HIV-2). 0042. While this invention has been particularly shown 0031. As used herein, “human virus' refers to a virus that and described with references to preferred embodiments infects human cells. In certain instances, a virus that infects thereof, it will be understood by those skilled in the art that human cells is also capable of infecting cells of other non various changes in form and details may be made therein human, animal cells. without departing from the scope of the invention encom 0032. As used herein, “infectivity” refers to the ability of a passed by the appended claims. In addition, the materials, virus to enter a cell. methods, and examples are illustrative only and not intended 0033. As used herein, “plant virus' refers to a virus that to be limiting. All publications, patent applications, patents, infects plant cells. and other references mentioned herein are incorporated by 0034. As used herein, “pharmaceutically acceptable reference in their entirety. excipient' includes solvents, dispersion media, diluents, coatings, antibacterial and antifungal agents, isotonic and DETAILED DESCRIPTION absorption delaying agents, etc., that are compatible with 0043. The present disclosure concerns the use of azide pharmaceutical administration. Use of these agents for phar modified biomolecules, such as fatty acids or carbohydrates, maceutically active Substances is well known in the art. for treating viral infections, as well as pharmaceutical com 0035. As used herein, “protein’ and “polypeptide' are positions containing an azide-modified biomolecule. AZide used in a generic sense to include polymers of amino acid modified fatty acids, azide-modified carbohydrates, and residues of any length. The term "peptide' is used herein to azide-modified isoprenoid lipids have been previously refer to polypeptides having less than 100 amino acid resi described as useful reagents for labeling and detecting pro dues, typically less than 10 amino acid residues. The terms teins of interest as part of a click chemistry reaction involving apply to amino acid polymers in which one or more amino a copper (I)-catalyzed cycloaddition reaction between an acid residues are an artificial chemical analogue of a corre azide and an alkyne. See CLICK-ITR) metabolic labeling sponding naturally occurring amino acid, as well as to natu reagents for proteins (Invitrogen, Carlsbad, Calif.); see also, rally occurring amino acid polymers. U.S. Patent Application Publication No. 2007/0249014 and 0036. As used herein, “reporter molecule” refers to any U.S. Patent Application Publication No. 20050222427, moiety capable of being attached to a modified post transla which disclosures are hereby incorporated by reference in its tionally modified protein of the present invention, and entirety. Applicants, however, have unexpectedly discovered detected either directly or indirectly. Reporter molecules that these azide-modified biomolecules have anti-viral activ include, without limitation, a chromophore, a fluorophore, a ity and can be used to treat viral infections. It was surprisingly fluorescent protein, a phosphorescent dye, a tandem dye, a discovered that these azide-modified biomolecules pro particle, a hapten, an enzyme and a radioisotope. Preferred foundly affect viral infectivity and that labeling viruses with reporter molecules include fluorophores, fluorescent pro these azide-modified biomolecules inhibited viral entry into teins, haptens, and enzymes. host cells. Without intending to be bound by any theory, it US 2012/0028335 A1 Feb. 2, 2012 appears that post-translational modification of viral proteins include, but are not limited to, glucose, galactose, mannose, with an azido-modified biomolecule at sites normally occu fucose, Xylose, N-acetylgalactosamine (GalNAc), N-acetyl pied by unmodified biomolecules, such as Saturated fatty glucosamine (GlcNAc) and N-acetylneuraminic acid acids (e.g., myristic acid and palmitic acid), may result in the (NANA, also known as sialic acid). N-acetyl-D-man inhibition of infectivity of the virus in a manner similar to the nosamine (ManNAc) is a precursor of the neuraminic acids, absence of these biomolecules at these sites. including NANA. Two of the same or different monosaccha 0044) Click Chemistry rides can join together to form a disaccharide. The addition of 0045 Azides and terminal or internal alkynes can undergo more monosaccharides results in the formation of oligosac a 1,3-dipolar cycloaddition (Huisgencycloaddition) reaction charides of increasing length. In addition, the Sugar moiety to give a 1,2,3-triazole. However, this reaction requires long can be a glycosyl group. reaction times and elevated temperatures. Alternatively, 0049. In glycoproteins the carbohydrates can be linked to azides and terminal alkynes can undergo Copper(I)-catalyzed the protein component by either N-glycosylation or O-glyco Azide-Alkyne Cycloaddition (CuAAC) at room temperature. Sylation. N-glycosylation commonly occurs through a nitro Such copper(I)-catalyzed azide-alkyne cycloadditions, also gen on an asparagine or arginine side chain, forming an known as click chemistry, is a variant of the Huisgen 1.3- N-glycosidic linkage via an amide group. O-glycosylation dipolar cycloaddition wherein organic azides and terminal commonly occurs at the hydroxy oxygen of hydroxylysine, alkynes react to give 1,4-regioisomers of 1,2,3-triazoles. hydroxyproline, serine, tyrosine or threonine side chains, Examples of click chemistry reactions are described by forming an O-glycosidic linkage. GalNAc and GlcNAc are Sharpless et al. (U.S. Patent Application Publication No. both O-linked carbohydrates. Sialic acid is found on both N 20050222427, PCT/US03/17311; Lewis W G, et al., and O-linked carbohydrates. Angewandte Chemie-Int’l Ed. 41 (6): 1053; method reviewed 0050 Protein glycosylation is one of the most abundant in Kolb, H. C., et al., Angew. Chem. Inst. Ed. 2001, 40:2004 post-translational modifications and plays a fundamental role 2021), which developed reagents that react with each other in in the control of biological systems. For example, glycosyla highyield and with few side reactions in a heteroatom linkage tion influences protein folding and can help to stabilize pro (as opposed to carbon-carbon bonds) in order to create librar teins and prevent their degradation. Glycosylation also can ies of chemical compounds. affect a protein's ability to bind to other molecules and medi 0046 Click chemistry has been used to label and detect ate intra- or inter-cellular signaling pathways. For example, proteins of interest. For example, the CLICK-ITR (Invitro carbohydrate modifications are important for host-pathogen gen, Carlsbad, Calif.) reaction is a two-step labeling tech interactions, inflammation, development, and malignancy nique involving the incorporation of a modified metabolic (Varki, A. Glycobiology 1993, 3,97-130; Lasky, L. A. Annu. precursor, Such as an azide-modified fatty acid, an azide Rev. Biochem. 1995, 64, 113-139. (c) Capila, I.: Linhardt, R. modified carbohydrate, oran azide-modified isoprenoid lipid, J. Angew. Chem., Int. Ed. 2002, 41,391-412: Rudd, P. M.: into proteins as a chemical “handle' followed by the Elliott, T., Cresswell, P.; Wilson, I. A.; Dwek, R. A. Science chemoselective ligation (or "click” reaction) between an 2001, 291, 2370-2376). One such covalent modification is azide and an alkyne. In the click reaction, the modified protein O-GlcNAc glycosylation, which is the covalent modification is detected with a corresponding azide- or alkyne-containing of serine and threonine residues by D-N-acetylglucosamine dye or hapten. The CLICK-ITR) metabolic labeling reagents (Wells, L.; Vosseller, K.: Hart, G. W. Science 2001, 291, have been used to monitor post translational modifications of 2376-2378; Zachara, N. E.; Hart, G. W. Chem. Rev. 2002, proteins, such as acylation, glycosylation, and prenylation, 102,431). The O-GlcNAc modification is found in all higher and include 1) azide-modified fatty acids, such as CLICK eukaryotic organisms from C. elegans to man and has been ITR palmitic acid azide (i.e., 15-azidopentadecanoic acid) shown to be ubiquitous, inducible and highly dynamic, Sug and CLICK-ITR myristic acid azide (i.e., 12-azidodode gesting a regulatory role analogous to phosphorylation. canoic acid), for labeling palmitoylated and myristoylated 0051 Fatty Acid Acylation proteins, respectively; 2) azide-modified carbohydrates, 0.052 Fatty acid acylation is an enzymatic process in including CLICK-ITR GalNAZ (tetraacetylated N-azi which fatty acids are attached to proteins in