HK1185542B - Anti- viral azide containing compounds - Google Patents

Description

Anti-viral azide-containing compounds

Background

Viral infections cause high morbidity and mortality in humans and animals. In addition, viral infections also result in substantial agricultural losses, with plant viruses estimated to cause crop yield losses worldwide every year at $ 600 billion. Although a large resource has been devoted to identifying compounds with antiviral properties, viral infections still pose significant risks to human health and agriculture.

In addition, the usefulness of most existing antiviral therapies is limited by the development of multidrug resistance, poor efficacy, and/or toxicity. In fact, many antiviral treatments are highly toxic and can cause serious side effects, including heart damage, renal failure, and osteoporosis. Other challenges include: creating a drug that is broadly applicable to combat many different types of viral infections is particularly important in the treatment of immunocompromised individuals.

One virus, specifically the Human Immunodeficiency Virus (HIV), remains prevalent worldwide despite the development of antiretroviral drugs targeting HIV. By 2007, it is estimated that over 3300 million people are infected with HIV, and that HIV-related diseases represent a significant worldwide health problem. HIV is a retrovirus that infects CD4 of the immune system⁺Cells, thereby disrupting or impairing their function. As the infection progresses, the immune system becomes weaker, making the infected person more susceptible to opportunistic infections and tumors, such as kaposi's sarcoma, cervical cancer, lymphoma, and neurological disorders. The most advanced stage of HIV infection is acquired immunodeficiency syndrome (AIDS). It takes 10-15 years for HIV infected people to develop AIDS. Certain antiretroviral drugs may even further delay this process.

Despite the extensive efforts that have been put into designing effective therapeutic agents against HIV, there are currently no curative antiretroviral drugs against HIV. Several stages of the HIV life cycle have been evaluated as targets for the development of therapeutic agents (Mitsuya, H. et al, 1991, FASEB J5: 2369-2381). One area of focus has been the HIV reverse transcriptase. Reverse transcriptase will replicate the single-stranded RNA genome of HIV into double-stranded viral DNA. The viral DNA is then integrated into the chromosomal DNA of the host, where cellular processes of the host (e.g., transcription and translation) are used to produce viral proteins, and ultimately new viral particles. Thus, interfering with reverse transcriptase inhibits the replication capacity of HIV. One class of reverse transcriptase inhibitors are nucleoside analogs such As Zidovudine (AZT), didanosine (ddI), zalcitabine (ddC) and stavudine (d4T), lamivudine (3TC), Abacavir (ABC), emtricitabine (FTC), entecavir (INN) and orecitabine (ATC) (Mitsuya, H. et al, 1991, Science 249: 1533-. Another class of reverse transcriptase inhibitors are nucleotide analogs such as tenofovir (tenofovir disoproxil fumarate) and adefovir (bis-POM PMPA) (Palmer et al, AIDS Res Hum Retroviruses, 2001, 17: 1167-73). These nucleoside and nucleotide compounds are analogs of naturally occurring deoxyribonucleotides, however, the analogs lack the 3' -hydroxyl group on the deoxyribose sugar. As a result, when the analog is incorporated into a growing viral DNA strand, the new deoxynucleotide does not form the phosphodiester bond with the analog required to extend the DNA strand. Thus, the analogs terminate viral DNA replication. Another class of reverse transcriptase inhibitors are non-nucleoside reverse transcriptase inhibitors such as efavirenz, nevirapine, delavirdine, and etravirine (El Safadi et al, apple Microbiol Biotechnol, 2007, 75: 723-37). They have a different mode of action than nucleoside and nucleotide inhibitors: bind to the reverse transcriptase and interfere with its function.

Late stages of HIV replication include: certain viral proteins are processed before final assembly of new virions. This late processing is dependent in part on the activity of viral proteases. Thus, another area of focus in the development of antiretroviral drugs is protease inhibitors such as saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir and atazanavir (Erickson, J., 1990, Science 249: 527-.

Other antiretroviral drugs target viral entry into cells, i.e. HIV-infectedThe earliest stage. In order for HIV to enter a cell, its surface gp120 protein binds to CD4, exposing a conserved region of gp120 that binds to the CCR5 or CXCR4 co-receptor. After gp120 binds to the co-receptor, the hydrophobic fusion peptide at the N-terminus of the gp41 envelope protein is exposed and inserted into the cell's membrane. Entry inhibitors act by interfering with any stage of the viral entry process. For example, it has been demonstrated that, for example, recombinant soluble CD4 inhibits CD4 from certain HIV-1 strains⁺Infection of T-cells (Smith, D.H. et al, 1987, Science 238: 1704-. Similarly, TNX-355 is a monoclonal antibody that binds to CD4 and inhibits binding to gp120 (Kuritzkes et al, JInfect Dis, 2004, 189: 286-91). BMS-806 binds to viral envelope proteins and inhibits binding to CD4 (Veazy et al, Nature2003, 438: 99-102). Coreceptor binding can be inhibited by several CCR5 inhibitors, including SCH-C and SCH-D, UK-427, 857, maraviroc, viriviroc and anti-CCR 5 antibody (PRO-140) (Emmelkamp et al, Eur J Med Res, 2007, 12: 409-17). Co-receptor binding can also be inhibited by the CXCR4 inhibitors AMD3100 and AMD070 (De Clerq, Nature Reviews Drug Discovery2003, 2: 581-87). Other compounds, such as enfuvirtide, bind gp41 and interfere with its ability to mediate membrane fusion and entry (La Bonte et al, Nature Reviews Drug Discovery2003, 2: 345-36).

While beneficial, these antiretroviral drugs often exhibit toxic side effects such as myelosuppression, emesis, and liver dysfunction. In addition, they are not curative, probably due to the rapid emergence of drug-resistant HIV mutants (Lander, B. et al, 1989, Science 243: 1731-1734). Due to the very high genetic variability of HIV, drug resistant HIV strains develop. This genetic variability arises from several factors, including the rapid replication cycle of HIV, where 10 are produced each day⁹To 10¹⁰A virion of approximately 3x10 nucleotides per nucleotide base in each replication cycle^-5High mutation rate, and recombinant gene properties of reverse transcriptase.

To combat the development of drug resistant HIV strains, a variety of drugs have been combined as part of highly effective antiretroviral therapy (HAART) (El Safadi et al, Appl Microbiol Biotechnol, 2007, 75: 723-37; Sharma et al, Cur Top Med Chem, 2004, 4: 895-. Currently, HAART generally includes: at least 3 drugs in combination, said drugs belonging to at least 2 classes of antiretroviral agents. As discussed above, these classes include nucleoside or nucleotide analog reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, and entry inhibitors.

Thus, despite the extensive efforts being directed to the design and testing of antiviral drugs, new and improved methods of treating viral infections (such as HIV) are still being sought.

Disclosure of Invention

The present disclosure provides methods of treating viral infections (such as HIV infection) or labeling proteins of a virus (such as HIV) using azide-modified biomolecules (such as azide-modified fatty acids, azide-modified carbohydrates, azide-modified isoprenoid lipids) or pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions containing the azide-modified biomolecules or pharmaceutically acceptable salts thereof.

One aspect of the present disclosure relates to a method of treating a subject infected with a plant, insect or animal virus and in need of treatment for the infection, the method comprising: administering to the subject a therapeutically effective amount of an azide-modified fatty acid, an azide-modified carbohydrate, an azide-modified isoprenoid lipid, or a pharmaceutically acceptable salt thereof.

In some embodiments, the azide-modified fatty acid or pharmaceutically acceptable salt thereof has the formula:

Y-CH₂-X-CO₂H [I]

wherein the content of the first and second substances,

y is H or azido; and

when Y is azido, X is a linear or branched carbon chain comprising 6-28 carbons, wherein one or more of the carbons may be independently replaced by oxygen, selenium, silicon, sulfur, SO₂Or NR₁Alternatively, or wherein one or more pairs of said carbons adjacent to each other may be connected to each other by a double or triple bond; or

When Y is H, X is a straight or branched carbon chain comprising 6-28 carbons, wherein one hydrogen on one of the carbons is replaced with an azido group, and wherein one or more of the carbons not having an azido group attached thereto may be independently replaced with oxygen, selenium, silicon, sulfur, SO₂Or NR₁Alternatively, or wherein one or more pairs of the carbons adjacent to each other and not having an azido group may be connected to each other by a double or triple bond;

wherein R is₁Is H or alkyl containing 1-6 carbons.

In some of these, Y is an azido group. In some of these, X is a linear carbon chain. In some of these, the linear carbon chain comprises 8 to 15 carbons. In some of these, the linear carbon chain is free of oxygen, selenium, silicon, sulfur, SO₂Or NR₁. In some of these, the carbon chain does not contain double or triple bonds. In some of these, the azide-modified fatty acid is 15-azidopentadecanoic acid, 12-azidododecanoic acid, or a pharmaceutically acceptable salt thereof.

In some embodiments, the azide-modified carbohydrate is an N-linked carbohydrate or an O-linked carbohydrate. In some embodiments, the azide-modified carbohydrate is tetraacetylated N-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine, or tetraacetylated N-azidoacetylglucosamine.

In some embodiments, the azide-modified isoprenoid lipid comprises a farnesyl group or a geranylgeranyl group. In some of these, the azide-modified isoprenoid lipid is azido farnesyl diphosphate, azido farnesol, azido geranylgeranyl diphosphate, or azido geranylgeraniol.

In some embodiments, the virus is a non-human animal virus or a human animal virus.

In some embodiments, the non-human animal virus is a picornavirus, pestivirus, arterivirus, coronavirus, paramyxovirus, orthomyxovirus, riovirus, porcine virus (porcine), circovirus, herpesvirus, african swine fever virus, retrovirus, flavivirus or rhabdovirus.

In some embodiments, the human animal virus is an adenovirus, a astrovirus, a hepadnavirus, a herpes virus, a papovavirus, a poxvirus, an arenavirus, a bunyavirus, a calicivirus (calcivirus), a coronavirus, a filovirus, a flavivirus, an orthomyxovirus, a paramyxovirus, a picornavirus, a rio virus, a retrovirus, a rhabdovirus, or a togavirus. In some of these, the retrovirus is a human immunodeficiency virus or a human T-cell lymphotrophic virus. In some of these, the retrovirus is the human immunodeficiency virus HIV-1.

In some embodiments, the virus is a plant virus. In some of these, the plant virus is alfalfa mosaic virus, green onion virus, alphalatent virus, avian paramyxovirus (anualvirus), apple north american scar viroid, golden virus, oat virus, avocado white spot viroid, baculovirus, bean golden mosaic virus, beet necrotic yellow vein virus, beta latent virus, betaflexviridae, brome mosaic virus, barley yellows mosaic virus, hairy virus, carnation latent virus, moschus mottle virus, cauliflower mosaic virus, cassava vein mosaic virus, cherry filing virus, wireform virus, coconut death viroid, coleus blumei viroid, cowpea mosaic virus, linear virus, cucumber mosaic virus, plant insect virus, qualitative rhabdovirus, carnation virus, bowled cone mosaic virus, umbellifery virus, and B-type satellite virus, fava bean virus, fava virus, canary virus, and B-type satellite virus, Fiji virus, fungal transmissible virus, barley virus, hop stunt viroid, raspberry virus, equiaxed unstable ringspot virus, sweet potato virus, yellow dwarf virus, maize chlorotic mottle virus, orange mosaic virus, maize reynaudian virus, maize streak virus, nano virus, necrosis virus, helminth polyhedrosis virus, nuclear rhabdovirus, oleavirus, citrus scale virus, rice virus, pannico virus, arachis clusterin virus, morning glory vein clearing-like virus, plant reovirus, potato leafroll virus, potato disease, potato spiny tuber viroid, potato virus X, potato virus Y, rio virus, rhabdovirus, lolium mosaic virus, wenzhou mandarin dwarf virus, SbCMV-like virus, companion virus, southern bean mosaic virus, parvovirus, TNsatV-like satellite virus, Tobacco mosaic virus, tomato false top virus, tomato spotted wilt virus, apple chlorosis leaf spot virus, wheat mosaic virus, rice degenerated baculovirus, turnip yellow mosaic virus, umbrella plant virus, megalovirus, grapevine virus or dwarfing virus.

In some embodiments, the virus is an insect virus. In some of these, the insect virus is a densovirus, an iridovirus, a green iridovirus, a baculovirus, a poly-DNA virus, an entomopoxvirus, a vesicular virus, an insect picornavirus, a calicivirus, or a nodavirus.

