WO2017011622A1

WO2017011622A1 - Compounds and compositions that induce rig-i-and other pattern recongnition receptors

Info

Publication number: WO2017011622A1
Application number: PCT/US2016/042198
Authority: WO
Inventors: Radhakrishnan P. Iyer; Seetharamaiyer Padmanabhan
Original assignee: Spring Bank Pharmaceuticals, Inc.
Priority date: 2015-07-14
Filing date: 2016-07-14
Publication date: 2017-01-19
Also published as: TW201717968A

Abstract

This invention relates to compounds and compositions for the induction of expression of a pattern recognition receptor (e.g., RIG-I, NOD2, or STING) and methods of use thereof.

Description

COMPOUNDS AND COMPOSITIONS THAT INDUCE RIG-I

AND OTHER PATTERN RECOGNITION RECEPTORS

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/192,438, filed July 14, 2015, the contents of which are herein incorporated by reference in their entirety.

FIELD OF INVENTION

This invention relates to compounds and compositions that induce expression of pattern recognition receptors (e.g., RIG-I) and methods of use thereof.

BACKGROUND OF INVENTION

A key feature of the innate immune system is the recognition and elimination of microbial pathogens. Identification of these pathogenic invaders occurs through host recognition of evolutionarily conserved microbial structures known as pathogen-associated molecular patterns (PAMPs) (Jensen, S. and Thomsen, A.R. Virol (2012) 86:2900-2910). These PAMPs include a wide array of molecular structures, such as nucleic acids, lipopolysaccharides, and glycoproteins that may be broadly shared by multiple microbial species and are critical to their survival and/or pathogenicity. Host recognition may occur by multiple pathways, such as activation of pattern recognition receptors (PRRs), which ultimately lead to downstream signaling events and culminate in the mounting of an immune response.

The retinoic acid-inducible gene-I (RIG-I) protein is a DNA helicase that also functions as a sensor of microbial-derived RNA. RIG-I is important factor in host recognition of RNA viruses from a variety of different viral families, including Flaviviridae (e.g., West Nile virus, Hepatitis C virus, Japanese encephalitis virus, Dengue virus), Paramyxoviridae (e.g., Sendai virus, Newcastle disease virus, Respiratory syncytial virus, Measles virus), Rhabdoviridae (e.g., Rabies virus), Orthomyxoviridae (e.g., influenza A virus, influenza B virus), and Arenaviridae (e.g., Lassa virus). In addition, recent work has shown that RIG-I functions as an efficient sensor for the Ebola virus (Cardenas, W.B. Virol (2006) 80:5168-5178), the Hepatitis B virus (Sato, S. et al, Immunity (2015) 42: 123-132), the human immunodeficiency virus (Berg, R.K. et al., PLoS One (2012) 7:e29291) and a number of bacteria (Dixit, E. and Kagan, J.C. Adv Immunol (2013) 117:99-125; Abdullah, Z. et al, EMBO J (2012) 31 :4153-4164), as well as a biomarker for the prediction of prognosis for certain types of cancer, such as hepatocellular carcinoma (Hou, J. et al, Cancer Cell (2014) 25:49-63). In addition to RIG-I, other PRRs also play a role in sensing microbial-derived nucleic acids, including NOD2, STING, LGP2, MDA5, and a number of Tolllike receptors (TLRs) that are expressed on the cell surface and within endosomal compartments. These PRRs are agnostic as to the viral genotype and recognize both with type and drug-resistant viral mutants. Recent evidence shows that activation of RIG-I can play an important role in eradicating latent HIV in reservoirs.

A major obstacle of many currently available antiviral therapies relates to the emergence of drug resistant variants that occurs upon extended use. In addition, many available treatments require persistent and long-term therapy, which often results in unwanted side effects and the risk of relapse upon treatment discontinuation. Further, many viruses can be subdivided into different genotypes, and certain drugs developed against one genotype may not be active against other genotypes. In contrast, the use of small molecule mimics of viral-derived RNA capable of PRR induction provides an alternate approach to the treatment of viral infection, as these compounds may be agnostic to genotype, may possess both direct antiviral activity as well as the ability to activate the host immune response, and potentially limit the development of drug resistance and toxicity.

Recent publications have highlighted the importance of RIG-I and STING as mediators of innate and adaptive immunity, and RIG-I and STING agonists have been recognized as immuno-oncology agents in cancer therapy (Li, X.Y. et al, Mol Cell Oncol (2014) l:e968016; Woo, S. R. Trends in Immunol (2015) 36:250-256). In particular, RIG-I is involved in the regulation of basic cellular processes such as hematopoietic proliferation and differentiation, maintenance of leukemic sternness, and tumorigenesis of hepatocellular carcinoma, indicating that RIG-I performs an essential function as a tumor suppressor. In addition, the STING

(stimulator of interferon genes) pathway of cytosolic DNA sensing has been shown to play an important mechanistic role in innate immune sensing, driving type I IFN production in cancer. As such, there exists a need for a new generation of therapies that induce expression of PRRs (e.g., RIG-I), for use in the treatment of disease and as diagnostic tools.

SUMMARY OF INVENTION In one aspect, the present invention features a method of inducing the expression of a pattern recognition receptor (PRR) in a subject, the method comprising administering to the subject a compound of Formula (I), wherein the compound is selected from:

Formula (la) Formula (lb) Formula (Ic) or a prodrug or pharmaceutically acceptable salt thereof to thereby treat the subject. In some embodiments, the prodrug of Formula (I) is a compound of Formula (II), wherein the compound is selected from:

Formula (Ila) Formula (lib)

Formula (lie)

or a pharmaceutically acceptable salt thereof. In some embodiments, the PRR comprises a RIG- I-like receptor or a NOD-like receptor. In some embodiments, the PRR comprises RIG-I, NOD2, MDA5, LPG2, or STING (e.g., RIG-I). In some embodiments, the PRR comprises RIG- I. In some embodiments, the PRR comprises NOD2. In some embodiments, the PRR comprises STING.

In some embodiments, the subject is administered a composition comprising a mixture of compounds of Formula (I), e.g., Formula (lb) and Formula (Ic). In some embodiments, the composition comprises Formula (lb) and comprises less than about 5% of Formula (Ic) (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1% of Formula (Ic)), or is substantially free of Formula (Ic). In some embodiments, the composition comprises Formula (Ic) and comprises less than about 5% of Formula (lb) (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1% of Formula (lb), or is substantially free of Formula (lb)).

In some embodiments, the subject is administered a composition comprising a mixture of compounds of Formula (II), e.g., Formula (lib) and Formula (lie). In some embodiments, the composition comprises Formula (lib) and comprises less than about 5% of Formula (lie) (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1% of Formula (lie), or is substantially free of Formula (lie)). In some embodiments, the composition comprises Formula (lie) and comprises less than about 5% of Formula (lib) (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1% of Formula (lib), or is substantially free of Formula (lib)).

In some embodiments, the induction of PRR expression in the subject comprises an increase, enhancement, or initiation of expression of a PRR (e.g., RIG-I or NOD2, e.g., in the liver). In some embodiments, the increase or enhancement of expression of a PRR (e.g., RIG-I or NOD2, e.g., in the liver) is between about 5% and about 95% compared with a reference standard or reference treatment (e.g., between about 10% and about 90%). In some

embodiments, the expression of a PRR (e.g., RIG-I or NOD2) is induced by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more relative to a reference standard. In some embodiments, the induction of expression of a PRR (e.g., RIG-I or NOD2) occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a compound of Formula (I) or Formula (II), or a

pharmaceutically acceptable salt thereof to a subject. In some embodiments, the induction of expression of a PRR (e.g., RIG-I or NOD2) continues after administration of a compound of Formula (I) or Formula (II) has ended.