a cell. This doacetylgalactosamine) for labeling O-linked glycoproteins, process can affect a protein's function as well as its cellular CLICK-ITR) ManNAZ (tetraacetylated N-azidoacetyl-D- location and is common to proteins of both cellular and viral mannosamine) for labeling sialic acid modified glycopro origin (Towler et al., Proc Natl AcadSci USA 1986, 83:2812 teins, and CLICK-ITR GlcNAZ (tetraacetylated N-azi 16). Myristic acid and palmitic acid are the two most common doacetylglucosamine) for labeling O-GlcNAZ-modified fatty acids that are attached to proteins (Olson et al., J Biol glycoproteins; and 3) azide-modified isoprenoid lipids, Such Chem 261 (5):2458–66). Generally myristic acid is attached to as CLICK-ITR) farnesyl alcohol azide and CLICK-ITR gera soluble and membrane proteins via an amide linkage to an nylgeranyl alcohol azide. As noted above, Applicants, have amino terminal glycine exposed during removal of an N-me unexpectedly found that these azide-modified biomolecules thionine residue, although it can also attach to other amino have anti-viral activity and can be used to treat viral infec acids. Myristoylation can also occur post-translationally, for tions. example, when a protease cleaves a polypeptide and exposes 0047 Glycosylation a glycine residue. Palmitic acid is attached to membrane 0048 Glycosylation is an enzymatic process in which car proteins via an ester or thioester linkage. Myristoylation and bohydrates are attached to proteins, lipids, or other organic palmitoylation appear to play a significant role in Subcellular molecules in a cell. Glycoproteins are biomolecules com trafficking of proteins between membrane compartments, as posed of proteins covalently linked to carbohydrates. Certain well as in modulating protein-protein interactions. post-translational modifications append a Sugar moiety (car 0053 Fatty acids have two distinct regions, a long hydro bohydrate) onto a protein, thereby forming a glycoprotein. phobic, hydrocarbon chain and a carboxylic acid group, The common monosaccharides found in glycoproteins which is generally ionized in solution (COO ), extremely US 2012/0028335 A1 Feb. 2, 2012 hydrophilic and readily forms esters and amides. Natural fatty 1919-26). Following infection, glycosylation is required for acids commonly have a chain of four to 28 carbons (usually cleavage of the envelope precursor protein (gp160) into unbranched and even numbered) and may be saturated or gp120 and gp41. Upon release of the virus from an infected unsaturated. Saturated fatty acids contain no double bonds in cell, glycosylation is also important for immune evasion as the hydrocarbon chain and include lauric acid, myristic acid, changes in envelope glycosylation significantly alter humoral palmitic acid, Stearic acid, and arachidic acid. Unsaturated immune responses to virus (Kwong et al., Nature 2002, 420: fatty acids contain at least one double bond in the hydrocar 678-82; Shi et al., J Gen Virol 2005, 86:3385-96). bon chain and include myristoleic acid, palmitoleic acid, 0059. The acylation of viral proteins is also important to sapienic acid, oleic acid, linoleic acid, C-linoleic acid, arachi HIV biology. HIV budding is a complex process involving the donic acid, eicosapentaenoic acid, erucic acid, and docosa coordination of many cellular and viral proteins (Resh Trends hexaenoic acid. Microbiol 2001, 9:57; Freed, J Virol 2002, 76:4679-87). HIV 0054 Prenylation budding is directed to an area of the plasma membrane 0055 Protein prenylation involves the attachment of an enriched in membrane rafts (Lindwasser et al., J Virol 2001, isoprenoid lipid, such as a farnesyl or a geranyl-geranyl moi 75:7913-24: Nguyen et al., J Virol 2000, 74:3264-72: Ono et ety, to a C-terminal cysteine(s) of the target protein (McTag al., Proc Natl AcadSci USA 2001, 98: 13925-30; Hermida gert, Cell Mol Life Sci 2006, 63:255-67). These reactions are Matsumoto et al., J Virol 2000, 74:8670-79), previously catalysed by farnesyltransferase, geranylgeranyltransferase, called lipidrafts (Pike et al., JLipid Res 2006, 47: 1597-98) by and Rab geranylgeranyltransferase. (Magee and Seabra, Bio myristoylation of the N-terminal glycine of the capsid protein chem J2003,376:e3-4). Due to the hydrophobic nature of the polyprotein precursor (pr55 gag) (Lindwasser et al., J Virol isoprenoid lipid, most prenylated proteins are associated with 2001, 75:7913-24: Nguyen et al., J Virol 2000, 74:3264-72: a membrane. Most farnesylated proteins are involved in cel Ono et al., Proc Natl AcadSci USA 2001,98:13925-30). The lular signaling where membrane association is important for gp120 protein is directed to membrane rafts by palmitoyla function. Isoprenoid lipids are also important for mediating tion (Yang et al., Proc Natl AcadSci USA 1995,92:9871-75). protein-protein binding through specialized prenyl-binding Membrane rafts play an important role in several cellular domains. processes including endocytosis, Vesicle transport, choles 0056 Post Translational Modifications in Viruses terol sorting, apoptosis, and signaling through the T cell 0057. Many viral proteins are extensively modified with receptor (Jordan et al., J Immunol 2003, 171:78-87; Viola et post translational modifications, including, but not limited to al., Apmis 1999, 107:615-23; Viola et al., Science 1999, 283: glycosylation, acylation, and prenylation. In many instances, 680-82; Bezombes et al., Curr Med Chem Anti-Canc Agents these post translational modifications are required for the 2002, 3:263-70; Kabouridis et al., Eur J Immunol 2000, virus to infect a host cell and/or evade the immune system. 30:954–63). Direction of HIV proteins to these regions may Post translational modifications are of particular importance allow the virions to more efficiently hijack these pathways, in Virology because, in general, viral genomes are Small and thus potentially explaining the complex pathogenicity asso thus there is heightened pressure for coding frugality. By ciated with disease progression in AIDS. In fact, the removal taking advantage of a host's post translational machinery, of cholesterol, an important membrane raft component, from viruses can exploit multiple pathways and function with mini HIV particles results in inactivation by at least two mecha mal genomes, as a single post translational modification can nisms, a loss of the ability to fuse to the target cell and the loss alter a protein's function or cellular location. of virion integrity resulting in permeabilization of the virus 0058 For example, in HIV and Simian Immunodeficiency (Guyader etal, J Virol 2002, 76:10356-64; Campbellet al., J Viruses (SIV), glycosylation plays an important role during Virol 2004, 78: 10556-65; Viard et al., J Virol 2002, 76:11584 multiple stages of the infectivity cycle. During infection, viral 595; Campbellet al., Aids 2002, 16:2253-61; Liao etal, AIDS glycoproteins influence the binding of viral proteins gp120 Res Hum Retroviruses 2003, 19:675-87; Graham et al., J Virol and gp41 to host cell CD4 receptor and CXCR4 and CCR5 2003, 77:8237-48). co-receptors (Chen et al., Virus Res 2001, 79.91-101). Gly 0060 Viruses can also use the host cell machinery to cosylation is responsible for the proper folding and process modify viral proteins by adding isoprenoid lipids, such as the ing of gp160 (the precursor to gp120 and gp41 (Land et al., farnesyl and geranylgeranyl groups. For example, prenyla Biochimie 2001, 83: 783-90) and can enhance the interactions tion plays an important role in the life cycle of the hepatitis of HIV and SIV with different cell types, including dendritic delta virus (HDV), the etiologic agent of acute and chronic cells (Geitenbeek et al., Curr Top Microbiol Immunol 2003, liver disease associated with hepatitis B virus. (Einav and 276:31-54). The normal role of gp120 in HIV biology is to Glenn, J Antimicrobial Chemotherapy 2003, 52:883-86). initiate viral binding to cells via CD4 receptor and CXCR4 One of the HDV proteins, the large delta antigen (LHDAg), is and CCR5 co-receptors expressed on the target cell. When critical for viralassembly and undergoesfarnesylation in both gp120 engages CD4, conformational changes occur in gp120 in vitro translation systems and in intact cells. (Einav and that expose co-receptor binding sites and trigger conforma Glenn, J Antimicrobial Chemotherapy 2003, 52:883-86). tional changes in gp41. The conformational changes in gp41, Inhibiting prenylation by using farnesyltransferase inhibitors in turn, expose a fusion peptide in gp41, that mediates fusion prevents HDV assembly and clears HDV viremia in a mouse between the viral envelope and the target cell (Chen et al., model of HDV, thus underscoring the importance of prenyla Virus Res 2001, 79.91-101). The change of one carbohydrate tion in the life cycle of certain viruses. (Einav and Glenn, J at a single residue (N197) in gp120 completely changes viral Antimicrobial Chemotherapy 2003, 52:883-86). tropism from CD4 tropic to CD4 independent (Kolchinksy et 0061 Similar to HIV. SIV, and HDV, other viruses rely on al., J Virol 2001, 75:3435-43). Changing the overall ratios of post translational modifications of viral proteins to mediate high mannose in comparison to complex type carbohydrates entry into host cells and/or to evade the host immune system. (sialic acid containing) present in gp120 affects the degree of Thus, the azide-modified fatty acids, azide-modified carbo viral binding to target cells (Fenouillet et al., J Gen Virol 1991, hydrates, and azide-modified isoprenoid lipids described US 2012/0028335 A1 Feb. 2, 2012