In some embodiments, the subject is a human animal.

Another aspect of the present disclosure relates to a method of inhibiting viral infectivity, the method comprising: contacting a virus-infected cell with an azide-modified fatty acid, an azide-modified carbohydrate, an azide-modified isoprenoid lipid, or a pharmaceutically acceptable salt thereof in an amount effective to inhibit viral infectivity.

In some embodiments, the azide-modified fatty acid or azide-modified fatty acidThe pharmaceutically acceptable salt thereof has the formula [ I ] as described above]. In some of these, Y is an azido group. In some of these, X is a linear carbon chain. In some of these, the linear carbon chain comprises 8 to 15 carbons. In some of these, the linear carbon chain is free of oxygen, selenium, silicon, sulfur, SO₂Or NR₁. In some of these, the carbon chain does not contain double or triple bonds. In some of these, the azide-modified fatty acid is 15-azidopentadecanoic acid, 12-azidododecanoic acid, or a pharmaceutically acceptable salt thereof.

In some embodiments, the azide-modified carbohydrate is an N-linked carbohydrate or an O-linked carbohydrate. In some embodiments, the azide-modified carbohydrate is tetraacetylated N-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine, or tetraacetylated N-azidoacetylglucosamine.

In some embodiments, the azide-modified isoprenoid lipid comprises a farnesyl group or a geranylgeranyl group. In some of these, the azide-modified isoprenoid lipid is azido farnesyl diphosphate, azido farnesol, azido geranylgeranyl diphosphate, or azido geranylgeraniol.

In some embodiments, the virus is a non-human animal virus or a human animal virus.

In some embodiments, the virus is a non-human animal virus. In some embodiments, the non-human animal virus is a picornavirus, pestivirus, arterivirus, coronavirus, paramyxovirus, orthomyxovirus, rioo virus, swine, circovirus, herpesvirus, african swine fever virus, retrovirus, flavivirus or rhabdovirus.

In some embodiments, the virus is a human animal virus. In some of these, the human animal virus is an adenovirus, a astrovirus, a hepadnavirus, a herpesvirus, a papovavirus, a poxvirus, an arenavirus, a bunyavirus, a calicivirus, a coronavirus, a filovirus, a flavivirus, an orthomyxovirus, a paramyxovirus, a picornavirus, a rio virus, a retrovirus, a rhabdovirus, or a togavirus. In some of these, the retrovirus is a human immunodeficiency virus or a human T-cell lymphotrophic virus. In some of these, the retrovirus is the human immunodeficiency virus HIV-1.

In some embodiments, the virus is a plant virus. In some of these, the plant virus is alfalfa mosaic virus, green onion virus, alphalatent virus, avian paramyxovirus, apple north american scar viroid, aurora virus, oat virus, avocado white spot viroid, baculovirus, bean golden mosaic virus, beet necrotic yellow vein virus, beta latent virus, beta flexviridae, brome mosaic virus, barley yellowing mosaic virus, hairy virus, carnation latent virus, moschus mottle virus, cauliflower mosaic virus, cassava vein mosaic virus, cherry rasped leaf virus, filovirus, coconut death viroid, coleus, cowpea mosaic virus, linear virus, cucumber virus, plant insect virus, rhabdovirus, carnation virus, bowled pea mosaic virus, umbellifere mosaic virus, and B-type satellite virus, fava bean virus, economy virus, alphavirus, betavirus, beta-type necrotic virus, beta-type mosaic virus, barley yellow mosaic virus, hairy virus, moso virus, carnation latent virus, moschata moso virus, a, Fungal infections, barley viruses, hop stunt viroids, raspberry viruses, equiaxed unstable ringspot viruses, sweet potato viruses, yellow dwarf viruses, maize chlorotic mottle viruses, orange mosaic viruses, maize reyadophtaviruses, maize streak viruses, nano-viruses, necrosis viruses, helminthic polyhedrosis viruses, nuclear rhabdoviruses, oleavirus, citrus scale viruses, rice viruses, panicoviridae viruses, peanut clusterin viruses, marjoram mosaic virus, plant reoviruses, potato leafroll viruses, potato spindle tuber viroids, potato virus X, potato virus Y, rio virus, rhabdoviruses, ryegrass mosaic viruses, wenzhou mandarin dwarf viruses, SbCMV-like viruses, morning glory mosaic viruses, lentivirus, parvovirus, TNsatV-like satellite viruses, motto-type viruses, and combinations thereof, Tobacco mosaic virus, tomato false top virus, tomato spotted wilt virus, apple chlorosis leaf spot virus, wheat mosaic virus, rice degenerated baculovirus, turnip yellow mosaic virus, umbrella plant virus, megalovirus, grapevine virus or dwarfing virus.

In some embodiments, the virus is an insect virus. In some of these, the insect virus is a densovirus, an iridovirus, a green iridovirus, a baculovirus, a poly-DNA virus, an entomopoxvirus, a vesicular virus, an insect picornavirus, a calicivirus, or a nodavirus.

In some embodiments, the cell is a human cell.

A third aspect of the present disclosure relates to a method of producing a virus labeled with an azide-modified fatty acid, an azide-modified carbohydrate, an azide-modified isoprenoid lipid, or a pharmaceutically acceptable salt thereof, the method comprising: contacting a virus-infected cell with the azide-modified fatty acid, the azide-modified carbohydrate, the azide-modified isoprenoid lipid, or pharmaceutically acceptable salt thereof, such that the azide-modified fatty acid, the azide-modified carbohydrate, the azide-modified isoprenoid lipid, or pharmaceutically acceptable salt thereof enters the cell and is incorporated into proteins of the virus, thereby producing a labeled virus.

In some embodiments, the method is a method of producing a human immunodeficiency virus labeled with an azide-modified fatty acid, an azide-modified carbohydrate, an azide-modified isoprenoid lipid, or pharmaceutically acceptable salt thereof, the method comprising: contacting a cell infected with the human immunodeficiency virus with the azide-modified fatty acid, the azide-modified carbohydrate, the azide-modified isoprenoid lipid, or pharmaceutically acceptable salt thereof, such that the azide-modified fatty acid, the azide-modified carbohydrate, the azide-modified isoprenoid lipid, or pharmaceutically acceptable salt thereof enters the cell and is incorporated into proteins of the virus, thereby generating a labeled virus.

In some embodiments, the azide-modified fatty acid or pharmaceutically acceptable salt thereof has the formula [ I ] as described above]. In some of these, Y is an azido group. In some of these, X is a linear carbon chain. In some of these, the linear carbon chain comprises 8 to 15 carbons. In some of these, the linear carbon chain is free of oxygen, selenium, silicon, sulfur, SO₂Or NR₁. In some of these, the carbon chain does not contain double or triple bonds. In some of these, the azide-modified fatty acid is 15-azidopentadecanoic acid, 12-azidododecanoic acid, or a pharmaceutically acceptable salt thereof.

In some embodiments, the azide-modified carbohydrate is an N-linked carbohydrate or an O-linked carbohydrate. In some embodiments, the azide-modified carbohydrate is tetraacetylated N-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine, or tetraacetylated N-azidoacetylglucosamine.

In some embodiments, the azide-modified isoprenoid lipid comprises a farnesyl group or a geranylgeranyl group. In some of these, the azide-modified isoprenoid lipid is azido farnesyl diphosphate, azido farnesol, azido geranylgeranyl diphosphate, or azido geranylgeraniol.

In some embodiments, the cell is a human cell.

In some embodiments, the virus is a human immunodeficiency virus, while in other embodiments, the virus is a baculovirus.

In some embodiments, the azide-modified carbohydrate, azide-modified fatty acid, azide-modified isoprenoid lipid, or pharmaceutically acceptable salt thereof is formulated with a pharmaceutically acceptable excipient.

In some embodiments, the method additionally comprises the steps of: administering to the cells the azide-modified carbohydrate, azide-modified fatty acid, azide-modified isoprenoid lipid, or pharmaceutically acceptable salt thereof formulated with a pharmaceutically acceptable excipient.

A fourth aspect of the present disclosure relates to a method of tracking a virus in vivo, the method comprising the steps of: contacting cultured cells or a subject with an azide-modified carbohydrate, an azide-modified fatty acid, an azide-modified isoprenoid lipid, or a pharmaceutically acceptable salt thereof; contacting the cultured cells or the subject with an alkyne-labeled reporter molecule; and tracking the reporter-labeled virus in the cultured cells or the subject.

In some embodiments, the cultured cells or the subject are contacted with an azide-modified fatty acid or a pharmaceutically acceptable salt thereof.

In some embodiments, the azide-modified fatty acid or pharmaceutically acceptable salt thereof has the formula [ I ] as described above]. In some embodiments, Y is an azido group. In some of these, X is a linear carbon chain. In some of these, the linear carbon chain comprises 8 to 15 carbons. In some of these, the linear carbon chain is free of oxygen, selenium, silicon, sulfur, SO₂Or NR₁. In some of these, the carbon chain does not contain double or triple bonds. In some of these, the azide-modified fatty acid is 15-azidopentadecanoic acid, 12-azidododecanoic acid, or a pharmaceutically acceptable salt thereof.

Another aspect of the disclosure relates to a pharmaceutical composition comprising an azide-modified fatty acid, an azide-modified carbohydrate, an azide-modified isoprenoid lipid, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

In some embodiments, the azide-modified fatty acid or pharmaceutically acceptable salt thereof has the formula [ I ] as described above]. In some of these, Y is an azido group. In some of these, X is a linear carbon chain. In some of these, the linear carbon chain comprises 8 to 15 carbons. In some of these, the linear carbon chain is free of oxygen, selenium, silicon, sulfur, SO₂Or NR₁. In some of these, the carbon chain does not contain double or triple bonds. In some of these, the azide-modified fatty acid is 15-azidopentadecanoic acid, 12-azidododecanoic acid, or a pharmaceutically acceptable salt thereof.

In some embodiments, the azide-modified carbohydrate is an N-linked carbohydrate or an O-linked carbohydrate. In some embodiments, the azide-modified carbohydrate is tetraacetylated N-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine, or tetraacetylated N-azidoacetylglucosamine.

In some embodiments, the azide-modified isoprenoid lipid comprises a farnesyl group or a geranylgeranyl group. In some of these, the azide-modified isoprenoid lipid is azido farnesyl diphosphate, azido farnesol, azido geranylgeranyl diphosphate, or azido geranylgeraniol.

In some embodiments, the composition further comprises at least one antiviral agent. In some of these, the antiviral agent is selected from: reverse transcriptase inhibitors, viral protease inhibitors, viral fusion inhibitors, viral integrase inhibitors, glycosidase inhibitors, viral neuraminidase inhibitors, M2 protein inhibitors, amphotericin B, hydroxyurea, alpha-interferon, beta-interferon, gamma-interferon, and antisense oligonucleotides. In some of these, the reverse transcriptase inhibitor is at least one of: zidovudine (AZT), didanosine (ddI), zalcitabine (ddC), ddA, stavudine (d4T), lamivudine (3TC), Abacavir (ABC), emtricitabine (FTC), entecavir (INN), Aliscitabine (ATC), atevirine, ribavirin, acyclovir, famciclovir, valacyclovir, ganciclovir, valacyclovir, tenofovir, adefovir, PMPA, cidofovir, efavirenz, nevirapine, delavirdine, or etravirine; wherein the viral protease inhibitor is at least one of: tipranavir, darunavir, indinavir, lopinavir, furinavir, atazanavir, saquinavir, ritonavir, indinavir, nelfinavir or amprenavir; wherein the viral fusion inhibitor is at least one of: a CD4 antagonist, a CCR5 antagonist, a CXCR4 antagonist, or an enfuvirtide; wherein the viral integrase is Latiravir; wherein the glycosidase inhibitor is at least one of: SC-48334 or MDL-28574; wherein the viral neuraminidase inhibitor is at least one of: oseltamivir, peramivir, zanamivir, and laninamivir; and wherein the M2 protein inhibitor is at least one of: amantadine or rimantadine (rimantadine).

In some embodiments, the composition further comprises a reagent for delivering the azide-modified fatty acid, the azide-modified carbohydrate, the azide-modified isoprenoid lipid, or pharmaceutically acceptable salt thereof to a cell. In some of these, the reagent for delivering the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid to the cell.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments of the invention and, together with the written description, serve to explain certain principles of the invention.