In some embodiments, the induction of PRR expression in the subject comprises an increase, enhancement, or initiation of expression of a PRR (e.g., STING). In some

embodiments, the induction of PRR expression (e.g., STING expression) occurs in the microenvironment of cancer cells (e.g., in the tumor microenvironment). In some embodiments, the induction of PRR expression (e.g., STING expression) occurs in antigen-presenting cells (e.g., dendritic cells) in the microenvironment of cancer cells (e.g., the tumor

microenvironment). In some embodiments, the increase or enhancement of expression of a PRR (e.g., STING, e.g., in the tumor microenvironment) is between about 5% and about 95% compared with a reference standard or reference treatment (e.g., between about 10% and about 90%). In some embodiments, the expression of a PRR (e.g., STING) is induced by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more relative to a reference standard. In some embodiments, the induction of expression of a PRR (e.g., STING) occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a compound of Formula (I) or Formula (II) or a

pharmaceutically acceptable salt thereof to a subject. In some embodiments, the induction of expression of a PRR (e.g., STING) continues after administration of a compound of Formula (I) or Formula (II) has ended.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments the subject is a non-human animal, e.g., a woodchuck (e.g., Eastern woodchuck) or a mouse.

In some embodiments, the subject is suffering from a microbial infection (e.g., a viral infection, a bacterial infection, a fungal infection, or a parasitic infection) or a cancer.

In some embodiments, the cancer is selected from a cancer of the breast, bone, brain, cervix, colon, gastrointestinal tract, eye, gall bladder, lymph nodes, blood, lung, liver, skin, mouth, prostate, ovary, penis, pancreas, uterus, testicles, stomach, thymus, thyroid, or other part of the body. In some embodiments, the cancer comprises a solid tumor (e.g., a carcinoma, a sarcoma, or a lymphoma). In some embodiments, the cancer comprises hepatocellular carcinoma. In some embodiments, the cancer comprises breast cancer, renal cell carcinoma, colon cancer, melanoma, ovarian cancer, head and neck squamous cell carcinoma, pancreatic cancer, prostate cancer, lung cancer, brain cancer, or gastrointestinal stromal cancer. In some embodiments, the cancer is a leukemia. In some embodiments, the cancer cells (e.g., tumor cells) comprise specific cancer-associated antigens that induce a T-cell response.

In some embodiments, the microbial infection (e.g., viral infection) comprises infection with an RNA virus or a DNA virus. In some embodiments, the RNA virus or DNA virus comprises a single-stranded virus (e.g., positive single- stranded or negative single- stranded) or a double-stranded virus. In some embodiments, the RNA virus or DNA virus comprises a virus in Group I, Group II, Group III, Group IV, Group V, Group VI, or Group VII class of virus.

In some embodiments, the RNA virus comprises a dsRNA virus, an ssRNA virus (e.g., a positive- strand ssRNA virus or a negative-strand ssRNA virus), or an ssRNA RT virus. In some embodiments, the dsRNA virus is a member of the Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae , Hypoviridae, Megabirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae, or Totiviridae families, or other family of dsRNA virus. In some embodiments, the positive- strand ssRNA virus is a member of the Arteriviridae, Coronaviridae, Mesoniviridae,

Roniviridae, Dicistroviridae , Iflaviridae, Marnaviridae, Piconaviridae, Secoviridae,

Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Tymoviridae, Alphatetraviridae, Alvernaviridae, Astroviridae, Barnaviridae, Bromoviridae, Caliciviridae, Carmotetraviridae, Closteroviridae, Flaviviridae, Leviviridae, Luteoviridae, Narnaviridae, Nodaviridae, Permutotetraviridae, Potyviridae, Togaviridae, or Virgaviridae families, or other family of positive- strand (sense) ssRNA virus. In some embodiments, the negative- strand ssRNA virus is a member of the Bornaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae , Nyamiviridae, Arenaviridae, Bunyaviridae, Ophioviridae, or Orthomyxoviridae families, or other family of negative-strand (antisense) ssRNA virus. In some embodiments, the ssRNA RT virus is a member of the Metaviridae, Pseudoviridae, or Retroviridae families, or other family of ssRNA RT virus. In some embodiments, the ssRNA RT virus is a member of the Lentivirus genus (e.g., human immunodeficiency virus 1 (HIV), or a subtype, species, or variant thereof). In some embodiments, the ssRNA RT virus is HIV or a subtype, species, or variant thereof.

In some embodiments, the DNA virus comprises a dsDNA virus, an ssDNA virus, or a dsDNA RT virus. In some embodiments, the dsDNA virus is a member of the Myoviridae, Podoviridae, Siphoviridae , Alloherpesviridae, Herpesviridae, Malacoherpesviridae,

Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae , Ascoviridae, Asfarviridae , Baculoviridae, Bicaudaviridae , Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae , Hytrosaviridae, Iridoviridae , Marseilleviridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Polydnaviruses, Polymaviridae, Poxviridae,

Sphaerolipoviridae, Tectiviridae , or Turriviridae families, or other family of dsDNA virus. In some embodiments, the ssDNA virus is a member of the Anelloviridae, Bacillariodnaviridiae, Bidnaviridae, Circoviridae , Geminiviridae, Inoviridae, Microviridae, Nanoviridae,

Parvoviridae, or Spiraviridae families, or other family of ssDNA virus. In some embodiments, the dsDNA RT virus is a member of the Hepadnaviridae, or Caulimoviridae families, or other family of dsDNA RT virus.

In some embodiments, the virus is in a latent stage. In some embodiments, the virus is an ssRNA retrovirus (ssRNA RT virus), e.g., a Group VI virus, and is latent, e.g., within a cell. In some embodiments, the virus is the human immunodeficiency virus 1 (HIV)), or a subtype, species, or variant thereof, and is latent, e.g., within a cell. In some embodiments, the subject is infected with the HIV virus and is asymptomatic.

In some embodiments, the bacterial infection comprises infection with a Gram-negative bacterium or a Gram-positive bacterium. In some embodiments, the bacterial infection comprises a bacterium selected from Listeria (e.g., Listeria monocytogenes), Francisella (e.g., Francisella tularensis), Mycobacteria (e.g., Mycobacteria tuberculosis), Brucella (e.g., Brucella abortis), Streptococcus (e.g., group B Streptococcus), Legionella (e.g., Legionella pneumophila), Escherichia (e.g., Escherichia coli), Pseudomonas (e.g., Psuedomonas aeruginosa), Salmonella (e.g., Salmonella typhi), Shigella (e.g., Shigella flexneri), Campylobacter (e.g., Campylobacter jejuni), Clostridium (e.g., Clostrodium botulinum), Enterococcus (e.g., Enterococcus faecalis), Vibrio (e.g., Vibrio cholera), Yersinia (e.g., Yersinia pestis), and Staphylococcus (e.g.,

Staphylococcus aureus), or other genera, species, subtypes, or variants thereof.