herein are expected to have abroad range of anti-viral activity or 12-azidododecanoic acid. In another embodiment, the (such as modulating activity by inhibiting or preventing azide-modified carbohydrate is an N-linked carbohydrate or reverse transcription of the HIV viral genome, late-stage pro an O-linked carbohydrate. In yet another embodiment, the cessing of certain viral proteins prior to final assembly of new azide-modified carbohydrate is N-azidoacetylgalactosamine, virons, or viral entry into the cell) and can be used to treat a N-azidoacetyl-D-mannosamine, or N-azidoacetylglu wide variety of viral infections. cosamine. The azide-modified carbohydrate optionally com 0062 Methods of Use prises a moiety that facilitates entry into the cell including, but 0063 1. Method of Treating a Viral Infection not limited to, a tetraacetyl moiety. Thus, in another embodi 0064. The present disclosure provides a method of treating ment, the azide-modified carbohydrate is tetraacetylated a plant or an animal infected with a virus, the method com N-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl prising administering to the plant or animal an effective D-mannosamine, or tetraacetylated N-azidoacetylglu amount of an azide-modified fatty acid, an azide-modified cosamine. In another embodiment, the isoprenoid lipid com carbohydrate, or an azide-modified isoprenoid lipid. In one prises a farnesyl group or a geranylgeranyl group and embodiment, the azide-modified fatty acid is a saturated fatty includes, but is not limited to, an azido farnesyl diphosphate, acid, Such as 15-azidopentadecanoic acid or 12-azidodode an azido farnesyl alcohol, an azido geranylgeranyl diphos canoic acid. In another embodiment, the azide-modified car phate, or an azido geranylgeranyl alcohol. bohydrate is an N-linked carbohydrate or an O-linked carbo 0069. The virus may be a plant virus, an animal virus, hydrate. In yet another embodiment, the azide-modified and/or a human virus. In certain embodiments, the cell is a carbohydrate is N-azidoacetylgalactosamine, N-azidoacetyl human cell and the virus is a human virus, such as an aden D-mannosamine, or N-azidoacetylglucosamine. The azide ovirus, an astrovirus, a hepadnavirus, a herpesvirus, a modified carbohydrate optionally comprises a moiety that papovavirus, a poxvirus, an arenavirus, a bunyavirus, a cal facilitates entry into the cell including, but not limited to, a civirus, a coronavirus, a filovirus, a flavivirus, an orthomyX tetraacetyl moiety. Thus, in another embodiment, the azide ovirus, a paramyxovirus, a picornavirus, a reovirus, a retro modified carbohydrate is tetraacetylated N-azidoacetylgalac virus, a rhabdovirus, or a togavirus. In one embodiment, the tosamine, tetraacetylated N-azidoacetyl-D-mannosamine, or animal is a human and the virus is a human immunodeficiency tetraacetylated N-azidoacetylglucosamine. In another virus. Preferably the human immunodeficiency virus is HIV embodiment, the isoprenoid lipid comprises a farnesyl group 1. or a geranylgeranyl group and includes, but is not limited to, 0070. Whether the azide-modified fatty acid, azide-modi an azido farnesyl diphosphate, an azido farnesyl alcohol, an fied carbohydrate, or azide-modified isoprenoid lipid is effec azido geranylgeranyl diphosphate, or an azido geranylgera tive to inhibit the infectivity of a virus can be determined nyl alcohol. using any of a variety of assays known in the art, including a 0065. The virus may be a plant virus, an animal virus, reporter gene assay, Such as the luciferase assay described in and/or a human virus. In certain embodiments, the animal is Example 2. a human and the virus is a human virus, Such as an adenovirus, (0071 3. Method of Labeling a Viral Protein an astrovirus, a hepadnavirus, a herpesvirus, a papovavirus, a 0072 An azide-modified fatty acid can also be used to poxvirus, an arenavirus, a bunyavirus, a calcivirus, a coro label viral proteins that are modified with lipids by post navirus, a filovirus, a flavivirus, an orthomyxovirus, a translational acylation including, but not limited to, palmi paramyxovirus, a picornavirus, a reovirus, a retrovirus, a toylation and myristoylation. In Such post-translational modi rhabdovirus, oratogavirus. In one embodiment, the animal is fications, an azide-modified fatty acid is used to label a viral a human and the virus is a human immunodeficiency virus. protein. If desired, the azide labeled viral protein can be Preferably the human immunodeficiency virus is HIV-1. coupled to an alkyne labeled reporter molecule using a click 0066. Whether the azide-modified fatty acid, azide-modi chemistry reaction to permit detection of the azide labeled fied carbohydrate, or azide-modified isoprenoid lipid is effec viral protein. tive to treat a viral infection can be determined using any of a 0073. Similarly, an azide-modified carbohydrate can be variety of assays known in the art. For example, existing used to label viral proteins that are modified with carbohy animal models or in vitro models of viral infection can be drates by post-translational glycosylation including, but not used to determine whether a given compound is effective to limited to, N-linked glycosylation and O-linked glycosyla reduce viral load. For HIV, by way of example, the in vitro, tion. In Such post-translational modifications, an azide-modi luciferase reporter assay described in Example 2, can be used fied carbohydrate is used to labela viral protein. If desired, the to measure the efficacy of an azide-modified compound. In a azide labeled viral protein can be coupled to an alkyne labeled human Subject, the compound's efficacy can be determined reporter molecule using click chemistry to permit the detec by measuring viral load and/or measuring one or more symp tion of the azide labeled viral protein. toms of a viral infection. Viral load can be measured by 0074 An azide-modified isoprenoid lipid can likewise be measuring the titer or level of virus in serum. These methods used to label viral proteins that are modified with lipids by include, but are not limited to, a quantitative polymerase post-translational prenylation including, but not limited to, chain reaction (PCR) and a branched DNA (bDNA) test. farnesylation and geranylgeranylation. In Such post-transla 0067 2. Method of Inhibiting Infectivity of a Virus tional modifications, an azide-modified isoprenoid lipid is 0068 Also provided is a method of inhibiting the infec used to label a viral protein. If desired, the azide labeled viral tivity of a virus, the method comprising contacting a cell protein can be coupled to an alkyne labeled reporter molecule infected with the virus with an azide-modified fatty acid, an using a click chemistry reaction to permit detection of the azide-modified carbohydrate, or an azide-modified iso azide labeled viral protein. prenoid lipid in an amount effective to inhibit the infectivity 0075. The azide labeled virus can used to track viral infec of the virus. In one embodiment, the azide-modified fatty acid tivity in vivo whereby the virus labeled with an azide-modi is a saturated fatty acid, such as 15-azidopentadecanoic acid fied carbohydrate, azide-modified fatty acid, or azide-modi US 2012/0028335 A1 Feb. 2, 2012