FIG. 1 shows the time course of azide-modified proteins in HIV-infected CEMx174 cells. Cells infected with CEMx174 were labeled with: (A) 15-azidopentadecanoic acid, (B) 12-azidododecanoic acid, (C) tetraacetylated N-azidoacetylgalactosamine or (D) tetraacetylated N-azidoacetyl-D-mannosamine, and harvested at 12, 24, 72 hours and 14 days post infection. Fig. 1(E) shows a representative gel after staining with the following total protein stain:protein stain (Sigma-Aldrich, st.

FIG. 2 shows gel electrophoresis of HIV-derived azide-modified proteins produced from chronically infected CEMx174 cells. FIG. 2(A) shows viral proteins labeled with tetraacetylated N-azidoacetyl-D-mannosamine (Man), tetraacetylated N-azidoacetylgalactosamine (GalNaz), 15-azidopentadecanoic acid (Palmitic), 12-azidododecanoic acid (Myristic), and labeled with TAMRA. FIG. 2(B) shows the useTotal protein staining of protein stain (Sigma-Aldrich, st.

FIG. 3 shows the results of a luciferase reporter assay (Applied biosystems luciferase reagent) for measuring the infectivity of unlabeled HIV (control) or HIV labeled with 15-azidopentadecanoic acid (PALM), 12-azidododecanoic acid (MYR), tetraacetylated N-azidoacetyl-D-Mannosamine (MAN), or tetraacetylated N-azidoacetylgalactosamine (GAL).

FIG. 4 shows the results of a luciferase reporter assay (Promega luciferase reagent) for measuring the infectivity of unlabeled HIV (control) or HIV labeled with 15-azidopentadecanoic acid (PALM), 12-azidododecanoic acid (MYR), tetraacetylated N-azidoacetyl-D-Mannosamine (MAN), or tetraacetylated N-azidoacetylgalactosamine (GAL).

Figure 5 shows the results of the effect of post-translational modification (PTM) analogue incorporation on the ability of BacMam to enter mammalian cells. The panels show the phase (next panel) and fluorescent GFP images (upper panel) of U2-OS cells infected with BacMam virus labeled with PTM analog.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In order that the invention may be more readily understood, certain terms are first defined. Other definitions are set forth in the detailed description. In case of conflict, the present specification, including definitions, will control.

As used herein, "azide-modified fatty acid" means a fatty acid that: which comprises an azido group and has the formula R-N₃Wherein R comprises a hydrocarbon chain having at least one carboxylic acid functional group, typically (although not necessarily) in a terminal position.

As used herein, "azide-modified carbohydrate" means a carbohydrate that: which comprises an azido group and has the formula R-N₃Wherein R is a carbohydrate.

As used herein, "azide-modified isoprenoid lipid" refers to an isoprene-containing lipid or derivative thereof. The azide-modified isoprenoid comprises an azide group and has the formula R-N₃Wherein R is an isoprene containing lipid, such as C₁₅Farnesyl isoprenoid lipid or C₂₀Geranylgeranyl isoprenoid lipids or derivatives thereof, including, but not limited to, azido farnesyl diphosphate, azido farnesol, azido geranylgeranyl diphosphate, or azido geranylgeraniol.

"animal virus" as used herein means a virus that infects cells of a non-human animal or human animal. Non-human animal viruses infect non-human animal cells. In some cases, viruses that infect non-human animal cells are also capable of infecting human animal cells. Human animal viruses infect human animal cells. In some cases, viruses that infect human animal cells are also capable of infecting non-human animal cells.

"biomolecule" as used herein means a protein, peptide, amino acid, glycoprotein, nucleic acid, nucleotide, nucleoside, oligonucleotide, sugar, oligosaccharide, lipid, hormone, proteoglycan, carbohydrate, polypeptide, polynucleotide, polysaccharide, which has the typical characteristics of a molecule found in a living organism and may be naturally occurring or may be artificial (not occurring in nature and being different from a molecule found in nature).

"click chemistry" as used herein means the Huisgen cycloaddition or the copper (I) -catalyzed variant of 1, 3-dipolar cycloaddition between azide and terminal alkyne (to form 1, 2, 4-triazole). Such chemical reactions can use, but are not limited to, simple heteroatom organic reactants, and are reliable, selective, stereospecific, and exothermic.

As used herein, "cycloaddition" refers to a chemical reaction: wherein 2 or more pi (pi) -electron systems (e.g., unsaturated molecules or unsaturated moieties of the same molecule) combine to form a cyclic product in which there is a net reduction in bond diversity. In cycloaddition, pi (pi) electrons are used to form new pi (pi) bonds. The products of cycloaddition are referred to as "addition compounds" or "cycloaddition compounds". Different types of cycloaddition are known in the art, including, but not limited to [3+2] cycloaddition and Diels-Alder reactions. The [3+2] cycloaddition (which is also known as 1, 3-dipolar cycloaddition) occurs between a1, 3-dipole and a dipole-philic species and is commonly used to construct 5-membered heterocycles. The term "[ 3+2] cycloaddition" also includes addition by Bertozzi et al, j.am.chem.soc., 2004, 126: 15046-15047 describes the "copper-free" [3+2] cycloaddition between azides and cyclooctynes and difluorocyclooctynes.

"DNA virus" as used herein means a virus having deoxyribonucleic acid (DNA) as its genetic material. DNA viruses are typically double-stranded, but may also be single-stranded.

"glycoprotein" as used herein means a protein that has been glycosylated, and those proteins that have been enzymatically modified in vivo or in vitro to contain a sugar group.

As used herein, "HIV" and "human immunodeficiency virus" refer to the human immunodeficiency viruses 1 and 2(HIV-1 and HIV-2).

"infectivity" as used herein refers to the ability of a virus to enter or leave a cell.

"insect virus" as used herein means a virus that infects insect cells. Certain insect viruses, e.g., unmodified baculoviruses or modified baculoviruses (BacMam), may also infect non-human animals and/or human animal cells.

"plant virus" as used herein means a virus that infects plant cells.

As used herein, "pharmaceutically acceptable excipients" include: pharmaceutically compatible solvents, dispersion media, diluents, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of these agents for pharmaceutically active substances is well known in the art.

As used herein, "protein" and "polypeptide" are used in a generic sense to include polymers of amino acid residues of any length. The term "peptide" is used herein to refer to a polypeptide that: it has less than 100 amino acid residues, typically less than 10 amino acid residues. The term applies to amino acid polymers: artificial chemical analogs in which one or more amino acid residues is a corresponding naturally occurring amino acid are also suitable for use in naturally occurring amino acid polymers.

As used herein, a "reporter molecule" refers to any moiety that is capable of being linked to and detected directly or indirectly from a post-translationally modified protein of the invention. Reporter molecules include, but are not limited to: chromophores, fluorophores, fluorescent proteins, phosphorescent dyes, tandem dyes, a-particles, haptens, enzymes, and radioisotopes. Preferred reporter molecules include fluorophores, fluorescent proteins, haptens, and enzymes.

"RNA virus" as used herein means a virus having ribonucleic acid (RNA) as its genetic material. RNA viruses are typically single-stranded, but may also be double-stranded.

The term "subject" as used herein is intended to include human and non-human animals, plants and insects. The subject may include a human patient having a viral infection, including, but not limited to, an HIV infection. The term "non-human animal" of the present invention includes all vertebrates such as non-human primates, sheep, dogs, cats, cows, goats, horses, chickens, pigs, amphibians, reptiles and the like.

"treatment" as used herein means either therapeutic or prophylactic measures. The treatment may be administered to a subject having a disorder (which may include, but is not limited to, a medical disorder where the subject is an animal) or may ultimately be afflicted with a disorder, in order to prevent, cure, delay, lessen the severity of and/or ameliorate one or more symptoms of the disorder or recurrent disorder, or in order to prolong the survival of the subject beyond that which would have been expected in the absence of such treatment.

As used herein, "therapeutically effective amount" or "effective amount" refers to the amount of such compound: when administered to a non-human animal or human animal, plant, insect or other subject for the treatment of a disease, it is sufficient to effect such treatment of the disease. The "effective amount" will vary with the compound, the disease and its severity, as well as the age, weight, etc., of the subject to be treated.

It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a virus" includes a plurality of viruses unless the context indicates otherwise.

While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Detailed Description

The present disclosure relates to: the use of azide-modified biomolecules, such as fatty acids or carbohydrates, for the treatment of viral infections, and pharmaceutical compositions containing the azide-modified biomolecules. Azide-modified fatty acids, azide-modified carbohydrates, and azide-modified isoprenoid lipids have been previously described as reagents useful for labeling and detecting target proteins as part of a click chemistry reaction that includes a copper (I) -catalyzed cycloaddition reaction between an azide and an alkyne. For metabolic labeling reagents for proteins, see(Invitrogen, Carlsbad, Calif.); see also: U.S. patent application publication No. 2007/0249014 and U.S. patent application publication No. 20050222427, the disclosures of which are hereby incorporated by reference in their entireties. However, applicants have surprisingly found that these azide-modified biomolecules have antiviral activity and can be used to treat viral infections. It was surprisingly found that these azide-modified biomolecules can profoundly affect viral infectivity, and that labeling viruses with these azide-modified biomolecules inhibits viral entry into host cells. Is not desired to be receivedWithout being bound by theory, post-translational modification of viral proteins with azido-modified biomolecules at sites normally occupied by unmodified biomolecules (such as saturated fatty acids, e.g., myristic acid and palmitic acid) appears to result in inhibition of viral infectivity in a manner similar to the absence of these biomolecules at these sites.

Click chemistry

The azide and terminal or internal alkyne can undergo a1, 3-dipolar cycloaddition (Huisgen cycloaddition) reaction to produce a1, 2, 3-triazole. However, the reaction requires a long reaction time and a high temperature. Alternatively, the azide and terminal alkyne can undergo a copper (I) -catalyzed azide-alkyne cycloaddition (CuAAC) at room temperature. Such copper (I) -catalyzed azide-alkyne cycloadditions (also known as click chemistry) are variants of Huisgen1, 3-dipolar cycloadditions, in which an organic azide and a terminal alkyne react to generate the 1, 4-position isomer of 1, 2, 3-triazole. Sharpless et al describe examples of click chemistry reactions (U.S. patent application publication No. 20050222427, PCT/US 03/17311; Lewis W G, et al, Angewandte Chemie-Int' Ed.41 (6): 1053; methods reviewed in Kolb, H.C., et al, Angew. chem. Inst. Ed.2001, 40: 2004-2021) that developed reagents that reacted with each other in high yield and with minimal side reactions in heteroatom linkages (as opposed to carbon-carbon bonds) in order to create libraries of chemical compounds.

Click chemistry has been used to label and detect target proteins. For example,the (Invitrogen, Carlsbad, CA) reaction is a two-step labeling technique that involves: modified metabolic precursors (such as azide-modified fatty acids, azide-modified carbohydrates, or azide-modified isoprenoid lipids) are incorporated into proteins as chemical "handles" followed by chemoselective ligation (or "click" reactions) between azides and alkynes. In a click reaction with the corresponding azide-or alkyne-containing dye orThe hapten detects the modified protein.Metabolic labeling reagents have been used to monitor post-translational modifications of proteins, such as acylation, glycosylation, and prenylation, and include: 1) azide-modified fatty acids, e.g.Palmitic acid azide (i.e., 15-azidopentadecanoic acid) andmyristic acid azide (i.e., 12-azidododecanoic acid), which is used to label palmitoylated and myristoylated proteins, respectively; 2) azide-modified carbohydrates including for labelling O-linked glycoproteins(tetraacetylated N-azidoacetylgalactosamine) for labeling sialic acid-modified glycoproteins(tetraacetylated N-azidoacetyl-D-mannosamine), and method for labeling O-GlcNAz-modified glycoproteins(tetraacetylated N-azidoacetylglucosamine); and 3) azide-modified isoprenoid lipids, such asFarnesol azides andgeranylgeraniol azide. As noted above, applicants have surprisingly found that these azide-modified biomolecules have antiviral activity and can be used to treat viral infections.