In some embodiments, the method described herein further comprises analyzing or receiving analysis of a biopsy specimen or biological tissue sample from the subject at least once prior to the end of treatment. In some embodiments, the biopsy specimen is a liver biopsy specimen. In some embodiments, the liver biopsy specimen is analyzed by

immunohistochemical staining. In some embodiments, the liver biopsy specimen is analyzed by immunohistochemical staining for the extent of induction of a PRR (e.g., RIG-I or NOD2). In some embodiments, the immunohistochemical staining indicates an increase in the distribution of PRR expression (e.g., RIG-I expression or NOD2 expression) in the liver over the course of treatment of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof. In some embodiments, the increase in the distribution of expression of a PRR (e.g., RIG-I or NOD2) in the liver is from the onset of treatment to the end of treatment is between about 5% and about 95%.

In some embodiments, the liver biopsy specimen is analyzed for the levels of one or more markers of microbial infection, e.g., viral DNA, viral RNA, viral antigens, cccDNA, bacterial load. In some embodiments, the liver biopsy specimen is analyzed for the expression level of interferon (e.g., interferon alfa or interferon beta), an interferon stimulating protein (e.g., ISG15, CXCL10, OAS 1), or other cytokines. In some embodiments, the liver biopsy specimen is analyzed for the reduction of liver inflammation, necrosis, steatosis, or fibrosis.

In some embodiments, the biopsy specimen is a cancer biopsy specimen (e.g., a tumor biopsy specimen). In some embodiments, the cancer biopsy specimen (e.g., tumor biopsy specimen) is analyzed by immunohistochemical staining. In some embodiments, the cancer biopsy specimen (e.g., tumor biopsy specimen) is analyzed by immunohistochemical staining for the extent of induction of a PRR (e.g., RIG-I or STING). In some embodiments, the immunohistochemical staining indicates an increase in the distribution of PRR expression (e.g., RIG-I expression or STING expression) in the cancer (e.g., tumor) over the course of treatment of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof. In some embodiments, the increase in the distribution of expression of a PRR (e.g., RIG-I or STING) in the cancer (e.g., tumor) is from the onset of treatment to the end of treatment is between about 5% and about 95%.

In some embodiments, the subject is treatment naive. In some embodiments, the subject has previously been treated for a microbial infection (e.g., a viral infection, a bacterial infection, a fungal infection, or a parasitic infection) or cancer. In some embodiments, the subject has previously been treated for HBV infection or HCV infection. In some embodiments, the subject has been previously treated for a bacterial infection or cancer.

In some embodiments, the compound of Formula (I) or Formula (II) is administered orally (e.g., the compound of Formula (II) is administered orally). In some embodiments, the compound of Formula (I) or Formula (II) is administered parenterally (e.g., the compound of Formula (II) is administered parenterally). In some embodiments, the method comprises daily administration of said compound. In some embodiments, the administration is once daily. In some embodiments, the method comprises administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof for a duration between about 1 week and 20 weeks. In some embodiments, the method comprises administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof for a duration between about 1 week and 12 weeks.

In some embodiments, the dosage of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof comprises about 0.5 mg/kg to about 100 mg/kg. In some embodiments, the dosage of a compound of Formula (I) or Formula (II) or a

pharmaceutically acceptable salt thereof comprises 0.5 mg/kg to about 50 mg/kg. In some embodiments, the dosage of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof comprises 5 mg/kg to about 50 mg/kg.

In some embodiments, the dosage comprises a liquid or a solid dosage form. In some embodiments, the liquid dosage form comprises a suspension, a solution, a linctus, an emulsion, a drink, an elixir, or a syrup. In some embodiments, the solid dosage form comprises a capsule, tablet, powder, dragee, or a microencapsulated dose form. In some embodiments, the method further comprises administration of a therapeutically effective amount of an additional agent. In some embodiments, the additional agent is an antiviral agent, an antibacterial agent, or an anticancer agent. In some embodiments, the antiviral agent comprises an interferon, a nucleoside analog, a non-nucleoside antiviral, or a small- molecule immune enhancer. In some embodiments, the antiviral agent comprises a capsid inhibitor, an entry inhibitor, a secretion inhibitor, a microRNA, an antisense RNA agent, an RNAi agent, or other agent designed to inhibit viral RNA or DNA. In some embodiments, the antiviral agent comprises entecavir, lamuvidine, adefovir, darunavir, sofosbuvir, telaprevir, tenofovir, zidovudine, and ribavirin. In some embodiments, the antibacterial agent comprises gentamicin, kanamycin, streptomycin, chloramphenicol, ceftobiprole, amoxicillin, penicillin, bacitracin, tetracycline, rifabutin, tigecycline, and vancomycin. In some embodiments, the anticancer agent is selected from methotrexate, 5-fluorouracil, doxorubicin, vincristine, bleomycin, vinblastine, dacarbazine, toposide, cisplatin, epirubicin, and sorafenib tosylate.

In another aspect, the present invention features a method of inducing the expression of a pattern recognition receptor in a subject suffering from a microbial infection or a cancer, the method comprising administering to the subject a compound of Formula (I), wherein the compound is selected from:

Formula (Ila) Formula (lib)

Formula (lie)

In some embodiments, the subject is administered a composition comprising a mixture of compounds of Formula (I), e.g., Formula (lb) and Formula (Ic). In some embodiments, the composition comprises Formula (lb) and comprises less than about 5% of Formula (Ic) (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1% of Formula (Ic)), or is substantially free of Formula (Ic). In some embodiments, the composition comprises Formula (Ic) and comprises less than about 5% of Formula (lb) (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1% of Formula (lb), or is substantially free of Formula (lb)). In some embodiments, the subject is administered a composition comprising a mixture of compounds of Formula (II), e.g., Formula (lib) and Formula (lie). In some embodiments, the composition comprises Formula (lib) and comprises less than about 5% of Formula (lie) (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1% of Formula (lie), or is substantially free of Formula (lie)). In some embodiments, the composition comprises Formula (lie) and comprises less than about 5% of Formula (lib) (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1% of Formula (lib), or is substantially free of Formula (lib)).

embodiments, the expression of a PRR (e.g., RIG-I or NOD2) is induced by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more relative to a reference standard. In some embodiments, the induction of expression of a PRR (e.g., RIG-I or NOD2) occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a compound of Formula (I) or Formula (II) or a

In some embodiments, the cancer is selected from a cancer of the breast, bone, brain, cervix, colon, gastrointestinal tract, eye, gall bladder, lymph nodes, blood, lung, liver, skin, mouth, prostate, ovary, penis, pancreas, uterus, testicles, stomach, thymus, thyroid, or other part of the body. . In some embodiments, the cancer comprises a solid tumor (e.g., a carcinoma, a sarcoma, or a lymphoma). In some embodiments, the cancer comprises hepatocellular carcinoma. In some embodiments, the cancer comprises breast cancer, renal cell carcinoma, colon cancer, melanoma, ovarian cancer, head and neck squamous cell carcinoma, pancreatic cancer, prostate cancer, lung cancer, brain cancer, or gastrointestinal stromal cancer. In some embodiments, the cancer is a leukemia. In some embodiments, the cancer cells (e.g., tumor cells) comprise specific cancer-associated antigens that induce a T-cell response. In some embodiments, the microbial infection (e.g., viral infection) comprises infection with an RNA virus or a DNA virus. In some embodiments, the RNA virus or DNA virus comprises a single-stranded virus (e.g., positive single- stranded or negative single- stranded) or a double-stranded virus. In some embodiments, the RNA virus or DNA virus comprises a virus in Group I, Group II, Group III, Group IV, Group V, Group VI, or Group VII class of virus.

Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Tymoviridae, Alphatetraviridae, Alvernaviridae, Astroviridae, Barnaviridae, Bromoviridae, Caliciviridae, Carmotetraviridae, Closteroviridae, Flaviviridae, Leviviridae, Luteoviridae, Narnaviridae, Nodaviridae,

Permutotetraviridae, Potyviridae, Togaviridae, or Virgaviridae families, or other family of positive- strand (sense) ssRNA virus. In some embodiments, the negative- strand ssRNA virus is a member of the Bornaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae , Nyamiviridae, Arenaviridae, Bunyaviridae, Ophioviridae, or Orthomyxoviridae families, or other family of negative-strand (antisense) ssRNA virus. In some embodiments, the ssRNA RT virus is a member of the Metaviridae, Pseudoviridae, or Retroviridae families, or other family of ssRNA RT virus. In some embodiments, the ssRNA RT virus is a member of the Lentivirus genus (e.g., human immunodeficiency virus 1 (HIV), or a subtype, species, or variant thereof). In some embodiments, the ssRNA RT virus is HIV or a subtype, species, or variant thereof.

Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae , Ascoviridae, Asfarviridae , Baculoviridae, Bicaudaviridae , Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae , Hytrosaviridae, Iridoviridae , Marseilleviridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Polydnaviruses, Polymaviridae, Poxviridae, Sphaerolipoviridae, Tectiviridae, or Turriviridae families, or other family of dsDNA virus. In some embodiments, the ssDNA virus is a member of the Anelloviridae, Bacillariodnaviridiae, Bidnaviridae, Circoviridae, Geminiviridae, Inoviridae, Microviridae, Nanoviridae,

In some embodiments, the biopsy specimen is a cancer biopsy specimen (e.g., a tumor biopsy specimen). In some embodiments, the cancer biopsy specimen (e.g., tumor biopsy specimen) is analyzed by immunohistochemical staining. In some embodiments, the cancer biopsy specimen (e.g., tumor biopsy specimen) is analyzed by immunohistochemical staining for the extent of induction of a PRR (e.g., RIG-I or STING). In some embodiments, the

immunohistochemical staining indicates an increase in the distribution of PRR expression (e.g., RIG-I expression or STING expression) in the cancer (e.g., tumor) over the course of treatment of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof. In some embodiments, the increase in the distribution of expression of a PRR (e.g., RIG-I or STING) in the cancer (e.g., tumor) is from the onset of treatment to the end of treatment is between about 5% and about 95%.

In some embodiments, the dosage comprises a liquid or a solid dosage form. In some embodiments, the liquid dosage form comprises a suspension, a solution, a linctus, an emulsion, a drink, an elixir, or a syrup. In some embodiments, the solid dosage form comprises a capsule, tablet, powder, dragee, or a microencapsulated dose form.

In some embodiments, the method further comprises administration of a therapeutically effective amount of an additional agent. In some embodiments, the additional agent is an antiviral agent, an antibacterial agent, or an anticancer agent. In some embodiments, the antiviral agent comprises an interferon, a nucleoside analog, a non-nucleoside antiviral, or a small- molecule immune enhancer. In some embodiments, the antiviral agent comprises a capsid inhibitor, an entry inhibitor, a secretion inhibitor, a microRNA, an antisense RNA agent, an RNAi agent, or other agent designed to inhibit viral RNA or DNA. In some embodiments, the antiviral agent comprises entecavir, lamuvidine, adefovir, darunavir, sofosbuvir, telaprevir, tenofovir, zidovudine, and ribavirin. In some embodiments, the antibacterial agent comprises gentamicin, kanamycin, streptomycin, chloramphenicol, ceftobiprole, amoxicillin, penicillin, bacitracin, tetracycline, rifabutin, tigecycline, and vancomycin. In some embodiments, the anticancer agent is selected from methotrexate, 5-fluorouracil, doxorubicin, vincristine, bleomycin, vinblastine, dacarbazine, toposide, cisplatin, epirubicin, and sorafenib tosylate.

In another aspect, the present invention features a method of activating a pattern recognition receptor (PRR) in a subject, the method comprising administering to the subject a compound of Formula (I), wherein the compound is selected from:

Formula (lie) or a pharmaceutically acceptable salt thereof. In some embodiments, the PRR comprises a RIG- I-like receptor or a NOD-like receptor. In some embodiments, the PRR comprises RIG-I, NOD2, MDA5, LPG2, or STING (e.g., RIG-I). In some embodiments, the PRR comprises RIG- I. In some embodiments, the PRR comprises NOD2. In some embodiments, the PRR comprises STING.

In some embodiments, the activation of a PRR (e.g., RIG-I or NOD2, e.g., in the liver) comprises activation of a downstream signaling protein (e.g., a pro -inflammatory cytokine, an anti-inflammatory cytokine, type I interferons (IFN-a, IFN-β), or an interferon stimulated gene) or downstream signaling pathway. In some embodiments, the activation of a PRR (e.g., RIG-I or NOD2, e.g., in the liver) is between about 5% and about 95% compared with a reference standard or reference treatment (e.g., between about 10% and about 90%). In some

embodiments, a PRR (e.g., RIG-I or NOD2) is activated by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more relative to a reference standard. In some embodiments, the activation of a PRR (e.g., RIG-I or NOD2) occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof to a subject.

In some embodiments, the activation of a PRR (e.g., RIG-I or NOD2) continues after administration of a compound of Formula (I) or Formula (II) has ended.

In some embodiments, the activation of a PRR (e.g., RIG-I or STING) comprises activation of a downstream signaling protein (e.g., a pro-inflammatory cytokine, an antiinflammatory cytokine, type I interferons (IFN-a, IFN-β), or an interferon stimulated gene) or downstream signaling pathway. In some embodiments, the activation of a PRR (e.g., RIG-I or STING) occurs in the microenvironment of cancer cells (e.g., in the tumor microenvironment). In some embodiments, the activation of a PRR (e.g., RIG-I or STING) occurs in antigen- presenting cells (e.g., dendritic cells) in the microenvironment of a cancer (e.g., the tumor microenvironment). In some embodiments, the activation of a PRR (e.g., RIG-I or STING) is between about 5% and about 95% compared with a reference standard or reference treatment (e.g., between about 10% and about 90%). In some embodiments, a PRR (e.g., RIG-I or STING) is activated by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more relative to a reference standard. In some embodiments, the activation of a PRR (e.g., RIG-I or STING) occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof to a subject. In some embodiments, the activation of a PRR (e.g., RIG-I or STING) continues after administration of a compound of Formula (I) or Formula (II) has ended.