fied isoprenoid lipid is used to infect cultured cells or 0080 1. Animal Viruses laboratory animals. Cells can be infected over a time course I0081. In methods directed to treating a viral infection or then fixed, permeabilized, and click-labeled with a fluores inhibiting viral infectivity in a non-human animal, the animal cent alkyne dye. The intracellular location (or transport over virus is preferably selected from a picornavirus, such as a a time course) of the fluorescent viral particles can be visu bovine enterovirus, a porcine enterovirus B, a foot-and alized, for example, by microscopy. Similarly, treatment of mouth disease virus, an equine rhinitis. A virus, a bovine small animals with azide-labeled virus can be performed and rhinitis B virus, a lungan virus, equine rhinitis B virus, an used to track virus bioavailability in different tissues includ aichi virus, a bovine kobuvirus, a porcine teschovirus, a por ing detection of the virus in circulating white blood cells by cine Sapelovirus, a simian Sapelovirus, an avian Sapelovirus, flow cytometry and cell/tissue section microscopy an avian encephalomyelitis virus, a duck hepatitis A virus, or a simian enterovirus A, a pestivirus, Such as border disease 0076. Thus, another aspect of the disclosure is directed to virus, a bovine virus diarrhea, or a classical Swine fever virus; a method of producing a human immunodeficiency virus an arterivirus, such as an equine arteritis virus, a porcine labeled with an azide-modified fatty acid, an azide-modified reproductive and respiratory syndrome virus, a lactate dehy carbohydrate, or an azide-modified isoprenoid lipid, the drogenase elevating virus, or a simian haemorrhagic fever method comprising contacting a cell infected with the human virus; a coronavirus, such as a bovine coronavirus, a porcine immunodeficiency virus with the azide-modified fatty acid, coronavirus, a feline coronavirus, or a canine coronavirus; a the azide-modified carbohydrate, or the azide-modified iso paramyxovirus, Such as a hendra virus, a nipah virus, a canine prenoid lipid so that the azide-modified fatty acid, the azide distemper virus, a rinderpest virus, a Newcastle disease virus, modified carbohydrate, or the azide-modified isoprenoid and a bovine respiratory syncytial virus; an orthomyxovirus, lipid enters the cell and is incorporated into a protein of the Such as an influenza A virus, an influenza B virus, or an virus, thereby producing the labeled virus. In one embodi influenza C virus; a reovirus, such as a bluetongue virus; a ment, the azide-modified fatty acid is a Saturated fatty acid, porcine circovirus, a herpesvirus, such as a pseudorabies Such as 15-azidopentadecanoic acid or 12-azidododecanoic virus or a bovine herpesvirus 1; an asfarvirus, Such as an acid. In another embodiment, the azide-modified carbohy African Swine fever virus; a retrovirus, such as a simian drate is an N-linked carbohydrate or an O-linked carbohy immunodeficiency virus, a feline immunodeficiency virus, a drate. In yet another embodiment, the azide-modified carbo bovine immunodeficiency virus, a bovine leukemia virus, a hydrate is N-azidoacetylgalactosamine, N-azidoacetyl-D- feline leukemia virus, a Jaag.siekte sheep retrovirus, or a mannosamine, or N-azidoacetylglucosamine. In another caprine arthritis encephalitis virus; a flavivirus, such as a embodiment, the isoprenoid lipid comprises a farnesyl group yellow fever virus, a West Nile virus, a dengue fever virus, a or a geranylgeranyl group and includes, but is not limited to, tick borne encephalitis virus, or a bovine viral diarrhea; or a an azido farnesyl diphosphate, an azido farnesyl alcohol, an rhabdovirus, such as a rabies virus. azido geranylgeranyl diphosphate, or an azido geranylgera 0082 2. Human Viruses nyl alcohol. The azide-modified carbohydrate optionally I0083. In methods directed to treating a viral infection or comprises a moiety that facilitates entry into the cell includ inhibiting viral infectivity in a human, the human virus is ing, but not limited to, a tetraacetyl moiety. Thus, in another preferably selected from an adenovirus, an astrovirus, a hep embodiment, the azide-modified carbohydrate is tetraacety adnavirus, a herpesvirus, a papovavirus, a poxvirus, an lated N-azidoacetylgalactosamine, tetraacetylated N-azi arenavirus, a bunyavirus, a calcivirus, a coronavirus, a filovi doacetyl-D-mannosamine, or tetraacetylated N-azidoacetyl rus, a flavivirus, an orthomyxovirus, a paramyxovirus, a glucosamine. In certain embodiments, the cell is a human picornavirus, a reovirus, a retrovirus, a rhabdovirus, or a cell. togavirus. 0.077 Viruses I0084. In preferred embodiments, the adenovirus includes, 0078. The azide-modified fatty acids, azide-modified car but is not limited to, a human adenovirus. In preferred bohydrates, or azide-modified isoprenoid lipids preferably embodiments, the astrovirus includes, but is not limited to, a target post translational modifications common to most mamastrovirus. In preferred embodiments, the hepadnavirus viruses and thus represent a new class of anti-viral agents with includes, but is not limited to, the hepatitis B virus. In pre potential for anti-viral activity against a broad spectrum of ferred embodiments, the herpesvirus includes, but is not lim viruses. In principle, these compounds may be used to treat a ited to, a herpes simplex virus type I, a herpes simplex virus plant or an animal infected with any virus. In some embodi type 2, a human cytomegalovirus, an Epstein-Barr virus, a ments, the virus is a plant virus. In other embodiments, the Varicella Zoster virus, a roseolovirus, and a Kaposi's sarcoma virus is an animal virus. In yet other embodiments, the virus associated herpesvirus. In preferred embodiments, the is a human virus. In one embodiment, the virus is one that papovavirus includes, but is not limited to, human papilloma infects a non-human mammal. Such as a mammalian live virus and a human polyomavirus. In preferred embodiments, stock animal, including, but not limited to, a cow, a horse, a the poxvirus includes, but is not limited to, a variola virus, a pig, a goat, or a sheep. vaccinia virus, a cowpox virus, a monkeypox virus, a small 0079. In other embodiments, the virus is a DNA virus. pox virus, a pseudocowpox virus, a papular stomatitis virus, a DNA viruses include, but are not limited to a virus belonging tanapox virus, ayaba monkey tumor virus, and a molluscum to one of the following families: adenovirus, astrovirus, hep contagiosum virus. In preferred embodiments, the arenavirus adnavirus, herpesvirus, papovavirus, and poxvirus. In other includes, but is not limited to lymphocytic choriomeningitis embodiments, the virus is an RNA virus. RNA viruses include virus, a lassa virus, a machupo virus, and a junin virus. In but are not limited to a virus belonging to one the following preferred embodiments, the bunyavirus includes, but is not families: arenavirus, bunyavirus, calcivirus, coronavirus, limited to, a hantavirus, a nairovirus, an orthobunyavirus, and filovirus, flavivirus, orthomyxovirus, paramyxovirus, picor a phlebovirus. In preferred embodiments, the calcivirus navirus, reovirus, retrovirus, rhabdovirus, and togavirus. includes, but is not limited to, a vesivirus, a norovirus, such as US 2012/0028335 A1 Feb. 2, 2012 the Norwalk virus and a sapovirus. In preferred embodiments, pharmaceutically acceptable derivative or prodrug thereof. the coronavirus includes, but is not limited to, a human coro The carbohydrate can be selected from a wide variety of navirus (etiologic agent of sever acute respiratory syndrome carbohydrates commercially available and/or widely known (SARS)). In preferred embodiments, the filovirus includes, to those skilled in the art. In preferred embodiments, the but is not limited to, an Ebola virus and a Marburg virus. In carbohydrate is selected to prevent, inhibit and/or retard viral preferred embodiments, the flavivirus includes, but is not infection of cells. Preferably the carbohydrate is naturally limited to, a yellow fever virus, a West Nile virus, a dengue occurring. It is appreciated that the azide-containing carbo fever virus, a hepatitis C virus, a tick borne encephalitis virus, hydrate, whether naturally occurring or not, may be modified, a Japanese encephalitis virus, a Murray Valley encephalitis for example, by short chain alkylation Such as methylation or virus, a St. Louisencephalitis virus, a Russian spring-Summer acetylation, esterification, as well as other derivitisations that encephalitis virus, a Omsk hemorrhagic fever virus, a bovine maintain antiviral activity. viral diarrhea virus, a Kyasanus Forest disease virus, and a I0089. In one embodiment, the carbohydrate is one that is Powassan encephalitis virus. In preferred embodiments, the attached directly or indirectly to a protein through a glycosy orthomyxovirus includes, but is not limited to, influenza virus lation reaction in a cell. In one embodiment, the