Glycosylation

Glycosylation is an enzymatic process in which carbohydrates are attached to proteins, lipids, or other organic molecules in a cell. Glycoproteins are biomolecules consisting of proteins covalently linked to carbohydrates. Certain post-translational modifications add sugar moieties (carbohydrates) to the protein, thereby forming a glycoprotein. Common monosaccharides found in glycoproteins include, but are not limited to: glucose, galactose, mannose, fucose, xylose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and N-acetylneuraminic acid (NANA, also known as sialic acid). N-acetyl-D-mannosamine (ManNAc) is a precursor of neuraminic acid, including NANA. 2 identical or different monosaccharides may be linked together to form a disaccharide. The addition of more monosaccharides results in the formation of oligosaccharides of increasing length. In addition, the sugar moiety may be a sugar group.

In glycoproteins, carbohydrates can be linked to the protein component by either N-glycosylation or O-glycosylation. N-glycosylation typically occurs through the nitrogen on the asparagine or arginine side chain, forming an N-glycosidic bond through the amide group. O-glycosylation typically occurs at the hydroxyl oxygen of the side chain of hydroxylysine, hydroxyproline, serine, tyrosine, or threonine, forming an O-glycosidic bond. GalNAc and GlcNAc are both O-linked carbohydrates. Sialic acids are present on both N-and O-linked carbohydrates.

Protein glycosylation is one of the most abundant post-translational modifications and plays an important role in the control of biological systems. For example, glycosylation can affect protein folding, and can help stabilize proteins and prevent their degradation. Glycosylation can also affect the ability of a protein to bind to other molecules and mediate intracellular or intercellular signaling pathways. For example, carbohydrate modifications are important for host-pathogen interactions, inflammation, development and malignancy (Varki, A. glycobiology 1993, 3, 97-130; Lasky, L.A. Annu. Rev. biochem.1995, 64, 113-. One such covalent modification is O-GlcNAc glycosylation, which is a covalent modification of serine and threonine residues by D-N-acetylglucosamine (Wells, L.; Vosseller, K.; Hart, G.W.Science2001, 291, 2376-. The O-GlcNAc modification is present in all higher eukaryotic organisms from caenorhabditis elegans to humans and has been shown to be ubiquitous, inducible and highly dynamic, suggesting a similar regulatory role to phosphorylation.

Fatty acid acylation

Fatty acid acylation is an enzymatic process in which fatty acids are linked to proteins in cells. This process can affect the function of the protein and its cellular localization and is common to proteins of both cellular and viral origin (Towler et al, Proc Natl Acad Sci USA1986, 83: 2812-16). Myristic acid and palmitic acid are the 2 most common fatty acids attached to proteins (Olson et al, J Biol Chem261 (5): 2458-66). Generally, myristic acid is linked via an amide bond to the amino-terminal glycine of soluble and membrane proteins (which is exposed during removal of the N-methionine residue), although it may also be linked to other amino acids. Myristoylation can also occur post-translationally, for example, when proteases cleave polypeptides and expose glycine residues. Palmitic acid is linked to membrane proteins via ester or thioester linkages. Myristoylation and palmitoylation appear to play an important role in subcellular transport of proteins between membrane compartments, as well as in regulating protein-protein interactions.

Fatty acids have 2 distinct regions: long hydrophobic hydrocarbon chains and carboxylic acid groups, which are typically ionized in solution (COO-), are extremely hydrophilic, and readily form esters and amides. Natural fatty acids typically have a chain of 4-28 carbons (usually straight, even limited), and may be saturated or unsaturated. Saturated fatty acids do not contain double bonds in the hydrocarbon chain and include lauric, myristic, palmitic, stearic, and arachidic acids. Unsaturated fatty acids contain at least one double bond in the hydrocarbon chain and include myristoleic acid, palmitoleic acid, hexadecen-6-oic acid, oleic acid, linoleic acid, alpha-linoleic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.

Prenylation

Protein prenylation involves the attachment of isoprenoid lipids (such as farnesyl or geranyl-geranyl moieties) to the C-terminal cysteine of a target protein (McTaggert, Cell Mol Life Sci 2006, 63: 255-67). These reactions are catalyzed by farnesyl transferase, geranylgeranyl transferase and Rab geranylgeranyl transferase (Magee and Seabra, Bioch em J2003, 376: e 3-4). Due to the hydrophobic nature of isoprenoid lipids, most prenylated proteins bind to membranes. Most farnesylated proteins are involved in cell signaling (where membrane binding is important for function). Isoprenoid lipids are also important for mediating protein-protein binding through a specialized prenyl binding domain.

Post-translational modification in viruses

Many viral proteins are extensively modified by post-translational modifications including, but not limited to, glycosylation, acylation, and prenylation. In many cases, these post-translational modifications are required for the virus to infect the host cell and/or to evade the immune system. Post-translational modifications are particularly important in virology because, in general, the viral genome is small and there is thus an enhanced frugal pressure to encode. By taking advantage of the host's post-translational mechanisms, viruses can develop multiple pathways and act with a minimal genome, as a single post-translational modification can alter the function of a protein or the location of a cell.

For example, in HIV and Simian Immunodeficiency Virus (SIV), glycosylation plays an important role in multiple stages of the infectious cycle. During infection, viral glycoproteins affect the binding of the viral proteins gp120 and gp41 to the host cell CD4 receptor and the CXCR4 and CCR5 co-receptors (Chen et al, Virus Res2001, 79: 91-101). Glycosylation is responsible for the proper folding and processing of gp160 (a precursor of gp120 and gp 41) (Land et al, Biochimie 2001, 83: 783-90) and can enhance the interaction of HIV and SIV with different cell types, including dendritic cells (Geijtenbeek et al, Curr Top Microbiol Immunol2003, 276: 31-54). The normal role of gp120 in HIV biology is to initiate viral binding to cells via the CD4 receptor and CXCR4 and CCR5 co-receptors expressed on target cells. When gp120 binds CD4, a conformational change occurs in gp120 that exposes the co-receptor binding site and triggers a conformational change in gp 41. Conformational changes in gp41 in turn reveal fusion peptides in gp41 that mediate fusion between the viral envelope and the target cell (Chen et al, Virus Res2001, 79: 91-101). A carbohydrate change at a single residue (N197) in gp120 will drastically change the viral tropism from CD4 tropism to CD4 independent (Kolchinsky et al, J Virol 2001, 75: 3435-43). Varying the overall ratio of high mannose versus complex carbohydrates (containing sialic acid) present in gp120 affects the extent of binding of the virus to the target cell (Fenouillet et al, JGen Virol 1991, 1919-26). Following infection, the envelope precursor protein (gp160) requires glycosylation for cleavage into gp120 and gp 41. Glycosylation is also important for immune evasion after release of the virus from infected cells, since changes in envelope glycosylation significantly alter the humoral immune response to the virus (Kwong et al, Nature2002, 420: 678-82; Shi et al, J Gen Virol2005, 86: 3385-96).

Acylation of viral proteins is also important for HIV biology. HIV budding is a complex process involving the coordination of many cellular and viral proteins (Resh trends Microbiol 2001, 9: 57; free, J Virol 2002, 76: 4679-87). HIV budding was directed to a region of the plasma membrane that is rich in membrane rafts (Lindwasser et al, J Virol 2001, 75: 7913-24; Nguyen et al, J Virol 2000, 74: 3264-72; Ono et al, Proc Natl Acad Sci USA 2001, 98: 13925-30; Hermida-Matsumoto et al, J Virol 2000, 74: 8670-79), previously known as lipid rafts (Pike et al, ipid Res2006, 47: 1597-98), by myristoylation of the N-terminal glycine of the capsid protein polyprotein precursor (pr55gag), JLl et al, J Virol, 75: 7913-24; Nguyen et al, J Virol 2000, 74: 3264-72; Ono et al, Proc Natl Acad Sci USA, 98: 13925). The gp120 protein is directed to membrane rafts by palmitoylation (Yang et al, Proc Natl Acad Sci USA 1995, 92: 9871-75). Membrane rafts play an important role in several cellular processes including endocytosis, vesicle trafficking, cholesterol sorting, apoptosis and signaling through T cell receptors (Jordan et al, JImmunol 2003, 171: 78-87; Viola et al, Apmis 1999, 107: 615-23; Viola et al, Science 1999, 283: 680-82; Bezombes et al, Curr Med Chem Anti-Cangene 2002, 3: 263-70; Kabouris et al, Eur J Immunol2000, 30: 954-63). The direction of HIV proteins to these regions may allow virions to hijack these pathways more effectively, thus possibly accounting for the complex pathogenicity associated with disease progression in AIDS. Indeed, the removal of cholesterol (an important membrane raft component) from HIV particles leads to inactivation by at least 2 mechanisms: loss of fusion with target cells, and loss of virion integrity (resulting in permeabilization of the virus) (Guyader et al, J Virol 2002, 76: 10356-64; Campbell et al, J Virol 2004, 78: 10556-65; Viard et al, J Virol 2002, 76: 11584-595; Campbell et al, Aids2002, 16: 2253-61; Liao et al, AIDS Res Hum Retroviruses 2003, 19: 675-87; Graham et al, J Vorol 2003, 77: 8237-48).

Viruses can also use host cell mechanisms to modify viral proteins by the addition of isoprenoid lipids such as farnesyl and geranylgeranyl. For example, prenylation plays an important role in the life cycle of the hepatitis virus (HDV), which is the causative agent of acute and chronic liver diseases associated with hepatitis B virus (Einav and Glenn, Japan biological chemistry 2003, 52: 883-86). One of the HDV proteins, the large antigen (LHDAg), is critical for virus assembly and farnesylation occurs in vitro translation systems and in intact cells (Einav and Glenn, J Antimicrobial Chemotherapy2003, 52: 883-86). Inhibition of prenylation by the use of farnesyl transferase inhibitors would prevent HDV assembly and clear HDV viremia in a mouse model of HDV, thus underscoring the importance of prenylation in the life cycle of certain viruses (Einav and Glenn, J Antimicrobial chemistry 2003, 52: 883-86).

Similar to HIV, SIV and HDV, other viruses rely on post-translational modification of viral proteins to mediate entry into host cells and/or to evade the host immune system. Thus, the azide-modified fatty acids, azide-modified carbohydrates, and azide-modified isoprenoid lipids described herein are expected to have broad spectrum antiviral activity (such as regulatory activity by inhibiting or preventing reverse transcription of the HIV viral genome, late processing of certain viral proteins prior to final assembly of new virions, or entry of the virus into cells), and may be useful in treating a variety of viral infections.

Application method

1. Methods of treating viral infections

The present disclosure provides a method of treating a plant, insect or animal infected with a virus, the method comprising: administering to the plant, insect or animal an effective amount of an azide-modified fatty acid, an azide-modified carbohydrate, or an azide-modified isoprenoid lipid. In one embodiment, the azide-modified fatty acid is a saturated fatty acid, such as 15-azidopentadecanoic acid or 12-azidododecanoic acid. In another embodiment, the azide-modified carbohydrate is an N-linked carbohydrate or an O-linked carbohydrate. In another embodiment, the azide-modified carbohydrate is N-azidoacetylgalactosamine, N-azidoacetyl-D-mannosamine, or N-azidoacetylglucosamine. The azide-modified carbohydrate optionally comprises a moiety that facilitates entry into a cell, including, but not limited to, a tetraacetyl moiety. Thus, in another embodiment, the azide-modified carbohydrate is tetraacetylated N-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine or tetraacetylated N-azidoacetylglucosamine. In another embodiment, the isoprenoid lipid comprises a farnesyl or geranylgeranyl group and includes, but is not limited to: azidofarnesyl diphosphate, azidofarnesol, azidogeranylgeranyl diphosphate or azidogeranylgeranyl alcohol.

The virus may be a plant virus, an insect virus or an animal virus. In certain embodiments, the animal is a human and the virus is a human virus, such as an adenovirus, astrovirus, hepadnavirus, herpesvirus, papovavirus, poxvirus, arenavirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, orthomyxovirus, paramyxovirus, picornavirus, riovirus, retrovirus, rhabdovirus, or togavirus. In one embodiment, the animal is a human and the virus is a human immunodeficiency virus. Preferably, the human immunodeficiency virus is HIV-1.

Whether the azide-modified fatty acid, azide-modified carbohydrate, or azide-modified isoprenoid lipid is effective to treat a viral infection can be determined using any of a variety of assays known in the art. For example, existing animal or in vitro models of viral infection can be used to determine whether a given compound is effective in reducing viral load. In the case of HIV, as an example, the in vitro luciferase reporter assay described in example 2 can be used to measure the potency of azide-modified compounds. In a human subject, the potency of a compound can be determined by measuring viral load and/or measuring one or more signs of viral infection. Viral load can be measured by measuring the titer or level of virus in serum. These methods include, but are not limited to, quantitative Polymerase Chain Reaction (PCR) and branched DNA (bDNA) assays.