In some embodiments, the activation of a PRR (e.g., RIG-I or NOD2) results in the induction of expression of a PRR (e.g., RIG-I or NOD2). In some embodiments, the induction of PRR expression in the subject comprises an increase, enhancement, or initiation of expression of a PRR (e.g., RIG-I or NOD2, e.g., in the liver). In some embodiments, the increase or

enhancement of expression of a PRR (e.g., RIG-I or NOD2, e.g., in the liver) is between about 5% and about 95% compared with a reference standard or reference treatment (e.g., between about 10% and about 90%). In some embodiments, the expression of a PRR (e.g., RIG-I or NOD2) is induced by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more relative to a reference standard. In some embodiments, the induction of expression of a PRR (e.g., RIG-I or NOD2) occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof to a subject. In some embodiments, the induction of expression of a PRR (e.g., RIG-I or NOD2) continues after administration of a compound of Formula (I) or Formula (II) has ended.

In some embodiments, the activation of a PRR (e.g., RIG-I or NOD2) does not result in the induction of expression of a PRR (e.g., RIG-I or NOD2).

In some embodiments, the activation of a PRR (e.g., RIG-I or STING) results in the induction of expression of a PRR (e.g., RIG-I or STING). In some embodiments, the induction of PRR expression in the subject comprises an increase, enhancement, or initiation of expression of a PRR (e.g., RIG-I or STING, e.g., in the microenvironment of a cancer, e.g., a tumor). In some embodiments, the increase or enhancement of expression of a PRR (e.g., RIG-I or STING, e.g., in the microenvironment of a cancer, e.g., a tumor) is between about 5% and about 95% compared with a reference standard or reference treatment (e.g., between about 10% and about 90%). In some embodiments, the expression of a PRR (e.g., RIG-I or STING) is induced by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more relative to a reference standard. In some embodiments, the induction of expression of a PRR (e.g., RIG-I or STING) occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof to a subject. In some embodiments, the induction of expression of a PRR (e.g., RIG-I or STING) continues after administration of a compound of Formula (I) or Formula (II) has ended.

In some embodiments, the activation of a PRR (e.g., RIG-I or STING) does not result in the induction of expression of a PRR (e.g., RIG-I or STING).

Sphaerolipoviridae, Tectiviridae , or Turriviridae families, or other family of dsDNA virus. In some embodiments, the ssDNA virus is a member of the Anelloviridae, Bacillariodnaviridiae, Bidnaviridae, Circoviridae, Geminiviridae , Inoviridae, Microviridae , Nanoviridae, Parvoviridae, or Spiraviridae families, or other family of ssDNA virus. In some embodiments, the dsDNA RT virus is a member of the Hepadnaviridae, or Caulimoviridae families, or other family of dsDNA RT virus.

immunohistochemical staining. In some embodiments, the liver biopsy specimen is analyzed by immunohistochemical staining for the extent of induction of a PRR (e.g., RIG-I or NOD2). In some embodiments, the immunohistochemical staining indicates an increase in the distribution of PRR expression (e.g., RIG-I expression or NOD2 expression) in the liver over the course of treatment of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof. In some embodiments, the increase in the distribution of expression of a PRR (e.g., RIG-I or NOD2) in the liver is from the onset of treatment to the end of treatment is between about 5% and about 95%. In some embodiments, the liver biopsy specimen is analyzed for the levels of one or more markers of microbial infection, e.g., viral DNA, viral RNA, viral antigens, cccDNA, bacterial load. In some embodiments, the liver biopsy specimen is analyzed for the expression level of interferon (e.g., interferon alfa or interferon beta), an interferon stimulating protein (e.g., ISG15, CXCL10, OAS 1), or other cytokines. In some embodiments, the liver biopsy specimen is analyzed for the reduction of liver inflammation, necrosis, steatosis, or fibrosis.

In another aspect, the present invention features a method of inducing the expression of a pattern recognition receptor in a subject suffering from a cancer, the method comprising administering to the subject a compound of Formula (I), wherein the compound is selected from:

Formula (Ila) Formula (lib)

Formula (lie)

In some embodiments, the induction of PRR expression in the subject comprises an increase, enhancement, or initiation of expression of a PRR (e.g., RIG-I, NOD2, or STING). In some embodiments, the induction of PRR expression (e.g., RIG-I expression, NOD2 expression, or STING expression) occurs in the microenvironment of cancer cells (e.g., in the tumor microenvironment). In some embodiments, the induction of PRR expression (e.g., STING expression) occurs in antigen-presenting cells (e.g., dendritic cells) in the microenvironment of cancer cells (e.g., the tumor microenvironment). In some embodiments, the increase or enhancement of expression of a PRR (e.g., RIG-I, NOD2, or STING) is between about 5% and about 95% compared with a reference standard or reference treatment (e.g., between about 10% and about 90%). In some embodiments, the expression of a PRR (e.g., RIG-I, NOD2, or STING) is induced by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more relative to a reference standard. In some embodiments, the induction of expression of a PRR (e.g., RIG- 1, NOD2, or STING) occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof to a subject. In some embodiments, the induction of expression of a PRR (e.g., RIG-I, NOD2, or STING) continues after administration of a compound of Formula (I) or Formula (II) has ended.

In some embodiments, the method described herein further comprises analyzing or receiving analysis of a biopsy specimen or biological tissue sample from the subject at least once prior to the end of treatment. In some embodiments, the biopsy specimen is a cancer biopsy specimen (e.g., a tumor biopsy specimen). In some embodiments, the cancer biopsy specimen (e.g., tumor biopsy specimen) is analyzed by immunohistochemical staining. In some embodiments, the cancer biopsy specimen (e.g., tumor biopsy specimen) is analyzed by immunohistochemical staining for the extent of induction of a PRR (e.g., RIG-I or STING). In some embodiments, the immunohistochemical staining indicates an increase in the distribution of PRR expression (e.g., RIG-I expression or STING expression) in the cancer (e.g., tumor) over the course of treatment of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof. In some embodiments, the increase in the distribution of expression of a PRR (e.g., RIG-I or STING) in the cancer (e.g., tumor) is from the onset of treatment to the end of treatment is between about 5% and about 95%.

In some embodiments, the subject is treatment naive. In some embodiments, the subject has previously been treated for a cancer. In some embodiments, the subject has relapsed. In some embodiments, the subject has relapsed more than once (e.g., twice, three times, four times). In some embodiments, the cancer is relapsed or refractory.

In some embodiments, the dosage comprises a liquid or a solid dosage form. In some embodiments, the liquid dosage form comprises a suspension, a solution, a linctus, an emulsion, a drink, an elixir, or a syrup. In some embodiments, the solid dosage form comprises a capsule, tablet, powder, dragee, or a microencapsulated dose form. In some embodiments, the method further comprises administration of a therapeutically effective amount of an additional agent. In some embodiments, the additional agent is an anticancer agent. In some embodiments, the anticancer agent is selected from methotrexate, 5- fluorouracil, doxorubicin, vincristine, bleomycin, vinblastine, dacarbazine, toposide, cisplatin, epirubicin, and sorafenib tosylate.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1-7 are slides depicting the induction of RIG-I expression in the livers of two chronically WHV-infected woodchucks upon administration of Formula (II). Induction of RIG-I expression was determined through evaluation of immunohistochemical staining of liver biopsy specimens taken over the course of Formula (II) treatment at week 0 (pre-treatment, FIG. 1 and FIG. 4), week 6 (midway through dosing, FIG. 5), week 12 (end of Formula (II) dosing, FIG. 2 and FIG. 6), and week 20 (end of recovery period, FIG. 3 and FIG. 7). FIGS 1-3 refer to specimens from woodchuck 1, and FIGS. 4-7 refer to specimens from woodchuck 2. All images are shown at a magnification of lOx. The evaluation of RIG-I expression levels was carried out by scoring of the color intensity and frequency of distribution during the histochemical staining analysis.