carbohydrate type A, influenza virus type B, and influenza virus type C. In is an N-linked carbohydrate or an O-linked carbohydrate. In preferred embodiments, the paramyxovirus includes, but is yet another embodiment, the carbohydrate is N-azidoacetyl not limited to, a parainfluenza virus, a rubula virus (mumps), galactosamine, N-azidoacetyl-D-mannosamine, or N-azi a morbillivirus (measles), a pneumovirus, such as a human doacetylglucosamine. respiratory syncytial virus, and a Subacute Sclerosing panen 0090. In certain embodiments, the azide-modified carbo cephalitis virus. In preferred embodiments, the picornavirus hydrate contains a moiety that facilitates entry into the cell includes, but is not limited to, a poliovirus, a rhinovirus, a including, but not limited to, a tetraacetyl moiety. Thus, in one coxsackievirus A, a coxsackievirus B, a hepatitis A virus, an embodiment, the azide-modified carbohydrate is a tetraacety echovirus, and an eneterovirus. In preferred embodiments, lated version of an N-linked carbohydrate or an O-linked the reovirus includes, but is not limited to, a Colorado tick carbohydrate. In yet another embodiment, the azide-modified fever virus and a rotavirus. In preferred embodiments, the carbohydrate is tetraacetylated N-azidoacetylgalactosamine, retrovirus includes, but is not limited to, a lentivirus. Such as tetraacetylated N-azidoacetyl-D-mannosamine, or tet a human immunodeficiency virus, and a human T-lym raacetylated N-azidoacetylglucosamine. photrophic virus (HTLV). In preferred embodiments, the 0091. In other embodiments, the azide-modified biomol rhabdovirus includes, but is not limited to, a lyssavirus. Such ecule is a fatty acid or a pharmaceutically acceptable deriva as the rabies virus, the vesicular stomatitis virus and the tive or prodrug thereof. The fatty acid can be selected from a infectious hematopoietic necrosis virus. In preferred embodi wide variety of fatty acids commercially available and/or ments, the togavirus includes, but is not limited to, an alphavi widely known to those skilled in the art. In preferred embodi rus, Such as a Ross river virus, an Onyongnyong virus, a ments, the fatty acid is selected to prevent, inhibit and/or Sindbis virus, a Venezuelan equine encephalitis virus, an retard viral infection of cells. Preferably the fatty acid is Eastern equine encephalitis virus, and a Western equine naturally occurring. encephalitis virus, and a rubella virus. 0092. In one embodiment, the fatty acid is saturated or 0085 3. Plant Viruses unsaturated and has a hydrocarbon chain with an even num I0086. In methods directed to treating a viral infection or ber of carbon atoms, such as 4-24 carbon atoms. Suitable inhibiting viral infectivity in a plant, the plant virus is selected unsaturated free fatty acids have a hydrocarbon chain with from an alfamovirus, an allexivirus, an alphacryptovirus, an 14-24 carbon atoms and include palmitoleic acid, oleic acid, anulavirus, an apscaviroid, an aureusvirus, an avenavirus, an linoleic acid, alpha and gamma linolenic acid, arachidonic aySunviroid, a badnavirus, a begomovirus, a beny virus, a acid, eicosapentaenoic acid and tetracosenoic acid. Suitable betacryptovirus, a betaflexiviridae, a bromovirus, abymovi saturated fatty acids have a hydrocarbon chain with 4-18 rus, a capillovirus, a carlavirus, a carmovirus, a caulimovirus, carbon atoms and are preferably selected from butyric or a cavemovirus, a cheravirus, a closterovirus, a cocadviroid, a isobutyric acid. Succinic acid, caproic acid, adipic acid, coleviroid, a comovirus, a crinivirus, a cucumovirus, a cur caprylic acid, capric acid, lauric acid, myristic acid, palmitic tovirus, a cytorhabdovirus, a dianthovirus, an enamovirus, an acid Stearic acid, and arachidic acid. It is appreciated that the umbravirus & B-type satellite virus, a fabavirus, a fijivirus, a azide-containing fatty acid, whether naturally occurring or furovirus, a hordeivirus, a hostuviroid, an idaeovirus, an not, may be modified by chemical Substitution including, but ilarvirus, an ipomovirus, a luteovirus, a machlomovirus, a not limited to, short chain alkylation Such as methylation or macluravirus, a marafivirus, a mastrevirus, a nanovirus, a acetylation, esterification, as well as other derivitisations that necrovirus, a nepovirus, a nucleorhabdovirus, an oleavirus, maintain antiviral activity. In addition, it is possible to replace an ophiovirus, an oryzavirus, a panicovirus, a pecluvirus, a the fatty acid in the azide-modified biomolecule with an petuvirus, a phytoreovirus, a polerovirus, a pomovirus, a alkyne, ketone, or other Small molecule that has been shown pospiviroid, a potexvirus, a potyvirus, a reovirus, a rhabdovi to be metabolically compatible. rus, a rymovirus, a sadwavirus, a SbCMV-like virus, a 0093. In one embodiment, the fatty acid is one that is sequivirus, a Sobemovirus, a tenuivirus, a TNsatV-like satel attached to a protein through an acylation reaction (e.g., lite virus, a tobamovirus, a topocuvirus, a tospovirus, a tri palmitoylation or myristoylation) in a cell. Thus, in one chovirus, a tritimovirus, a tungrovirus, a tymovirus, an embodiment, the azide-modified fatty acid is a saturated fatty umbravirus, a varicosavirus, a vitivirus, or a waikavirus. acid, such as 15-azidopentadecanoic acid (palmitic acid, 0087 Azide-Modified Biomolecules azide) or 12-azidododecanoic acid (myristic acid, azide). 0088. The azide-modified biomolecules described herein 0094. In yet another embodiment, the azide-modified bio represent a new class of anti-viral agents. In certain embodi molecule is an azide-modified isoprenoid lipid or a pharma ments, the azide-modified biomolecule is a carbohydrate or a ceutically acceptable derivative or prodrug thereof. The iso US 2012/0028335 A1 Feb. 2, 2012 prenoid lipid can be selected from a wide variety of modified fatty acid, the azide-modified carbohydrate, or the isoprenoid lipids commercially available and/or widely azide-modified isoprenoid lipid. The additional anti-viral known to those skilled in the art. In preferred embodiments, agent may include at least one reverse transcriptase inhibitor, the isoprenoid lipid is selected to prevent, inhibit and/or retard a virus protease inhibitor, a viral fusion inhibitor, a viral viral infection of cells. Preferably the isoprenoid lipid is integrase inhibitor, a glycosidase inhibitor, a viral neuramini naturally occurring. It is appreciated that the azide-containing dase inhibitor, an M2 protein inhibitor, an amphotericin B, isoprenoid lipid, whether naturally occurring or not, may be hydroxyurea, C.-interferon, B-interferon, y-interferon, and an modified, for example, by short chain alkylation Such as antisense oligonucleotide. methylation or acetylation, esterification, as well as other 0100. The at least one reverse transcriptase inhibitor derivitisations that maintain antiviral activity. includes, but is not limited to, one or more nucleoside ana 0095. In one embodiment, the isoprenoid lipid is one that logs, such as Zidovudine (AZT), Didanosine (ddI), Zalcitab is attached to a protein through a prenylation reactionina cell. ine (ddC). Stavudine (d4T), Lamivudine (3TC), Abacavir In one embodiment, the isoprenoid lipid, in the presence of (ABC), Emtricitabine (FTC), Entecavir (INN), Apricitabine the catalytic activity of a farnesyltransferase or a geranylgera (ATC), Atevirapine, ribavirin, acyclovir, famciclovir, Valacy nyltransferase, is attached to a protein in a cell. In another clovir, ganciclovir, and Valganciclovir, one or more nucle embodiment, the isoprenoid lipid comprises a farnesyl group otide analogs, such as Tenofovir (tenofovir disoproxil fuma or a geranylgeranyl group and includes, but is not limited to, rate), Adefovir (bis-POM PMPA), PMPA, and cidofovir; or an azido farnesyl diphosphate, an azido farnesyl alcohol, an one or more non-nucleoside reverse transcriptase inhibitors, azido geranylgeranyl diphosphate, or an azido geranylgera such as Efavirenz, Nevirapine, Delavirdine, and Etravirine. nyl alcohol. 