2. Method for inhibiting viral infectivity

Also provided is a method of inhibiting viral infectivity, the method comprising: contacting a virus-infected cell with an azide-modified fatty acid, azide-modified carbohydrate, or azide-modified isoprenoid lipid in an amount effective to inhibit viral infectivity. In one embodiment, the azide-modified fatty acid is a saturated fatty acid, such as 15-azidopentadecanoic acid or 12-azidododecanoic acid. In another embodiment, the azide-modified carbohydrate is an N-linked carbohydrate or an O-linked carbohydrate. In another embodiment, the azide-modified carbohydrate is N-azidoacetylgalactosamine, N-azidoacetyl-D-mannosamine, or N-azidoacetylglucosamine. The azide-modified carbohydrate optionally comprises a moiety that facilitates entry into a cell, including, but not limited to, a tetraacetyl moiety. Thus, in another embodiment, the azide-modified carbohydrate is tetraacetylated N-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine or tetraacetylated N-azidoacetylglucosamine. In another embodiment, the isoprenoid lipid comprises a farnesyl or geranylgeranyl group and includes, but is not limited to: azidofarnesyl diphosphate, azidofarnesol, azidogeranylgeranyl diphosphate or azidogeranylgeranyl alcohol.

The virus may be a plant virus, an insect virus or an animal virus. In certain embodiments, the cell is a human cell and the virus is a human virus, such as an adenovirus, astrovirus, hepadnavirus, herpesvirus, papovavirus, poxvirus, arenavirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, orthomyxovirus, paramyxovirus, picornavirus, riovirus, retrovirus, rhabdovirus, or togavirus. In one embodiment, the animal is a human and the virus is a human immunodeficiency virus. Preferably, the human immunodeficiency virus is HIV-1.

Whether the azide-modified fatty acid, azide-modified carbohydrate, or azide-modified isoprenoid lipid is effective to inhibit viral infectivity can be determined using any of a variety of assays known in the art, including reporter assays, such as the luciferase assay described in example 2.

3. Method for labeling viral proteins

Azide-modified fatty acids can also be used to label viral proteins that are lipid-modified by post-translational acylation (including, but not limited to, palmitoylation and myristoylation). In such post-translational modifications, azide-modified fatty acids are used to label viral proteins. If desired, the azide-labeled viral protein may be conjugated to an alkyne-labeled reporter molecule using a click chemistry reaction to allow detection of the azide-labeled viral protein.

Similarly, azide-modified carbohydrates may be used to label viral proteins that are carbohydrate-modified by post-translational glycosylation (including, but not limited to, N-linked glycosylation and O-linked glycosylation). In such post-translational modifications, azide-modified carbohydrates are used to label viral proteins. If desired, the azide-labeled viral protein may be conjugated to an alkyne-labeled reporter molecule using click chemistry to allow detection of the azide-labeled viral protein.

Azide-modified isoprenoid lipids can also be used to label viral proteins that are lipid-modified by post-translational prenylation (including, but not limited to, farnesylation and geranylgeranylation). In such post-translational modifications, azide-modified isoprenoid lipids are used to label viral proteins. If desired, the azide-labeled viral protein may be conjugated to an alkyne-labeled reporter molecule using a click chemistry reaction to allow detection of the azide-labeled viral protein.

Any virus labeled as described above may be conjugated to an alkyne-labeled reporter molecule or carrier molecule. The term "alkyne" includes, but is not limited to, terminal alkynes and internal alkynes, for example, by Agard et al, j.am.chem.soc., 2004, 126 (46): 15046-15047, dibenzocyclooctynes described by Boon et al, WO2009/067663 a1(2009), and by debts et al, chem.comm., 2010, 46: 97-99 to described azadibenzocyclooctynes. Reporter molecules for use in the methods and compositions described herein can contain, but are not limited to: chromophores, fluorophores, fluorescent proteins, phosphorescent dyes, tandem dyes, nanocrystalline particles, haptens, enzymes, and radioisotopes. In certain embodiments, such reporter molecules include fluorophores, fluorescent proteins, haptens, and enzymes.

Azide-labeled viruses may be used to track viral infectivity in vivo, wherein cultured cells or a subject are infected with an azide-modified carbohydrate, azide-modified fatty acid, or azide-modified isoprenoid lipid-labeled virus. Cells can be infected for a period of time, then fixed, permeabilized, and labeled with a click-label with a fluorescent alkyne dye. Intracellular localization (or transport over time) of fluorescent viral particles can be visualized, for example, by microscopy. Similarly, small animals can be treated with azide-labeled virus and used to follow viral bioavailability in different tissues, including detection of virus in circulating white blood cells by flow cytometry and cell/tissue slice microscopy. In some embodiments, the method of tracking a virus in vivo comprises the steps of: contacting cultured cells or a subject with an azide-modified carbohydrate, an azide-modified fatty acid, an azide-modified isoprenoid lipid, or a pharmaceutically acceptable salt thereof; contacting the cultured cells or the subject with an alkyne-labeled reporter molecule; and tracking the reporter-labeled virus in the cultured cells or the subject. In some embodiments, the method of tracking a virus in vivo comprises the steps of: contacting the cultured cells or subject with an azide-modified fatty acid or a pharmaceutically acceptable salt thereof; contacting the cultured cells or the subject with an alkyne-labeled reporter molecule; and tracking the reporter-labeled virus in the cultured cells or the subject.

Thus, another aspect of the present disclosure relates to a method of producing an azide-modified fatty acid, azide-modified carbohydrate, or azide-modified isoprenoid lipid-labeled human immunodeficiency virus, the method comprising: contacting a cell infected with the human immunodeficiency virus with the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid such that the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid enters the cell and is incorporated into proteins of the virus, thereby generating a labeled virus. In one embodiment, the azide-modified fatty acid is a saturated fatty acid, such as 15-azidopentadecanoic acid or 12-azidododecanoic acid. In another embodiment, the azide-modified carbohydrate is an N-linked carbohydrate or an O-linked carbohydrate. In another embodiment, the azide-modified carbohydrate is N-azidoacetylgalactosamine, N-azidoacetyl-D-mannosamine, or N-azidoacetylglucosamine. In another embodiment, the isoprenoid lipid comprises a farnesyl or geranylgeranyl group and includes, but is not limited to: azidofarnesyl diphosphate, azidofarnesol, azidogeranylgeranyl diphosphate or azidogeranylgeranyl alcohol. The azide-modified carbohydrate optionally comprises a moiety that facilitates entry into a cell, including, but not limited to, a tetraacetyl moiety. Thus, in another embodiment, the azide-modified carbohydrate is tetraacetylated N-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine or tetraacetylated N-azidoacetylglucosamine. In certain embodiments, the cell is a human cell.

Virus

The azide-modified fatty acids, azide-modified carbohydrates, or azide-modified isoprenoid lipids preferably target post-translational modifications common to most viruses and thus represent a new class of antiviral agents with the potential for antiviral activity against a broad spectrum of viruses. In general, these compounds may be used to treat plants, insects, or animals infected with any virus. In some embodiments, the virus is a plant virus. In some embodiments, the virus is an insect virus. In other embodiments, the virus is an animal virus. In other embodiments, the virus is a human virus. In one embodiment, the virus is a virus that infects a non-human mammal (e.g., a mammalian livestock animal including, but not limited to, cattle, horses, pigs, goats, or sheep).

In other embodiments, the virus is a DNA virus. DNA viruses include, but are not limited to, viruses belonging to the following families: adenovirus, astrovirus, hepadnavirus, herpesvirus, papovavirus and poxvirus. In other embodiments, the virus is an RNA virus. RNA viruses include, but are not limited to, viruses belonging to the following families: arenaviruses, bunyaviruses, caliciviruses, coronaviruses, filoviruses, flaviviruses, orthomyxoviruses, paramyxoviruses, picornaviruses, rioviruses, retroviruses, rhabdoviruses, and togaviruses.

1. Non-human animal virus

In the methods directed to treating a viral infection or inhibiting viral infectivity in a non-human animal, the animal virus is preferably selected from the group consisting of: small RNA virus genus, such as bovine enterovirus, porcine enterovirus B, foot and mouth disease virus, equine rhinitis a virus, bovine rhinovirus B virus, ljungan virus, equine rhinitis B virus, alphavirus, bovine kobuvirus, porcine teschovirus, porcine sapporovirus, simian sapporovirus, avian encephalomyelitis virus, duck hepatitis a virus or simian enterovirus a; pestiviruses, such as border disease virus, bovine viral diarrhea virus or classical swine fever virus; an arterivirus, such as equine arteritis virus, porcine reproductive and respiratory syndrome virus, lactate dehydrogenase activity virus, or simian hemorrhagic fever virus; coronaviruses such as bovine coronavirus, porcine coronavirus, feline coronavirus, or canine coronavirus; paramyxoviruses such as hendra virus, nipah virus, canine distemper virus, rinderpest virus, newcastle disease virus, and bovine respiratory syncytial virus; orthomyxoviruses, such as influenza a virus, influenza B virus, or influenza C virus; rio virus, such as bluetongue virus; porcine circovirus, herpesvirus, such as pseudorabies virus or bovine herpesvirus type 1; african swine fever virus genus, such as african swine fever virus; a retrovirus, such as simian immunodeficiency virus, feline immunodeficiency virus, bovine leukemia virus, feline leukemia virus, ovine lung adenoma retrovirus, or caprine arthritis encephalitis virus; flaviviruses, such as yellow fever virus, west nile virus, dengue virus, tick-borne encephalitis virus, or bovine viral diarrhea virus; or rhabdovirus, such as rabies virus.

2. Human animal virus

In the method directed to treating a viral infection or inhibiting viral infectivity in a human, said human virus is preferably selected from the group consisting of: adenovirus, astrovirus, hepadnavirus, herpesvirus, papovavirus, poxvirus, arenavirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, orthomyxovirus, paramyxovirus, picornavirus, riovirus, retrovirus, rhabdovirus, or togavirus.

In a preferred embodiment, the adenovirus includes, but is not limited to, a human adenovirus. In a preferred embodiment, the astrovirus includes, but is not limited to, a mammalian astrovirus. In a preferred embodiment, the hepadnavirus includes, but is not limited to, hepatitis b virus. In preferred embodiments, the herpes virus includes, but is not limited to, herpes simplex virus type I, herpes simplex virus type 2, human cytomegalovirus, epstein-barr virus, varicella zoster virus, roseola virus and kaposi sarcoma-related herpes virus. In a preferred embodiment, the milk-foamy virus includes, but is not limited to, human papilloma virus and human polyoma virus. In preferred embodiments, the poxvirus includes, but is not limited to, variola virus, vaccinia virus, monkeypox virus, variola virus, pseudovaccinia virus, papulostomatitis virus, tanapoxvirus, yabavirus and molluscum contagiosum virus. In preferred embodiments, the arenaviruses include, but are not limited to, lymphocytic choriomeningitis virus, lassa virus, machupo virus, and junin virus. In preferred embodiments, the bunyavirus includes, but is not limited to, hantavirus, nairovirus, bunyavirus, and phlebovirus. In preferred embodiments, the caliciviruses include, but are not limited to, bursa viruses, norovirus, such as norwalk viruses and sapporo viruses. In a preferred embodiment, the coronavirus includes, but is not limited to, a human coronavirus (the causative agent of Severe Acute Respiratory Syndrome (SARS)). In preferred embodiments, the filoviruses include, but are not limited to, epstein-barr virus and marburg virus. In preferred embodiments, the flavivirus includes, but is not limited to, yellow fever virus, West Nile virus, dengue virus, hepatitis C virus, tick-borne encephalitis virus, Japanese encephalitis virus, Murray Valley encephalitis virus, St.Louis encephalitis virus, Russian spring summer encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Kosarnoulli forest disease virus, and Powassan encephalitis virus. In preferred embodiments, the orthomyxovirus includes, but is not limited to, influenza virus type a, influenza virus type B, and influenza virus type C. In preferred embodiments, the paramyxovirus includes, but is not limited to, parainfluenza virus, mumps virus (mumps), measles virus (measles), pneumoviruses such as human respiratory syncytial virus and subacute sclerosing panencephalitis virus. In preferred embodiments, the picornaviruses include, but are not limited to, poliovirus, rhinovirus, coxsackievirus a, coxsackievirus B, hepatitis a, echovirus, and enterovirus. In preferred embodiments, the rio virus includes, but is not limited to, Colorado tick fever virus and rotavirus. In a preferred embodiment, the retrovirus includes, but is not limited to, lentiviruses, such as human immunodeficiency virus and human T-cell lymphotrophic virus (HTLV). In a preferred embodiment, the rhabdovirus includes, but is not limited to, rabies virus (lyssavirus), such as rabies virus (rabies virus), vesicular stomatitis virus, and infectious hematopoietic necrosis virus. In a preferred embodiment, the togavirus includes, but is not limited to, alphaviruses such as ross river virus, aortosis virus, Sindbis virus, venezuelan equine encephalitis virus, eastern equine encephalitis virus, and western equine encephalitis virus, and rubella virus.