FIGS. 8A-8B show agarose gels depicting the siRNA-mediated silencing of RIG-I and NOD2 in human lung epithelial (HLE) cells. The induction level of NOD2 and RIG-I in HLE cells transfected with either NOD2 siRNA, RIG-I siRNA, or control siRNA was monitored by RT-PCR.

FIG. 9 is a graph illustrating the connection between expression of NOD2 and RIG-I and interferon-β (IFN-β) induction. HLE cells were transfected with either NOD2 siRNA, RIG-I siRNA, or control siRNA, and then treated with Formula (II) 24 hours after transfection. After 12 hours, the levels of IFN-β were determined by ELISA analysis.

FIGS. 10A-10B are graphs depicting the dose-dependent binding of a compound of Formula (I) to either RIG-I (FIG. 10A) or STING (FIG. 10B). RIG-I or STING was

immobilized to a microplate, followed by incubation of different doses of biotinylated Formula (I). The samples were then exposed to HRP-conjugated streptavidin, and the difference in absorbance at 450 nm (OD 450 nm) was determined.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of inducing the expression of a PRR (e.g., RIG- I, NOD2, or STING) in a subject. In some embodiments, the method comprises administration of a compound of Formula (I) or a prodrug (e.g., a compound of Formula (II)) or

pharmaceutically acceptable salt thereof.

Definitions

As used herein, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

"About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.

As used herein, the term "acquire" or "acquiring" as the terms are used herein, refer to obtaining possession of a physical entity (e.g. , a sample, e.g. , blood sample or liver biopsy specimen), or a value, e.g. , a numerical value, by "directly acquiring" or "indirectly acquiring" the physical entity or value. "Directly acquiring" means performing a process (e.g. , an analytical method) to obtain the physical entity or value. "Indirectly acquiring" refers to receiving the physical entity or value from another party or source (e.g. , a third party laboratory that directly acquired the physical entity or value). Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance, e.g. , performing an analytical process which includes a physical change in a substance, e.g. , a sample, performing an analytical method, e.g. , a method as described herein, e.g. , by sample analysis of bodily fluid, such as blood by, e.g. , mass spectroscopy, e.g. LC-MS.

As used herein, the terms "induce" or "induction of refer to the increase or enhancement of a function, e.g., the increase or enhancement of the expression of a pattern recognition receptor (e.g, RIG-I, NOD2, or STING). In some embodiments, "induction of PRR expression" refers to induction of transcription of PRR RNA, e.g., RIG-I RNA, NOD2 RNA, or STING RNA (e.g., mRNA) (e.g., an increase or enhancement of), or the translation of a PRR protein, e.g., the RIG-I protein, NOD2 protein, or STING protein (e.g., an increase or enhancement of). In some embodiments, induction of PRR expression (e.g., RIG-I expression, NOD2 expression, or STING expression) refers to the increase or enhancement of the concentration of a PRR RNA, e.g., RIG-I RNA, NOD2 RNA, or STING RNA (e.g., mRNA) or the RIG-I protein, NOD2 protein, or STING protein, e.g., in a cell. In some embodiments, induction of PRR expression (e.g., RIG-I expression, NOD2 expression, or STING expression) refers to the increase of the number of copies of PRR RNA, e.g., RIG-I RNA, NOD2 RNA, STING RNA (e.g., mRNA) or PRR protein, e.g., the RIG-I protein, NOD2 protein, or STING protein, e.g., in a cell. In some embodiments, to induce expression of a PRR (e.g., RIG-I, NOD2, or STING) may refer to the initiation of PRR RNA (e.g., RIG-I RNA. NOD2 RNA, or STING RNA (e.g., mRNA)) transcription or PRR protein (e.g., RIG-I protein, NOD2 protein, or STING protein) translation. In some embodiments, to induce expression of a PRR (e.g., RIG-I, NOD2, or STING) may refer to an increase in the rate of PRR RNA (e.g., RIG-I RNA, NOD2 RNA, or STING RNA (e.g., mRNA)) transcription or an increase in the rate of PRR protein (e.g., RIG-I protein, NOD2 protein, or STING protein) expression.

As used herein, the terms "activate" or "activation" refer to the stimulation or triggering of a function, e.g., of a downstream pathway, e.g., a downstream signaling pathway. In some embodiments, activation of a pattern recognition receptor (PRR) (e.g., RIG-I, NOD2, STING) refers to the stimulation of a specific protein or pathway, e.g., through interaction with a downstream signaling partner (e.g., IFN-β promoter stimulator 1 (IPS-1), IRF3, IRF7, NF-KB, interferons (e.g., IFN-a or IFN-β) and/or cytokines). In some embodiments, activation is distinct from the induction of expression of a PRR. In some embodiments, a PRR may be activated without resulting in an induction of PRR expression (e.g., expression of RIG-I, expression of NOD2, expression of STING). In some embodiments, activation may include induction of expression of a PRR (e.g., RIG-I, NOD2, STING). In some embodiments, activation of a PRR may trigger the induction of expression of a PRR (e.g., RIG-I, NOD2, STING) by about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more compared to a reference standard (e.g., basal expression levels of a PRR (e.g., RIG-I, NOD2, STING)). As used herein, an amount of a compound, conjugate, or substance effective to treat a disorder (e.g. , a disorder described herein), "therapeutically effective amount," "effective amount" or "effective course" refers to an amount of the compound, substance, or composition which is effective, upon single or multiple dose administration(s) to a subject, in treating a subject, or in curing, alleviating, relieving or improving a subject with a disorder (e.g. , a microbial infection) beyond that expected in the absence of such treatment.

As used herein, the term "latent" refers to a microbial infection (e.g., a viral infection) in which the microbe has entered a dormant stage (e.g., in a cell) and is no longer substantially replicating. In some embodiments, viral latency refers to the lysogenic portion of the viral replication cycle. In some embodiments, latency may refer to microbial infection of a host (e.g., a subject described herein). In these cases, the infected subject may experience symptoms related to the infection, or alternatively may be substantially asymptomatic.

As used herein, the terms "prevent" or "preventing" as used in the context of a disorder or disease, refer to administration of an agent to a subject, e.g. , the administration of a compound of the present invention (e.g., compound of Formula (I) or a prodrug (e.g., a compound of Formula (II)) to a subject, such that the onset of at least one symptom of the disorder or disease is delayed as compared to what would be seen in the absence of administration of said agent.

As used herein, the terms "reference treatment" or "reference standard" refer to a standardized level or standardized treatment that is used as basis for comparison. In some embodiments, the reference standard or reference treatment is an accepted, well known, or well characterized standard or treatment in the art. In some embodiments, the reference standard describes an outcome of a method described herein. In some embodiments, the reference standard describes a level of a marker (e.g., a level of induction of a PRR, e.g., RIG-I, NOD2, STING) in a subject or a sample, e.g., prior to initiation of treatment, e.g., with a compound or composition described herein. In some embodiments, the reference standard describes a measure of the presence of, progression of, or severity of a disease or the symptoms thereof, e.g., prior to initiation of treatment, e.g., with a compound or composition described herein.