0101 The at least one viral protease inhibitor includes, but 0096. The azide-modified carbohydrates, azide-modified is not limited to, tipranavir, darunavir, indinavir, lopinavir, fatty acids, and azide-modified isoprenoid lipids described fosamprenavir, atazanavir, saquinavir, ritonavir, indinavir, herein can be prepared using methods known in the art, nelfinavir, and amprenavir. including those disclosed in U.S. Patent Application Publica 0102 Theat least one viral fusion inhibitor includes, but is tion No. 20050222427, U.S. Patent Application Publication not limited to a CD4 antagonist, such as soluble CD4 or an No. 2007/0249014, and Hang, H. C. et al., JAm Chem Soc antibody that binds to CD4, such as TNX-355, BMS-806; a 2007, 129:2744-45, the disclosures of which are incorporated CCR5 antagonist, such as SCH-C, SCH-D, UK-427,857, by reference in their entirety. maraviroc, Vicriviroc, or an antibody that binds to CCR5, 0097. Combination Therapy such as PRO-140; a CXCR4 antagonist, such as, AMD3100 0098. In one embodiment, a pharmaceutical composition or AMD070; or an antagonist of gp41, such as enfuvirtide. comprising an azide-modified fatty acid, an azide-modified 0103) The at least one viral integrase inhibitor includes, carbohydrate, or an azide-modified isoprenoid lipid and at but is not limited to, raltegravir. least one anti-viral agent is administered in combination 0104. The at least one glycosidase inhibitor includes, but therapy. The therapy is useful for treating viral infections, is not limited to, SC-48334 or MDL-28574. including, but not limited to, an HIV infection. The term “in 0105. The at least one viral neuraminidase inhibitor combination' in this context means that the azide-modified includes, but is not limited to, Oseltamivir, peramivir, Zan fatty acid, azide-modified carbohydrate, or the azide-modi amivir, and laninamivir. Neuraminidase is a protein on the fied isoprenoid lipid and the anti-viral agent are given Sub surface of influenza viruses that mediates the virus release stantially contemporaneously, either simultaneously or from an infected cell. (Bossart-Whitaker et al., J Mol Biol, sequentially. In one embodiment, if given sequentially, at the 1993, 232:1069-83). The influenza virus attaches to the cell onset of administration of the second compound, the first of membrane using the viral hemagglutinin protein. The hemag the two compounds is still detectable at effective concentra glutinin protein binds to sialic acid moieties found on glyco tions at the site of treatment. In another embodiment, if given proteins in the host cell's membranes. In order for the virus to sequentially, at the onset of administration of the second be released from the cell, neuraminidase must enzymatically compound, the first of the two compounds is not detectable at cleave the Sialic acid groups from the host glycoproteins. effective concentrations at the site of treatment. Thus, inhibiting neuraminidase prevents the release of the 0099 For example, the combination therapy can include influenza virus from an infected cell. an azide-modified fatty acid, an azide-modified carbohydrate, 0106. The at least one M2 inhibitor includes, but is not or an azide-modified isoprenoid lipid co-formulated with, limited to, amantadine and rimantidine. M2 is an ion channel and/or co-administered with, at least one additional anti-viral protein found in the viral envelope of the influenza virus agent. Although specific examples of anti-viral agents are (Henckel et al., J Biol Chem, 1998, 273:6518-24). The M2 provided, in principle, the azide-modified fatty acid, azide protein plays an important role in controlling the uncoating of modified carbohydrate, or an azide-modified isoprenoid lipid the influenza virus, leading to the release of the virion con can be combined with any pharmaceutical composition use tents into the host cell cytoplasm. Blocking M2 inhibits viral ful for treating a viral infection. Such combination therapies replication. may advantageously use lower dosages of the administered 0107 An azide-modified fatty acid, an azide-modified car therapeutic agents, thus avoiding possible toxicities or com bohydrate, or an azide-modified isoprenoid lipid disclosed plications associated with the various monotherapies. More herein can be used in combination with other therapeutic over, the additional anti-viral agents disclosed herein act on agents to treat specific viral infections as discussed in further pathways or stage of viral infection in addition to or that differ detail below. from the pathway stage of viral infection affected by the 0108) Non-limiting examples of agents for treating an azide-modified fatty acid, the azide-modified carbohydrate, HIV infection, with which the azide-modified fatty acid, the or the azide-modified isoprenoid lipid, and thus are expected azide-modified carbohydrate, or the azide-modified iso to enhance and/or synergize with the effects of the azide prenoid lipid can be combined include at least one of the US 2012/0028335 A1 Feb. 2, 2012

following: Zidovudine (AZT), Didanosine (ddI), Zalcitabine carbohydrate. In yet another embodiment, the azide-modified (ddC). Stavudine (d4T), Lamivudine (3TC), Abacavir carbohydrate is tetraacetylated N-azidoacetylgalactosamine, (ABC), Emtricitabine (FTC), Entecavir (INN), Apricitabine tetraacetylated N-azidoacetyl-D-mannosamine, or tet (ATC), Tenofovir (tenofovir disoproxil fumarate), Adefovir raacetylated N-azidoacetylglucosamine. In another embodi (bis-POM PMPA) Efavirenz, Nevirapine, Delavirdine, ment, the isoprenoid lipid comprises a farnesyl or a gera Etravirine, tipranavir, darunavir, indinavir, lopinavir, foSam nylgeranyl group and includes, but is not limited to, an azido prenavir, atazanavir, saquinavir, ritonavir, indinavir, nelfi farnesyl diphosphate, an azido farnesyl alcohol, an azido navir, amprenavir, a CD4 antagonist, Such as soluble CD4 or geranylgeranyl diphosphate, oran azido geranylgeranyl alco an antibody that binds to CD4, such as TNX-355, BMS-806, hol. The pharmaceutical compositions may also be included a CCR5 antagonist, such as SCH-C, SCH-D, UK-427,857, maraviroc, Vicriviroc, or an antibody that binds to CCR5, in a container, pack, or dispenser together with instructions such as PRO-140, a CXCR4 antagonist, such as, AMD3100 for administration. or AMD070, or an antagonist of gp41, such as enfuvirtide. 0116. A pharmaceutical composition is formulated to be 0109 Specific examples of combination therapy that can compatible with its intended route of administration. Meth be used to treat HIV infection include, but are not limited to, ods to accomplish the administration are known to those of an azide-modified fatty acid, azide-modified carbohydrate, or ordinary skill in the art. Pharmaceutical compositions may be the azide-modified isoprenoid lipid combined with: 1) teno topically or orally administered, or capable of transmission fovir, emitricitabine, and efavirenz; 2) lopinavir and ritonavir, across mucous membranes. Examples of administration of a 3) lamivudine and Zidovudine; 4) abacavir, lamivudine, and pharmaceutical composition include oral ingestion or inhala zidovudine; 5) lamivudine and abacavir; or 6) tenofovir and tion. Administration may also be intravenous, intraperitoneal, emtricitabine. intramuscular, intracavity, Subcutaneous, cutaneous, or trans 0110. Non-limiting examples of agents for a herpesvirus dermal. infection with which the azide-modified fatty acid, the azide 0117 Solutions or suspensions used for intradermal or modified carbohydrate, or the azide-modified isoprenoid Subcutaneous application typically include at least one of the lipid can be combined include acyclovir, famciclovir, Valacy following components: a sterile diluent such as water, Saline clovir, cidofovir, foscarnet, ganciclovir, and Valganciclovir. Solution, fixed oils, polyethylene glycol, glycerine, propylene 0111 Non-limiting examples of agents for an influenza glycol, or other synthetic solvent; antibacterial agents such as virus infection with which the azide-modified fatty acid, the benzyl alcohol or methyl parabens; antioxidants such as azide-modified carbohydrate, or the azide-modified iso ascorbic acid or sodium bisulfite; chelating agents such as prenoid lipid can be combined include amantadine, rimanti ethylenediaminetetraacetic acid (EDTA); buffers such as dine, oseltamivir, peramivir, Zanamivir, and laninamivir. acetate, citrate, or phosphate; and tonicity agents such as 0112 Non-limiting examples of agents for a respiratory sodium chloride or dextrose. The pH can be adjusted with synctial virus infection with which the azide-modified fatty acids or bases. Such preparations may be enclosed in acid, the azide-modified carbohydrate, or the azide-modified ampoules, disposable syringes, or multiple dose vials. isoprenoid lipid can be combined include ribavirin. 