3. Plant virus

In a method directed to treating a viral infection or inhibiting viral infectivity in a plant, said plant virus is selected from the group consisting of: alfalfa mosaic virus, green onion virus, alphacryptovirus, avian paramyxovirus, apple North American scar viroid, golden virus, oat virus, avocado white spot viroid, baculovirus, bean golden mosaic virus, beet necrotic yellow vein virus, beta latent virus, beta flexviridae, brome mosaic virus, barley yellowing mosaic virus, hairy virus, carnauba latent virus, moschus caryophyllata mottle virus, cauliflower mosaic virus, cassava vein mosaic virus, cherry filing virus, closterovirus, coconut death viroid, coleus, cowpea mosaic virus, linear virus, cucumber mosaic virus, plant insect virus, rhabdovirus, carnation virus, bowled ear mosaic virus, umbelliferyl virus and satellite type B virus, broad bean virus, feiji virus, fungal baculovirus, barley virus, maize virus, hop stunt viroids, rubus corchorifolius, equiaxed unstable ringspot viruses, sweet potato viruses, yellow dwarf viruses, maize chlorotic mottle viruses, orange mulberry mosaic viruses, maize reyadophora viruses, maize streak viruses, nano-viruses, necrosis viruses, helminthic polyhedrosis viruses, nuclear rhabdoviruses, oleavirus, citrus scale virus, rice viruses, panicoviridae viruses, peanut cluster viruses, morning mosaic vein clearing-like viruses, plant reoviruses, potato leaf roll viruses, potato spindle tuber viruses, potato virus X, potato virus Y, rio viruses, rhabdoviruses, lodamycin mosaic viruses, wenzhou mandarin dwarf viruses, SbCMV-like viruses, companion viruses, southern bean mosaic viruses, teniviruses, TNsatV-like satellite viruses, tobacco mosaic viruses, tomato kojic top viruses, Tomato spotted wilt virus, apple chlorosis leaf spot virus, wheat mosaic virus, rice regression baculovirus, turnip yellow mosaic virus, umbelliferyl plant virus, megalovirus, grapevine virus or dwarf virus.

4. Insect virus

In the method directed to marking an insect virus, treating an insect virus infection, or inhibiting an insect virus infectivity, the insect virus is preferably selected from the group consisting of: densovirus such as Kallima Kallissima densovirus, Bombyx mori densovirus, Aedes aegypti densovirus, or Blattella fuliginosa densovirus; iridovirus, such as iridovirus type 6; green iridovirus, baculovirus, such as nuclear polyhedrosis virus or granulovirus; a poly-DNA virus genus, such as a Simplex virus or a Coccida virus; entomopoxviruses, such as entomopoxvirus a, entomopoxvirus B, or entomopoxvirus C; vesiculoviruses, such as spodoptera frugiperda vesiculovirus type 1a, spodoptera litura vesiculovirus type 2a, or geminivirus apis mellifera vesiculovirus type 4 a; insect picornavirus, such as bee acute paralysis virus, drosophila P, C or a virus, bee X virus, or silkworm softening disease virus; calicivirus genus; nodavirus, such as black cockroach virus, avian hut virus, nodavirus, pariacoto virus or gypsy moth virus.

Azide-modified biomolecules

The azide-modified biomolecules described herein represent a new class of antiviral agents. In certain embodiments, the azide-modified biomolecule is a carbohydrate or a pharmaceutically acceptable derivative or prodrug thereof. The carbohydrate may be selected from a variety of carbohydrates that are commercially available and/or widely known to those skilled in the art. In a preferred embodiment, carbohydrates are selected that will prevent, inhibit and/or delay viral infection of a cell. Preferably, the carbohydrate is naturally occurring. It will be appreciated that the azide-containing carbohydrate (whether or not naturally occurring) may be modified, for example, by short chain alkylation such as methylation or acetylation, esterification, and other derivatizations that maintain antiviral activity.

In one embodiment, the carbohydrate is a carbohydrate that is linked to the protein directly or indirectly by a glycosylation reaction in the cell. In one embodiment, the carbohydrate is an N-linked carbohydrate or an O-linked carbohydrate. In another embodiment, the carbohydrate is N-azidoacetylgalactosamine, N-azidoacetyl-D-mannosamine, or N-azidoacetylglucosamine.

In certain embodiments, the azide-modified carbohydrate contains a moiety that facilitates entry into a cell, including, but not limited to, a tetraacetyl moiety. Thus, in one embodiment, the azide-modified carbohydrate is a tetra-acetylated form of an N-linked carbohydrate or an O-linked carbohydrate. In another embodiment, the azide-modified carbohydrate is tetraacetylated N-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine or tetraacetylated N-azidoacetylglucosamine.

In other embodiments, the azide-modified biomolecule is a fatty acid or a pharmaceutically acceptable derivative or prodrug thereof. The fatty acid may be selected from a variety of fatty acids that are commercially available and/or widely known to those skilled in the art. In a preferred embodiment, the fatty acids are selected to prevent, inhibit and/or delay viral infection of the cells. Preferably, the fatty acid is naturally occurring.

In one embodiment, the fatty acid is saturated or unsaturated and has a hydrocarbon chain with an even number of carbon atoms, such as from 4 to 24 carbon atoms. Suitable unsaturated free fatty acids have a hydrocarbon chain containing 14 to 24 carbon atoms and include palmitoleic acid, oleic acid, linoleic acid, alpha and gamma linolenic acid, arachidonic acid, eicosapentaenoic acid and tetracosenoic acid. Suitable saturated fatty acids have a hydrocarbon chain containing from 4 to 18 carbon atoms and are preferably selected from butyric or isobutyric acid, succinic acid, caproic acid, adipic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid and arachidic acid. It is understood that azide-containing fatty acids (whether or not naturally occurring) may be modified by chemical substitution, including, but not limited to, short-chain alkylation such as methylation or acetylation, esterification, and other derivatizations that maintain antiviral activity. In addition, it is possible to replace the fatty acid in the azide-modified biomolecule with an alkyne, ketone, or other small molecule (which has been shown to be metabolically compatible).

In some embodiments, the azide-modified fatty acid or pharmaceutically acceptable salt thereof has the formula: Y-CH₂-X-CO₂H, wherein Y is H or azido; and when Y is azido, X is a linear or branched carbon chain comprising 6-28 carbons, wherein one or more of the carbons may be independently replaced by oxygen, selenium, silicon, sulfur, SO₂Or NR₁Alternatively, or wherein one or more pairs of said carbons adjacent to each other may be connected to each other by a double or triple bond; or when Y is H, X is a straight or branched carbon chain comprising 6-28 carbons, wherein one hydrogen on one of the carbons is replaced with an azido group, and wherein one or more of the carbons not having an azido group attached thereto may be independently replaced with oxygen, selenium, silicon, sulfur, SO₂Or NR₁Alternatively, or wherein one or more pairs of the carbons adjacent to each other and not having an azido group may be connected to each other by a double or triple bond; wherein R is₁Is H or alkyl containing 1-6 carbons.

In one embodiment, the fatty acid is a fatty acid linked to a protein by an acylation reaction (e.g., palmitoylation or myristoylation) in a cell. Thus, in one embodiment, the azide-modified fatty acid is a saturated fatty acid, such as 15-azidopentadecanoic acid (palmitic acid, azide) or 12-azidododecanoic acid (myristic acid, azide). The compounds used in the methods of the invention may be present in the form of pharmaceutically acceptable salts. For use in medicine, the salts of the compounds of the present invention refer to non-toxic pharmaceutically acceptable salts.

Pharmaceutically acceptable salts of these compounds include acid addition salts and base addition salts. The term "pharmaceutically acceptable salts" includes salts commonly used to form alkali metal salts and to form addition salts of the free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically acceptable.

Suitable pharmaceutically acceptable acid addition salts of these compounds may be prepared from inorganic or organic acids. Examples of such inorganic acids are hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, and phosphoric acid. Suitable organic acids may be selected from the classes of aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, maleic, pamoic (pamoic), methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, methanesulfonic, cyclohexylsulfamic, stearic, alginic, β -hydroxybutyric, malonic, hemi-lactic and galacturonic acids.

Pharmaceutically acceptable acidic/anionic salts also include: acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camphorsulfonate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, etonate, ethanesulfonate, fumarate, glucoheptonate (glycoeptate), gluconate, glutamate, glycollylalonate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, malonate, mandelate, methanesulfonate, methylsulfate, mucate, naphthalenesulfonate, nitrate, pamoate, pantothenate, phosphate/biphosphate (diphosphonate), polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, polygalacturonate, salicylate, stearate, subacetate, succinate, camphorsulfonate, carbonate, chloride, gluconate, chloride, hydrochloride, dihydrochloride, hydrochloride, acetate, Hydrogen sulfate, tannate, tartrate, 8-chlorotheophylline, tosylate and triethyliodide.

Suitable pharmaceutically acceptable base addition salts of the disclosed compounds include, but are not limited to: metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc, or organic salts made from N, N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methyl reduced glucamine, lysine, arginine and procaine. All of these salts can be prepared in a conventional manner from the corresponding compounds represented by the disclosed compounds, for example, by treating the disclosed compounds with an appropriate acid or base. Pharmaceutically acceptable basic/cationic salts also include diethanolamine, ammonium, ethanolamine, piperazine and triethanolamine salts.

Such salts may be formed according to methods known to those skilled in the art.

In another embodiment, the azide-modified biomolecule is an azide-modified isoprenoid lipid or a pharmaceutically acceptable derivative or prodrug thereof. The isoprenoid lipid may be selected from a variety of isoprenoid lipids that are commercially available and/or widely known to those of skill in the art. In preferred embodiments, the isoprenoid lipid is selected that will prevent, inhibit and/or delay viral infection of a cell. Preferably, the isoprenoid lipid is naturally occurring. It is understood that the azide-containing isoprenoid lipids (whether or not naturally occurring) may be modified, for example, by short chain alkylation such as methylation or acetylation, esterification, and other derivatizations that maintain antiviral activity.

In one embodiment, the isoprenoid lipid is one that is linked to a protein by an isoprenylation reaction in a cell. In one embodiment, the isoprenoid lipid is linked to a protein in a cell in the presence of catalytic activity of farnesyl transferase or geranylgeranyl transferase. In another embodiment, the isoprenoid lipid comprises a farnesyl or geranylgeranyl group and includes, but is not limited to: azidofarnesyl diphosphate, azidofarnesol, azidogeranylgeranyl diphosphate or azidogeranylgeranyl alcohol. Methods known in the art are used, including those described in U.S. patent application publication No. 20050222427, U.S. patent application publication No. 2007/0249014, and Hang, h.c. et al, J Am Chem Soc 2007, 129: 2744-45 (the disclosure of which is incorporated by reference in its entirety), the azide-modified carbohydrates, the azide-modified fatty acids, and the azide-modified isoprenoid lipids described herein can be prepared.

Combination therapy

In one embodiment, the pharmaceutical composition comprising an azide-modified fatty acid, an azide-modified carbohydrate, or an azide-modified isoprenoid lipid and at least one antiviral agent is administered in a combination therapy. The treatment may be used to treat viral infections, including, but not limited to, HIV infections. In this context, the term "in combination" means: administering the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid and the antiviral agent substantially simultaneously (simultaneously or sequentially). In one embodiment, if administered sequentially, the effective concentration of the first of the two compounds at the treatment site is still detectable at the beginning of administration of the second compound. In another embodiment, if administered sequentially, the effective concentration of the first of the two compounds at the treatment site is undetectable at the beginning of administration of the second compound.