As used herein, the term "subject" is intended to include human and non-human animals. Exemplary human subjects include a human patient having a disorder, e.g. , a disorder described herein, or a normal subject. The term "non-human animals" includes all vertebrates, e.g. , non- mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, domesticated and/or agriculturally useful animals, e.g. , sheep, dogs, cats, cows, pigs, etc. In exemplary embodiments of the invention, the subject is a woodchuck (e.g., an Eastern woodchuck (Marmota monax)).

As used herein, the terms "treat" or "treating" a subject having a disorder or disease refer to subjecting the subject to a regimen, e.g. , the administration of a compound of Formula (I) or a prodrug (e.g., a compound of Formula (II)) or pharmaceutically acceptable salt thereof, or a composition comprising Formula (I) or a prodrug (e.g., a compound of Formula (II)) or pharmaceutically acceptable salt thereof, such that at least one symptom of the disorder or disease is cured, healed, alleviated, relieved, altered, remedied, ameliorated, or improved.

Treating includes administering an amount effective to alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder or disease, or the symptoms of the disorder or disease. The treatment may inhibit deterioration or worsening of a symptom of a disorder or disease.

Numerous ranges, e.g., ranges for the amount of a drug administered per day, are provided herein. In some embodiments, the range includes both endpoints. In other embodiments, the range excludes one or both endpoints. By way of example, the range can exclude the lower endpoint. Thus, in such an embodiment, a range of 250 to 400 mg/day, excluding the lower endpoint, would cover an amount greater than 250 that is less than or equal to 400 mg/day.

Pattern Recognition Receptors

The disclosure presented herein features methods for the activation and induction of PRR expression (e.g., RIG-I expression, NOD2 expression, or STING expression) in a subject, e.g., a subject with a microbial infection (e.g., a viral infection, bacterial infection, fungal infection, or parasitic infection). Pattern recognition receptors (PRRs) are a broad class of proteins which recognize pathogen-associated molecular patterns (PAMPs) conserved within pathogenic invaders. PAMPs are typically products of bio synthetic pathways that are essential to the survival and/or infectivity of the pathogen, e.g., lipopolysaccharides, glycoproteins, and nucleic acids. Recognition of PAMPs by their cognate PRRs activates signaling pathways that result in the production of immune defense factors such as pro-inflammatory and anti-inflammatory cytokines, type I interferons (IFN-a, IFN-β), and/or interferon stimulated genes (ISGs). It is well known that induction of innate immune signaling also results in the activation of T cell responses as well as the induction of adaptive immunity. These downstream immune effects are essential for clearance of the virus through apoptosis and killing of infected cells through cytotoxic T lymphocytes and other defense mechanisms. It is also well known that interferons act on IS RE (interferon response elements) that can trigger the production of ISGs, which play an important role in antiviral cellular defense.

RIG-I is the founding member of a family of PRRs termed RIG-I-like receptors (RLRs), which primarily detect RNA derived from foreign sources. It is a critical sensor of microbial infection (e.g., viral infection) in most cells and is constitutively expressed at low levels in the cytosol. After ligand binding, the expression of RIG-I is rapidly enhanced, leading to increased RIG-I concentrations in the cell (Jensen, S. and Thomsen, A.R. Virol (2012) 86:2900-2910; Yoneyama M. et al. Nat Immunol (2004) 5:730-737). RIG-I is an ATP-dependent helicase containing a central DExD/H box ATPase domain and tandem N-terminal caspase-recruiting domains (CARDs) that mediate downstream signaling. The C-terminus of RIG-I comprises an ssRNA/dsRNA-binding domain that when unbound acts to silence CARD function at the N- terminus. Without wishing to be bound by theory, it is believed that upon recognition of target RNA structures, two N-terminal CARDs are exposed, allowing for interaction with the CARD of a downstream binding partner, IFN-β promoter stimulator 1 (IPS-1), also known as

mitochondrial antiviral signaling molecule (MAVS) and CARD IF. This interaction in turn triggers further downstream signaling, such as induction of IRF3, IRF7, NF-κΒ, IFNS, and cytokine production that results in the initiation of the host immune response.

RIG-I binds to RNA structures, specifically double- stranded RNA (dsRNA) and single- stranded RNA (ssRNA) bearing an uncapped 5 '-triphosphate group (Jensen, S. and Thomsen, A.R. Virol (2012) 86:2900-2910). RIG-I is able to distinguish foreign ssRNA from self- ssRNAs, such as mRNA and tRNA, primarily through the presence of the uncapped 5'- triphosphate group in the foreign ssRNA, as self-ssRNAs typically do not possess exposed triphosphates on the 5 '-end. In addition, self-mRNA is posttranscriptionally modified with a 5'- 7-methylguanosine cap, which is further subject to 2'-0-methylation. This 2'-0-methylation is suggested to function in order to prevent RIG-I recognition (Daffis, S. et al, Nature (2010) 468:452-456). Similarly, self-tRNA undergoes 5' cleavage and a series of base modifications, and is also often associated with the ribosome, which prevents binding to RIG-I (Jensen, S. and Thomsen, A.R. Virol (2012) 86:2900-2910). Recent work further suggest that RIG-I ssRNA ligands possess internal polyuridine-rich sequences in addition to the uncapped 5 '-triphosphate, and that in certain cases, a 3'-phosphoryl group may be capable of replacing the 5 '-triphosphate (Malathi, K. et al, RNA (2010) 16:2108-2119).

Other RLRs are homologous to RIG-I and function in a similar manner, including MDA5, LGP2, and RNase L. MDA5 is highly homologous to RIG-I, and has been shown to be crucial for triggering a cytokine response upon infection with picornaviruses (e.g.,

encephalomyocarditis virus (EMCV), Theiler's virus, and Mengo virus), Sendai virus, rabies virus, West Nile virus, rabies virus, rotavirus, murine hepatitis virus, and murine norovirus. LPG2 lacks a CARD domain found in RIG-I and MDA5, which is responsible for direct interaction with IPS-1 to initiate downstream signaling. As such, LPG2 is believed to behave as a modulator of the innate immune response in conjunction with other CARD-bearing RLRs such as RIG-I and MDA5.