0118 Solutions or suspensions used for intravenous 0113 Another aspect of the present invention accordingly administration include a carrier Such as physiological saline, relates to kits for carrying out the combined administration of bacteriostatic water, Cremophor ELTM (BASF, Parsippany, the azide-modified fatty acid, the azide-modified carbohy N.J.), ethanol, or polyol. In all cases, the composition must be drate, or the azide-modified isoprenoid lipid with other thera sterile and fluid for easy syringability. Proper fluidity can peutic agents. In one embodiment, the kit comprises the often be obtained using lecithin or Surfactants. The composi azide-modified fatty acid, the azide-modified carbohydrate, tion must also be stable under the conditions of manufacture or the azide-modified isoprenoid lipid formulated in a phar and storage. Prevention of microorganisms can be achieved maceutical excipient, and at least one anti-viral agent, formu with antibacterial and antifungal agents, e.g., parabens, chlo lated as appropriate in one or more separate pharmaceutical robutanol, phenol, ascorbic acid, thimerosal, etc. In many preparations. cases, isotonic agents (Sugar), polyalcohols (mannitol and 0114 Pharmaceutical Compositions and Methods of sorbitol), or sodium chloride may be included in the compo Administration sition. Prolonged absorption of the composition can be 0115 This disclosure provides compositions that are suit accomplished by adding an agent which delays absorption, able for pharmaceutical use and administration to patients. e.g., aluminum monostearate and gelatin. The pharmaceutical compositions comprise an azide-modi 0119 Oral compositions include an inert diluent or edible fied fatty acid, an azide-modified carbohydrate, or an azide carrier. The composition can be enclosed in gelatin or com modified isoprenoid lipid and a pharmaceutically acceptable pressed into tablets. For the purpose of oral administration, excipient. In one embodiment, the azide-modified fatty acid is the azide-modified fatty acid, the azide-modified carbohy a saturated fatty acid, Such as 15-azidopentadecanoic acid or drate, or the azide-modified isoprenoid lipid can be incorpo 12-azidododecanoic acid. In another embodiment, the azide rated with excipients and placed in tablets, troches, or cap modified carbohydrate is an N-linked carbohydrate or an Sules. Pharmaceutically compatible binding agents or O-linked carbohydrate. In yet another embodiment, the adjuvant materials can be included in the composition. The azide-modified carbohydrate is N-azidoacetylgalactosamine, tablets, troches, and capsules, may contain (1) a binder Such N-azidoacetyl-D-mannosamine, or N-azidoacetylglu as microcrystalline cellulose, gum tragacanth or gelatin; (2) cosamine. In another embodiment, the azide-modified carbo an excipient such as starch or lactose, (3) a disintegrating hydrate contains a moiety that facilitates entry into the cell agent such as alginic acid, Primogel, or corn starch; (4) a including, but not limited to, a tetraacetyl moiety. Thus, in one lubricant Such as magnesium Stearate; (5) a glidant Such as embodiment, the azide-modified carbohydrate is a tetraacety colloidal silicon dioxide; or (6) a Sweetening agent or a fla lated version of an N-linked carbohydrate or an O-linked Voring agent. US 2012/0028335 A1 Feb. 2, 2012

0120. The composition may also be administered by a ug/kg to 100 ug/kg, 100 ug/kg to 1 mg/kg, 250 ug/kg to 2 transmucosal or transdermal route. Transmucosal administra mg/kg, 250 ug/kg to 1 mg/kg, 500 ug/kg to 2 mg/kg, 500 tion can be accomplished through the use of lozenges, nasal g/kg to 1 mg/kg, 1 mg/kg to 2 mg/kg, 1 mg/kg to 5 mg/kg, 5 sprays, inhalers, or Suppositories. Transdermal administra mg/kg to 10 mg/kg, 10 mg/kg to 20 mg/kg, 15 mg/kg to 20 tion can also be accomplished through the use of a composi mg/kg, 10 mg/kg to 25 mg/kg, 15 mg/kg to 25 mg/kg, 20 tion containing ointments, salves, gels, or creams known in mg/kg to 25 mg/kg, and 20 mg/kg to 30 mg/kg (or higher). the art. For transmucosal or transdermal administration, pen These dosages may be administered daily, weekly, biweekly, etrants appropriate to the barrier to be permeated are used. monthly, or less frequently, for example, biannually, depend 0121 For administration by inhalation, the azide-modi ing on dosage, method of administration, disorder or symp fied fatty acid, the azide-modified carbohydrate, or the azide tom(s) to be treated, and individual Subject characteristics. modified isoprenoid lipid are delivered in an aerosol spray Dosages can also be administered via continuous infusion from a pressured container or dispenser, which contains a (such as through a pump). The administered dose may also propellant (e.g., liquid or gas) or a nebulizer. In certain depend on the route of administration. For example, Subcu embodiments, the azide-modified fatty acid, the azide-modi taneous administration may require a higher dosage than fied carbohydrate, or the azide-modified isoprenoid lipid is intravenous administration. prepared with a carrier to protect the compounds against rapid 0.126 In certain circumstances, it may be advantageous to elimination from the body. Biodegradable polymers (e.g., formulate compositions in dosage unit form for ease of ethylene vinyl acetate, polyanhydrides, polyglycolic acid, administration and uniformity of dosage. Dosage unit form as collagen, polyorthoesters, polylactic acid) are often used. used herein refers to physically discrete units suited for the Methods for the preparation of such formulations are known patient. Each dosage unit contains a predetermined quantity by those skilled in the art. of azide-modified fatty acid, the azide-modified carbohy 0122. In other embodiments, the composition comprises a drate, or the azide-modified isoprenoid lipid calculated to delivery agent for delivering the azide-modified fatty acid, the produce a therapeutic effect in association with the carrier. azide-modified carbohydrate, or the azide-modified iso The dosage unit depends on the characteristics of the azide prenoid lipid to a cell including but not limited to, a liposome. modified fatty acid, the azide-modified carbohydrate, or the Liposomes (also known as lipid vesicles) are colloidal par azide-modified isoprenoid lipid and the particular therapeutic ticles that are prepared from polar lipid molecules derived effect to be achieved. either from natural sources or chemical synthesis. Such I0127 Toxicity and therapeutic efficacy of the composition spherical, closed structures composed of curved lipid bilay can be determined by standard pharmaceutical procedures in ers, are typically used to entrap drugs, which are often cyto cell cultures or experimental animals, e.g., determining the toxic, in order to reduce toxicity and/or increase efficacy. LDs (the dose lethal to 50% of the population) and the EDs Liposome-entrapped drug preparations are often provided in (the dose therapeutically effective in 50% of the population). a dry (e.g. freeze-dried) form, which is Subsequently recon The dose ratio between toxic and therapeutic effects is the stituted with an aqueous solution immediately prior to admin therapeutic index and it can be expressed as the ratio LDso/ istration. This is done in order to minimize the possibility of EDso. leakage of e.g. cytotoxic drug into aqueous Solution and I0128. The data obtained from the cell culture assays and thereby reducing the entrapping effect of the liposome. animal studies can be used to formulate a dosage range in 0123 Examples of formulations comprising inter alia humans. The dosage of these compounds may lie within the liposome-encapsulated active ingredients are discussed in range of circulating concentrations of the azide-modified U.S. Pat. No. 4,427,649, U.S. Pat. No. 4,522,811, U.S. Pat. fatty acid, azide-modified carbohydrate, or azide-modified No. 4,839,175, U.S. Pat. No. 5,569,464, EP 249 561, WO isoprenoid lipid in the blood, that includes an EDs with little 00/38681, WO 88/01862, WO 98/58629, WO 98/00111, WO or no toxicity. The dosage may vary within this range depend 03/105805, U.S. Pat. No. 5,049,388, U.S. Pat. No. 5,141,674, ing upon the dosage composition form employed and the U.S. Pat. No. 5,498,420, U.S. Pat. No. 5,422,120, WO route of administration. For any azide-modified fatty acid, 87/01586, WO 2005/039533, US 2005/0112199 and U.S. azide-modified carbohydrate, or azide-modified isoprenoid Pat. No. 6.228,393, all of which are hereby incorporated by lipid used in the methods described herein, the therapeutically reference in their entirety. effective dose can be estimated initially using cell culture 0.124. The azide-modified fatty acid, azide-modified car assays. A dose may be formulated in animal models to bohydrate, or azide-modified isoprenoid lipid containing achieve a circulating plasma concentration range that compositions are administered in therapeutically effective includes the ICs (i.e., the concentration of antibody which amounts as described. Therapeutically effective amounts achieves a half-maximal inhibition of symptoms). The effects may vary with the Subject's age, condition, sex, and severity of any particular dosage can be monitored by a suitable bio of medical condition. Appropriate dosage may be determined assay. by a physician based on clinical indications. The azide-modi I0129. The compositions may also contain other active fied fatty acid, azide-modified carbohydrate, or azide-modi compounds providing Supplemental, additional, or enhanced fied isoprenoid lipid containing composition may be given as therapeutic functions. In one embodiment, the composition a bolus dose to maximize the circulating levels of the azide further comprises at least one anti-viral agent, such as a modified fatty acid, azide-modified carbohydrate, or the reverse transcriptase inhibitor, a virus protease inhibitor, a azide-modified isoprenoid lipid for the greatest length of viral fusion inhibitor, a viral integrase inhibitor, a glycosidase time. Continuous infusion may also be used after the bolus inhibitor, an amphotericin B. hydroxyurea, C.