For example, combination therapy may include: co-formulating and/or co-administering an azide-modified fatty acid, an azide-modified carbohydrate, or an azide-modified isoprenoid lipid with at least one other antiviral agent. Although specific examples of antiviral agents are provided, in general, the azide-modified fatty acids, azide-modified carbohydrates, or azide-modified isoprenoid lipids may be combined with any pharmaceutical composition useful for treating viral infections. Such combination therapy may advantageously reduce the dose of therapeutic agent administered, thereby avoiding possible toxicity or complications associated with each monotherapy. In addition, the viral infection pathway or stage in which other antiviral agents disclosed herein act is additive to or different from the viral infection pathway or stage affected by the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid, and thus would be expected to enhance and/or synergize with the effect of the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid. The additional antiviral agents may include at least one reverse transcriptase inhibitor, viral protease inhibitor, viral fusion inhibitor, viral integrase inhibitor, glycosidase inhibitor, viral neuraminidase inhibitor, M2 protein inhibitor, amphotericin B, hydroxyurea, interferon-alpha, interferon-beta, interferon-gamma, and antisense oligonucleotide.

The at least one reverse transcriptase inhibitor includes, but is not limited to: one or more nucleoside analogs such As Zidovudine (AZT), didanosine (ddI), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), Abacavir (ABC), emtricitabine (FTC), entecavir (INN), Alecitabine (ATC), atevirine, ribavirin, acyclovir, famciclovir, valacyclovir, ganciclovir, and valacyclovir; one or more nucleotide analogs such as tenofovir (tenofovir disoproxil fumarate), adefovir (bis-POM PMPA), PMPA, and cidofovir; or one or more non-nucleoside reverse transcriptase inhibitors, such as efavirenz, nevirapine, delavirdine, and etravirine.

The at least one viral protease inhibitor includes, but is not limited to: tipranavir, darunavir, indinavir, lopinavir, furinavir, atazanavir, saquinavir, ritonavir, indinavir, nelfinavir and amprenavir.

The at least one viral fusion inhibitor includes, but is not limited to: CD4 antagonists, such as soluble CD4, or antibodies that bind to CD4, such as TNX-355, BMS-806; CCR5 antagonists such as SCH-C, SCH-D, UK-427, 857, maraviroc, viriviroc, or antibodies that bind to CCR5 such as PRO-140; CXCR4 antagonists, for example, AMD3100 or AMD 070; or an antagonist of gp41, such as enfuvirtide.

The at least one viral integrase inhibitor includes, but is not limited to, raltegravir.

The at least one glycosidase inhibitor includes, but is not limited to, SC-48334 or MDL-28574.

The at least one viral neuraminidase inhibitor includes, but is not limited to oseltamivir, peramivir, zanamivir, and ranimivir. Neuraminidase is a protein on the surface of influenza virus that mediates release of the virus from infected cells (Bossart-Whitaker et al, J Mol Biol, 1993, 232: 1069-83). Influenza viruses use viral hemagglutinin proteins to attach to cell membranes. The hemagglutinin protein will bind to sialic acid moieties present on glycoproteins in the host cell membrane. In order to release the virus from the cell, neuraminidase must enzymatically cleave sialic acid groups from host glycoproteins. Thus, inhibition of neuraminidase prevents the release of influenza virus from infected cells.

The at least one M2 inhibitor includes, but is not limited to, amantadine and rimantadine. M2 is an ion channel protein found in the viral envelope of influenza virus (Henckel et al, J biol chem, 1998, 273: 6518-24). The M2 protein plays an important role in the control of uncoating of influenza viruses, resulting in the release of virion contents into the host cell cytoplasm. Blocking M2 inhibited viral replication.

The azide-modified fatty acids, azide-modified carbohydrates, or azide-modified isoprenoid lipids disclosed herein can be used in combination with other therapeutic agents for the treatment of particular viral infections, discussed in further detail below.

Non-limiting examples of agents useful for treating HIV infection (to which the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid may be combined) include at least one of: zidovudine (AZT), didanosine (ddI), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), Abacavir (ABC), emtricitabine (FTC), entecavir (INN), Aliscitabine (ATC), tenofovir (tenofovir dipivoxil fumarate), adefovir (bis-POM PMPA) efavirenz, nevirapine, delavirdine, etravirine, tipranavirine, darunavir, indinavir, lopinavir, furamevir, atazanavir, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, CD4 antagonists, such as CD4, or antibodies that bind to CD4, such as TNX-355, BMS-806, CCR5 antagonists, such as SCH-2-D, UK-3875-5, SCH 857, mavir, or soluble antibody that binds to CDCR 4, such as PRO-140, CXCR4 antagonists, e.g., AMD3100 or AMD070, or gp41 antagonists, such as enfuvirtide.

Specific examples of combination therapies that may be used to treat HIV infection include, but are not limited to, azide-modified fatty acids, azide-modified carbohydrates, or azide-modified isoprenoid lipids in combination with: 1) tenofovir, emtricitabine and efavirenz; 2) lopinavir and ritonavir; 3) lamivudine and zidovudine; 4) abacavir, lamivudine, and zidovudine; 5) lamivudine and abacavir; or 6) tenofovir and emtricitabine.

Non-limiting examples of agents useful for treating herpes virus infections (to which the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid may be combined) include: acyclovir, famciclovir, valacyclovir, cidofovir, foscarnet, ganciclovir and valacyclovir.

Non-limiting examples of agents useful for treating influenza virus infection (to which the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid may be combined) include: amantadine, rimantadine, oseltamivir, peramivir, zanamivir, and ranimivir.

Non-limiting examples of agents useful for treating respiratory syncytial virus infection with which the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid may be combined include ribavirin.

Thus, another aspect of the invention relates to a kit for effecting the combined administration of the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid and an additional therapeutic agent. In one embodiment, the kit comprises: the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid formulated in a pharmaceutically acceptable excipient, and at least one antiviral agent formulated in one or more separate pharmaceutical formulations as appropriate.

Pharmaceutical compositions and methods of administration

The present disclosure provides compositions suitable for pharmaceutical use and administration to a patient. The pharmaceutical composition comprises an azide-modified fatty acid, an azide-modified carbohydrate, an azide-modified isoprenoid lipid, or any of the compounds described herein and a pharmaceutically acceptable excipient. In one embodiment, the azide-modified fatty acid is a saturated fatty acid, such as 15-azidopentadecanoic acid or 12-azidododecanoic acid. In another embodiment, the azide-modified carbohydrate is an N-linked carbohydrate or an O-linked carbohydrate. In another embodiment, the azide-modified carbohydrate is N-azidoacetylgalactosamine, N-azidoacetyl-D-mannosamine, or N-azidoacetylglucosamine. In another embodiment, the azide-modified carbohydrate contains a moiety that facilitates entry into a cell, including, but not limited to, a tetraacetyl moiety. Thus, in one embodiment, the azide-modified carbohydrate is a tetra-acetylated form of an N-linked carbohydrate or an O-linked carbohydrate. In another embodiment, the azide-modified carbohydrate is tetraacetylated N-azidoacetylgalactosamine, tetraacetylated N-azidoacetyl-D-mannosamine or tetraacetylated N-azidoacetylglucosamine. In another embodiment, the isoprenoid lipid comprises a farnesyl or geranylgeranyl group and includes, but is not limited to: azidofarnesyl diphosphate, azidofarnesol, azidogeranylgeranyl diphosphate or azidogeranylgeranyl alcohol. The pharmaceutical composition may also be contained in a container, package or dispenser together with instructions for administration.

The pharmaceutical composition is formulated to be compatible with its intended route of administration. Methods of achieving administration are known to those of ordinary skill in the art. The pharmaceutical composition may be administered topically or orally, or may be capable of transmucosal delivery. Examples of administration of pharmaceutical compositions include oral or inhalation. Administration can also be intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, dermal, or transdermal.

Solutions or suspensions for intradermal or subcutaneous administration typically include at least one of the following components: sterile diluents such as water, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate or phosphate; and tonicity agents such as sodium chloride or dextrose. The pH can be adjusted with an acid or a base. Such formulations may be enclosed in ampoules, single use syringes or multi-dose vials.

Solutions or suspensions for intravenous administration include: carriers such as physiological saline, bacteriostatic water, cremophor EL^TM(BASF, Parsippany, NJ), ethanol or a polyol. In all cases, the composition must be sterile and easily injectable fluid. Lecithin or surfactants are often used to obtain proper fluidity. The composition must also be stable under the conditions of manufacture and storage. Microorganisms can be prevented by using antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents (sugars), polyols (mannitol and sorbitol), or sodium chloride may be included in the composition. Prolonged absorption of the composition can be brought about by the addition of agents which delay absorption, for example, aluminum monostearate and gelatin.

Oral compositions include an inert diluent or an edible carrier. The composition may be encapsulated in gelatin or compressed into tablets. For oral administration purposes, the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid may be blended with excipients and formulated into tablets, lozenges, or capsules. Pharmaceutically suitable binders or excipients may be included in the composition. The tablets, lozenges and capsules may contain: (1) binders such as microcrystalline cellulose, gum tragacanth or gelatin; (2) excipients such as starch or lactose, (3) disintegrants such as alginic acid, Primogel or corn starch; (4) lubricants such as magnesium stearate; (5) glidants such as colloidal silicon dioxide; or (6) a sweetener or a flavoring agent.

The compositions may also be administered by transmucosal or transdermal routes. Transmucosal administration can be achieved through the use of lozenges, nasal sprays, inhalers, or suppositories. Transdermal administration may also be achieved by using ointments, salves, gels or creams as known in the art containing the compositions. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used.

For administration by inhalation, the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid is delivered as an aerosol spray from a pressurized container or dispenser containing a propellant (e.g., a liquid or gas) or a nebulizer. In certain embodiments, the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid is prepared with a carrier to protect the compound from rapid elimination from the body. Biodegradable polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid) are often used. Methods for preparing such formulations are known to those skilled in the art.

In other embodiments, the composition comprises a delivery agent, including but not limited to liposomes, for delivering the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid to a cell. Liposomes (also known as lipid vesicles) are colloidal particles made from polar lipid molecules, which are derived from natural sources or chemically synthesized. Such spherical closed structures, consisting of a curved lipid bilayer, are commonly used to entrap drugs (which are often cytotoxic) in order to reduce toxicity and/or increase efficacy. Liposome-entrapped pharmaceutical formulations are often provided in a dried (e.g., lyophilized) form, which is later reconstituted with an aqueous solution immediately prior to administration. This is done to minimize the possibility of leakage of e.g. cytotoxic drugs in aqueous solution and thereby reduce the trapping effect of the liposomes.

Examples of formulations comprising liposome-encapsulated active ingredients and other agents are disclosed in U.S. patent No. 4,427,649, U.S. patent No. 4,522,811, U.S. patent No. 4,839,175, U.S. patent No. 5,569,464, EP 249561, WO 00/38681, WO 88/01862, WO 98/58629, WO 98/00111, WO 03/105805, U.S. patent No. 5,049,388, U.S. patent No. 5,141,674, U.S. patent No. 5,498,420, U.S. patent No. 5,422,120, WO 87/01586, WO 2005/039533, US 2005/0112199, and U.S. patent No. 6,228,393, all of which are hereby incorporated by reference in their entirety.

Compositions containing azide-modified fatty acids, azide-modified carbohydrates, or azide-modified isoprenoid lipids are administered in therapeutically effective amounts as described. The therapeutically effective amount may vary with the age, condition, sex, and severity of the medical condition of the subject. The appropriate dosage may be determined by a physician based on the clinical indication. The composition containing the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid may be administered as a single dose (bolus dose) to maximize and maintain the circulating level of the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid for the longest period of time. Continuous infusion may also be used after a single dose.

Examples of dosage ranges that may be administered to a subject may be selected from: 1 to 20mg/kg, 1 to 10mg/kg, 1 to 1mg/kg, 10 to 100 μ g/kg, 100 to 1mg/kg, 250 to 2mg/kg, 250 to 1mg/kg, 500 to 2mg/kg, 500 to 1mg/kg, 1 to 2mg/kg, 1 to 5mg/kg, 5 to 10mg/kg, 10 to 20mg/kg, 15 to 20mg/kg, 10 to 25mg/kg, 15 to 25mg/kg, 20 to 25mg/kg, And 20mg/kg to 30mg/kg (or higher). These doses may be administered daily, weekly, every 2 weeks, monthly, or less frequently (e.g., every half year), depending on the dose, method of administration, condition or symptom to be treated, and individual subject characteristics. The dose may also be administered by continuous infusion, such as by a pump. The dosage administered may also depend on the route of administration. For example, subcutaneous administration may require higher doses than intravenous administration.