-interferon, dose. B-interferon, y-interferon, and an antisense oligonucleotide. 0.125 Examples of dosage ranges that can be administered 0.130. In one embodiment, the at least one reverse tran to a subject can be chosen from: 1 ug/kg to 20 mg/kg, 1 ug/kg Scriptase inhibitor includes, but is not limited to, one or more to 10 mg/kg, 1 g/kg to 1 mg/kg, 10 ug/kg to 1 mg/kg, 10 nucleoside analogs, such as Zidovudine (AZT), Didanosine US 2012/0028335 A1 Feb. 2, 2012

(ddI), Zalcitabine (ddC). Stavudine (d4T), Lamivudine 0.139. No apparent effects of acute or chronic replication (3TC), Abacavir (ABC), Emtricitabine (FTC), Entecavir of HIV on host cellular protein modifications were observed (INN), Apricitabine (ATC), Atevirapine, ribavirin, acyclovir, (FIG. 1, compare label to controls). The azide-modified bio famciclovir, Valacyclovir, ganciclovir, and Valganciclovir, molecules, however, did label viral proteins and permit their one or more nucleotide analogs, such as Tenofovir (tenofovir detection at the expected molecular weights for HIV viral disoproxil fumarate), Adefovir (bis-POM PMPA), PMPA, proteins: 55 KDa (gag-myristoylated); 41 KDa (gp41-palmi and cidofovir, or one or more non-nucleoside reverse tran toylated) and 120 KDa (gp120-N-glycosylated) at 14 days in scriptase inhibitors, such as Efavirenz, Nevirapine, Delavir chronically infected cells (FIG. 1). dine, and Etravirine. 0131. In other embodiments, the at least one viral protease Example 2 inhibitor includes, but is not limited to, tipranavir, darunavir, indinavir, lopinavir, fosamprenavir, atazanavir, saquinavir, Inhibiting Infectivity of HIV ritonavir, indinavir, nelfinavir, and amprenavir. 0132. In other embodiments, the at least one viral fusion 0140. To examine the effect of the azide-modified biomol inhibitor includes, but is not limited to a CD4 antagonist, such ecules on the innate biology of the virus, the azide-labeled as soluble CD4 or an antibody that binds to CD4, such as virus from the transfected cells was isolated and tested in cell TNX-355, BMS-806; a CCR5 antagonist, such as SCH-C, infection studies. Unlabeled HIV, at a concentration 100 SCH-D, UK-427.857, maraviroc, Vicriviroc, or an antibody times less than the test samples, was used as a control. Viral that binds to CCR5, such as PRO-140; a CXCR4 antagonist, loads were normalized to p24 abundance and virus incubated such as, AMD3100 or AMD070; or an antagonist of gp41, for 12 hours on a reporter cell line (TZM/BI). TZM/BI is a such as enfuvirtide. genetically engineered HeLa cell line that expresses CD4. 0133. In other embodiments, the at least one viral inte CXCR4 and CCR5 and contains Tat-inducible luciferase and grase inhibitor includes, but is not limited to, raltegravir. B-Gal reporter genes. Viral infectivity was determined by 0134. In other embodiments, the at least one glycosidase measuring cellular luciferase activities with two different inhibitor includes, but is not limited to, SC-48334 or MDL luciferase reagents. The results using a single cycle replica 28574. tion system showed that virus labeled with the azide-modified 0135 Reference will now be made in detail to various biomolecules, particularly 12-azidododecanoic acid, and to a exemplary embodiments. It is to be understood that the fol lesser extent, 15-azidopentadecanoic acid and tetraacetylated lowing detailed description is provided to give the reader a N-azidoacetylgalactosamine, had a profound impact on the fuller understanding of certain embodiments, features, and infectivity of the virus (FIGS. 3 and 4). The level of inhibition details of aspects of the invention, and should not be inter of viral entry observed was comparable to the level of inhi preted as a limitation of the scope of the invention. bition observed in cells pre-treated with an anti-retroviral agent, Such as a fusion inhibitor or a nucleoside analogue. Example 1 Example 3 Labeling HIV with Azide-Modified Biomolecules 0.136 CEMX 174 cells were transfected with HIV in a Toxicity Profile T-150 flask and the virus production was monitored by reverse transcriptase activity until peak virus production 0141 Little to no toxicity has been observed when using occurred (usually 7 to 9 days post transfection). Prior to these azide-modified biomolecules in various eukaryotic cell transfection, the CEMX174 cells were spiked with the follow lines, suggesting that these compounds have a minimal tox ing azide-modified biomolecules: 15-azidopentadecanoic icity profile and Supporting their use in a therapeutic setting. acid (50-100 uM), 12-azidododecanoic acid (50-100 uM), tetraacetylated N-azidoacetylgalactosamine (20-40 uM), and What is claimed: tetraacetylated N-azidoacetyl-D-mannosamine (20-40 uM). 1. A method of producing a human immunodeficiency 0.137 Infected cells were harvested at 12, 24, 72 hours, virus labeled with an azide-modified fatty acid, an azide and 14 days. Harvested cells were isolated and lysed. Cell modified carbohydrate, or an azide-modified isoprenoid lipid, lysates were then mixed with the CLICK-ITR) detection the method comprising contacting a cell infected with the reagent, tetramethylrhodamine (TAMRA) alkyne and the human immunodeficiency virus with the azide-modified fatty CLICK-ITR Protein Reaction Buffer Kit (Invitrogen, Carls acid, the azide-modified carbohydrate, or the azide-modified bad, Calif.). Cell lysate samples were run on a one-dimen isoprenoid lipid so that the azide-modified fatty acid, the sional gel to monitor changes in the azide labeled proteins azide-modified carbohydrate, or the azide-modified iso over time (FIG. 1). prenoid lipid enters the cell and is incorporated into a protein 0138 Labeled virus was also obtained from the trans fected cells. More specifically, virus-containing Supernatants of the virus, thereby producing the labeled virus. were collected and virus was purified through 20% sucrose as 2. The method of claim 1, wherein the azide-modified fatty previously described (Graham, D. R. et al., Proteomics 2008, acid is a Saturated fatty acid. 8:4919-30). The purified virus was then mixed with the 3. The method of claim 1, wherein the azide-modified fatty CLICK-ITR) detection reagent, tetramethylrhodamine acid is 15-azidopentadecanoic acid. (TAMRA) alkyne and the CLICK-ITR Protein Reaction 4. The method of claim 1, wherein the azide-modified fatty Buffer Kit (Invitrogen, Carlsbad, Calif.). Virus samples were acid is 12-azidododecanoic acid. run on a one-dimensional gel to reveal azide-labeled viral 5. The method of claim 1, wherein the azide-modified proteins (FIG. 2). Virus levels were normalized p24 content carbohydrate is an N-linked carbohydrate or an O-linked and by one-dimensional gel electrophoresis. carbohydrate. US 2012/0028335 A1 Feb. 2, 2012

6. The method of claim 1, wherein the azide-modified 8. The method of claim 7, wherein the azide-modified carbohydrate is tetraacetylated N-azidoacetylgalactosamine, isoprenoid lipid is an azido farnesyl diphosphate, an azido tetraacetylated N-azidoacetyl-D-mannosamine, or tet farnesyl alcohol, an azido geranylgeranyl diphosphate, or an raacetylated N-azidoacetylglucosamine. azido geranylgeranyl alcohol. 9. The method of any one of claims 1-8, wherein the cell is 7. The method of claim 1, wherein the azide-modified a human cell. isoprenoid lipid comprises a farnesyl group or a geranylgera nyl group.