In certain instances, it may be advantageous to formulate the compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, "dosage unit form" refers to physically discrete units suitable for use in a patient. Each dosage unit contains a predetermined amount of an azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid calculated to produce a therapeutic effect in combination with a carrier. The dosage unit depends on the identity of the azide-modified fatty acid, the azide-modified carbohydrate, or the azide-modified isoprenoid lipid and the particular therapeutic effect to be achieved.

Toxicity and therapeutic efficacy of the compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD₅₀(dose lethal to 50% of the population) and ED₅₀(therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀。

Data from cell culture assays and animal studies can be used to formulate dose ranges in humans. The dosage of these compounds may be within the circulating concentration of the azide-modified fatty acid, azide-modified carbohydrate, or azide-modified isoprenoid lipid in blood, including the ED with little or no toxicity₅₀. The dosage may vary within this range depending upon the dosage composition form employed and the route of administration. For any azide-modified fatty acid, azide-modified carbohydrate, or azide-modified isoprenoid lipid used in the methods described herein, a fine particle is usedCell culture assays allow for an initial estimate of the therapeutically effective dose. The dose can be formulated in animal models to include the IC₅₀(i.e., the concentration of antibody that achieves half-maximal inhibition of symptoms). The effect of any particular dose can be monitored by a suitable bioassay.

The compositions may also contain other active compounds that provide supplemental, additional, or enhanced therapeutic functions. In one embodiment, the composition additionally comprises at least one antiviral agent, such as reverse transcriptase inhibitors, viral protease inhibitors, viral fusion inhibitors, viral integrase inhibitors, glycosidase inhibitors, amphotericin B, hydroxyurea, interferon-alpha, interferon-beta, interferon-gamma and antisense oligonucleotides.

In one embodiment, the at least one reverse transcriptase inhibitor includes, but is not limited to: one or more nucleoside analogs such As Zidovudine (AZT), didanosine (ddI), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), Abacavir (ABC), emtricitabine (FTC), entecavir (INN), Alecitabine (ATC), atevirine, ribavirin, acyclovir, famciclovir, valacyclovir, ganciclovir, and valacyclovir; one or more nucleotide analogs such as tenofovir (tenofovir disoproxil fumarate), adefovir (bis-POM PMPA), PMPA, and cidofovir; or one or more non-nucleoside reverse transcriptase inhibitors, such as efavirenz, nevirapine, delavirdine, and etravirine.

In other embodiments, the at least one viral protease inhibitor includes, but is not limited to: tipranavir, darunavir, indinavir, lopinavir, furinavir, atazanavir, saquinavir, ritonavir, indinavir, nelfinavir and amprenavir.

In other embodiments, the at least one viral fusion inhibitor includes, but is not limited to: CD4 antagonists, such as soluble CD4, or antibodies that bind to CD4, such as TNX-355, BMS-806; CCR5 antagonists such as SCH-C, SCH-D, UK-427, 857, maraviroc, viriviroc, or antibodies that bind to CCR5 such as PRO-140; CXCR4 antagonists, for example, AMD3100 or AMD 070; or an antagonist of gp41, such as enfuvirtide.

In other embodiments, the at least one viral integrase inhibitor includes, but is not limited to, raltegravir.

In other embodiments, the at least one glycosidase inhibitor includes, but is not limited to, SC-48334 or MDL-28574.

Reference will now be made in detail to various exemplary embodiments. It should be understood that the following detailed description is provided to give the reader a fuller understanding of certain embodiments, features and details of the various aspects of the invention, and should not be construed as limiting the scope of the invention.

Example 1: labelling of HIV with azide-modified biomolecules

Use of HIV in T-150 flasks_NL4-3CEMx174 cells were transfected and viral production was monitored by reverse transcriptase activity until peak viral production was reached (typically 7-9 days post transfection). Before transfection, CEMx174 cells were stimulated (spike) with the following azide-modified biomolecules: 15-azidopentadecanoic acid (50-100. mu.M), 12-azidododecanoic acid (50-100. mu.M), tetraacetylated N-azidoacetylgalactosamine (20-40. mu.M), and tetraacetylated N-azidoacetyl-D-mannosamine (20-40. mu.M).

Infected cells were harvested at 12, 24, 72 hours and 14 days. The harvested cells were isolated and lysed. The cell lysate is then reacted withDetection reagent, Tetramethylrhodamine (TAMRA) alkyne andprotein reaction buffer reagentThe cassettes (Invitrogen, Carlsbad, Calif.) were mixed together. Cell lysate samples were run on one-dimensional gels to monitor the changes in azide-labeled proteins over time (figure 1).

The labeled virus was also obtained from transfected cells. More specifically, the virus-containing supernatant was collected and the virus was purified by 20% sucrose as previously described (Graham, D.R. et al, Proteomics 2008, 8: 4919-30). The purified virus is then reacted withDetection reagent, Tetramethylrhodamine (TAMRA) alkyne andprotein reaction buffer kits (Invitrogen, Carlsbad, CA) were mixed together. The virus sample was run on a one-dimensional gel to reveal the azide-labeled viral proteins (figure 2). Virus levels were normalized to p24 content and were run by one-dimensional gel electrophoresis.

No significant effect of acute or chronic replication of HIV on host cell protein modification was observed (figure 1, markers compared to control). However, the azide-modified biomolecules do label the viral proteins and allow their detection at 14 days in chronically infected cells at the expected molecular weight of HIV viral proteins: 55kDa (gag-myristoylated), 41kDa (gp 41-palmitoylated) and 120kDa (gp 120-N-glycosylated) (FIG. 1).

Example 2: inhibiting HIV infectivity

To examine the effect of the azide-modified biomolecule on the innate biology of the virus, the azide-labeled virus was isolated from transfected cells and tested in cell infection studies. Unlabeled HIV (1/100 concentration of the test sample) was used as a control. Viral load was normalized to p24 abundance and virus was incubated on the reporter cell line (TZM/BI) for 12 hours. TZM/BI is a genetically engineered HeLa cell line expressing CD4, CXCR4, and CCR5, and containing Tat-inducible luciferase and β -Gal reporter genes. Viral infectivity was determined by measuring cellular luciferase activity with 2 different luciferase reagents. The results using the single-cycle replication system show that virus labeled with the azide-modified biomolecules (specifically, 12-azidododecanoic acid, and to a lesser extent, 15-azidopentadecanoic acid and tetraacetylated N-azidoacetylgalactosamine) has a profound effect on viral infectivity (fig. 3 and 4). The level of inhibition observed for viral entry is comparable to that observed in cells pretreated with an anti-retroviral agent, such as a fusion inhibitor or nucleoside analog.

Example 3: toxicity Spectrum

Little or no toxicity was observed in different eukaryotic cell lines using these azide-modified biomolecules, suggesting that these compounds have a minimal toxicity profile and supporting their use in therapeutic settings.

Example 4: inhibiting baculovirus infectivity of insect cells

Inhibition of insect cell baculovirus infectivity with azido fatty acid analogs: the BacMam system uses a modified insect cell virus (baculovirus) as a vehicle to efficiently deliver and express genes in mammalian cells. The Nuclear-GFP bacmam2.0 expression system (Invitrogen, C10602) was used as a model to determine whether PTM analogue markers of the virus would affect mammalian cell infectivity. The virus was labeled with different PTM analogs and used to infect mammalian cells. Infectivity was determined by the expression of nuclear-GFP, as expression of GFP protein requires viral entry into the cell.

Labeling and amplification of the Nuclear-GFP BacMam2.0 virus: for labeling, amplification and enrichment of BacMam virus containing azide/alkyne post-translational modifications (PTM) analogues, 20ml of Sf9 insect cells (concentration 1.5E6 cells/ml) in Sf-900II SFM insect cell culture medium were infected with Nuclear-GFP BacMam2.0 at a multiplicity of infection (MOI) of 0.1 virus/cell. Simultaneously adding different PTM analogs in DMSO or 100% ethanol to insect cells to a final concentration of 50uM, said PTM analogs comprising: palmitic acid azide (15-azidopentadecanoic acid) (Invitrogen, C10265), myristic acid azide (12-azidododecanoic acid)) (Invitrogen, C10268), fucose alkyne (Invitrogen, C10264), ManNAz (tetraacetylated N-azidoacetyl-d-mannosamine) (Invitrogen, C33366), and GalNAz (tetraacetylated N-azidoacetylgalactosamine) (Invitrogen, C33365). The cultures were shaken (120rpm) in the dark at 27 ℃ for 4 days. BacMam baculovirus was harvested by centrifugation at 1000xg for 15 minutes. The resulting supernatant was filtered through a 0.22um sterile filter into separate sterile amber 50ml erlenmeyer flasks and stored at 4 ℃.

Characterization of BacMam virus production: to verify, quantify and normalize the amount of enriched virus obtained from insect cell supernatants, samples of each virus were lysed in SDS Sample Preparation Buffer (SPB), sonicated with a probe tip sonicator, and heated at 90 ℃ to completely solubilize the viral proteins. The lysate is then assayed for viral protein concentration. Viral lysate samples were separated by 1D SDS-PAGE and then analyzed by Western blotting using antibodies gp64[ eBiosciences, mouse monoclonal antibodies, 14-69995-85) and VSV-G (Sigma, rabbit polyclonal antibody, V4888) ] directed against virus-specific proteins. Viral addition in subsequent viral transduction experiments was normalized using the relative viral concentrations obtained from protein analysis and western blotting.

Mammalian cell infection protocol: to determine whether the PTM analog-labeled virus retained the ability to infect mammalian cells, 50,000U 2-OS cells (human osteosarcoma cell line) were plated on a 6-well chamber glass bottom plate. 20 or 50uL of enriched virus was added to a final volume of 2ml of medium + serum (McCoy's + 10% FBS). Then will beThe plates were maintained at 5% CO₂Incubated at 37 ℃ overnight. The following day, cells were photographed at 10 or 20X magnification on an AMG EVOS fluorescence microscope using white light and GFP filters (fig. 5).

To determine the effect of PTM analog incorporation on BacMam's ability to enter mammalian cells, a BacMam construct expressing nuclear GFP was used. Since BacMam virus replicates in insect cells but not in mammalian cells, sugar and fatty acid analogue tagged viruses were produced in SF9 insect cells and then used to determine infectivity in U2-OS cells (human osteosarcoma cell line). Nuclear GFP expression occurs in U2-OS cells only if the virus is able to enter the cell. FIG. 5 is a panel showing the phase (lower panel) and fluorescent GFP image (upper panel) of U2-OS cells infected with BacMam virus labeled with PTM analog. In these panels, cells treated with myristate-azide and palmitate-azide labeled viruses showed no nuclear-GFP expression, while control cells (no virus) and cells treated with sugar-labeled viruses showed significant nuclear GFP expression.

Claims (8)

1. An in vitro method of producing a labeled virus, the method comprising: contacting a cell infected with a virus with an azide-modified fatty acid or a pharmaceutically acceptable salt thereof, wherein the azide-modified fatty acid is 12-azidododecanoic acid or 15-azidopentadecanoic acid, such that the azide-modified fatty acid or pharmaceutically acceptable salt thereof enters the cell and is incorporated into proteins of the virus, thereby generating a labeled virus, wherein the labeled virus has reduced or inhibited infectivity.

2. The method of claim 1, wherein the cell is a human cell.

3. The method of claim 1, wherein the virus is a human immunodeficiency virus.

4. The method of claim 1, wherein the cell is an insect cell.

5. The method of claim 1, wherein the virus is a baculovirus.

6. The method of claim 1, wherein the azide-modified fatty acid or pharmaceutically acceptable salt is formulated with a pharmaceutically acceptable excipient.

7. The method of claim 6, further comprising the steps of: administering to the cell the azide-modified fatty acid or pharmaceutically acceptable salt formulated with a pharmaceutically acceptable excipient.

8. Use of an azide-modified fatty acid or a pharmaceutically acceptable salt thereof, wherein the azide-modified fatty acid is 12-azidododecanoic acid or 15-azidopentadecanoic acid, and wherein the labeled virus has reduced or inhibited infectivity, in the preparation of a reagent for in vivo tracking of labeled virus.