Another class of PRRs encompasses the nucleotide-binding and oligomerization domain (NOD)-like receptors, or NLR family (Caruso, R. et al, Immunity (2014) 41:898-908), which includes the microbial sensor NOD2. NOD2 is composed of an N-terminal CARD, a centrally- located nucleotide-binding oligomerization domain, and a C-terminal leucine rich repeat domain that is responsible for binding microbial PAMPs, such as bacterial peptidoglycan fragments and microbial nucleic acids. Ligand binding activates NOD2 and is believed to drive interaction with the CARD-containing kinase RIPK2, which in turn activates a number of downstream proteins including NF-κΒ, MAPK, IRF7, and IRF3, the latter of which results in the induction of type 1 interferons. NOD2 is expressed in a diverse set of cell types, including macrophages, dendritic cells, paneth cells, epithelial cells (e.g., lung epithelial cells, intestinal epithelia), and osteoblasts. NOD2 has been established as a sensor of infection by variety of pathogenic invaders, such as protozoa (e.g., Toxoplasma gondii and Plasmodium berghei), bacteria (e.g., Bacillus anthracis, Borrelia burgdorferi, Burkholderia pseudomallei, Helicobacter hepaticus, Legionella pneumophilia, Mycobacterium tuberculosis, Propionibacterium acne, Porphyromonas gingivalis, Salmonella enterica, and Streptococcus pneumonia), and viruses (e.g., respiratory syncytial virus and murine norovirus-1) (Moreira, L. O. and Zamboni, D. S. Front Immunol (2012) 3: 1-12). Recent work has shown that mutation of NOD2 may contribute to inflammatory diseases such as Crohn's disease, resulting in an aberrant inflammatory response upon stimulation. In addition to those described above, other PRRs play a critical role in host immunity against microbial invaders. The stimulator of interferon genes (STING) is a cytosolic microbial- derived DNA sensor that has been shown to be particularly sensitive to double-stranded DNA (Burdette, D. L. and Vance, R. E. (2013) Nat Immunol 14: 19-26). Two molecules of STING form a homodimer mediated by an a-helix present in the C-terminal dimerization domain, and molecular binding studies have revealed that each STING dimer binds one molecule of microbial nucleic acids. Upon ligand binding, STING activates the innate immune response through interaction with RIG-I and IPS-1, resulting in interferon production (e.g., IFN-a and IFN-β) and other downstream signaling events. Since its discovery, STING has been shown to function as a critical sensor of viruses (e.g., adenovirus, herpes simplex virus, hepatitis B virus, vesicular stomatitis virus, hepatitis C virus), bacteria (e.g., Listeria monocytogenes, Legionella

pneumopholia, Mycobacterium tuberculosis) and protozoa (Plasmodium falciparum,

Plasmodium berghei). In addition, STING has been shown to play a major role in the innate immune response against tumor antigens, driving dendritic cell activation and subsequent T cell priming in several cancers. (Woo, S.R. et al. Trends in Immunol (2015) 36:250-256).

Compounds

The present disclosure features methods for the induction of PRR expression (e.g., RIG-I expression, NOD2 expression, or STING expression) in a subject (e.g., a subject with a microbial infection, e.g., a viral infection, a bacterial infection, a fungal infection, or a parasitic infection), comprising administration of a compound of Formula (I) or a prodrug or

pharmaceutically acceptable salt thereof. The active agent is Formula (I), which may be described by any one of Formula (la), Formula (lb), and Formula (Ic), or a combination thereof:

Formula (la) Formula (lb) Formula (Ic)

The composition of the present invention may comprise a prodrug of Formula (I), wherein said prodrug is a compound of Formula (II). The prodrug (e.g., the compound of Formula (II)) may be described by any one of Formula (Ila), Formula (lib), and Formula (lie), or a combination thereof:

Formula (lie)

Formula (I) and its prodrug Formula (II) are small molecule nucleic acid hybrid

(dinucleotide) compounds that combine both antiviral and immune modulating activities. The latter activity mediates, for example, controlled apoptosis of virus-infected hepatocytes via stimulation of the innate immune response, similar to what is also achieved by IFN-a therapy in patients suffering from a viral infection.

Without wishing to be bound by theory, the mechanism of action of Formula (I) and its prodrug Formula (II) may be dissected into two components. The first component entails the host immune stimulating activity of Formula (I), which induces endogenous IFNs via the activation of PRRs, e.g., RIG-I, NOD2, and STING (Takeuchi, O. and Akira S. Cell (2010) 140:805-820; Sato, S. et al, Immunity (2015) 42: 123-132; Sabbah, A. et al, Nat Immunol (2009) 10: 1073- 1080). Activation may occur by binding of Formula (I) to the nucleotide binding domain of PRRs (e.g., RIG-I, NOD2, or STING), as described previously, and may further result in the induction of PRR expression (e.g., RIG-I expression, NOD2 expression, or STING expression).

The second component of the mechanism of action of Formula (I) and its prodrug Formula (II) involves its direct antiviral activity, which inhibits the synthesis of viral nucleic acids by steric blockage of the viral polymerase. The block may be achieved by interaction of Formula (I) with a PRR (e.g., RIG-I, NOD2, or STING) as described earlier that then in turn may prevent the polymerase enzyme from engaging with the nucleic acid template for replication (e.g., viral-derived RNA). The cytotoxic potential of Formula (I) has been initially evaluated using a panel of cell lines. Similar to the parental drug, Formula (II) demonstrated an excellent safety profile, with a 50% cytotoxic concentration (CC₅₀) of greater than 1000 μΜ (Coughlin, J.E. et al. Bioorg Med Chem Lett (2010) 20: 1783-1786). Formula (II) has been further evaluated for antiviral activity, such as anti-HBV activity in a cell-based assay against wild-type HBV and lamivudine-(3TC) and adefovir-(ADV) resistant mutant HBV, as well as anti-HCV activity.

In some embodiments, the method described herein comprises administration of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In other embodiments, the method described herein comprises administration of prodrug of Formula (I) (e.g., a compound of Formula (II)) or a pharmaceutically acceptable salt thereof. In other embodiments, the method herein describes administration of a composition comprised of a combination of a compound of Formula (I) and a compound of Formula (II) or pharmaceutically acceptable salts thereof. It is well established that the prodrug Formula (I) has been shown to be converted to the active drug Formula (I) (e.g., the Rp- and Sp-Formula (I) isomers, Formula (lb) and Formula (Ic)) upon administration.

The compounds provided herein may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included within the scope. Unless otherwise indicated when a compound is named or depicted by a structure without specifying the stereochemistry and has one or more chiral centers, it is understood to represent all possible stereoisomers of the compound. The compounds provided herewith may also contain linkages (e.g., carbon-carbon bonds, phosphorus-oxygen bonds, or phosphorus-sulfur bonds) or substituents that can restrict bond rotation, e.g. restriction resulting from the presence of a ring or double bond.

Methods of Use

The present disclosure relates to methods for inducing the expression of PRRs (e.g., RIG-I, NOD2, STING) in a subject through administration of Formula (I) or the prodrug Formula (II), or a pharmaceutically acceptable salt thereof. In some embodiments, the subject may be suffering from a condition described below, e.g., a viral infection (e.g., viral latency), a bacterial infection, or a cancer.

Treatment of Viral Infections

Pattern recognition receptors such as RIG-I, NOD2, MDA5, and STING have been shown to be an important factor in host recognition of a large number of RNA viruses from a variety of different viral families. In some embodiments, the methods of inducing expression of PRRs (e.g., RIG-I, NOD2, STING) disclosed herein comprise administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof to a subject infected with a microbial infection. In some embodiments, the microbial infection is a virus. In some embodiments, the virus is a RNA virus (e.g., a double- stranded RNA (dsRNA) virus, a single- stranded RNA (ssRNA) virus (e.g., a positive- strand (sense) ssRNA virus or a negative- strand (antisense) ssRNA virus), or a ssRNA retrovirus) or a DNA virus (e.g., a dsDNA virus, ssDNA virus, or a dsDNA retrovirus). In some embodiments, the virus may be a Group I, Group II, Group III, Group IV, Group V, Group VI, or Group VII class of virus, e.g., according to the Baltimore classification system.

In some embodiments, the virus is dsRNA virus, e.g., a Group III virus. In some embodiments, expression of one or more PRRs (e.g., RIG-I, NOD2, STING) is induced through host-produced or viral-derived RNA. In some embodiments, the virus is a dsRNA virus, and is a member of the Birnaviridae, Chrysoviridae, Cystoviridae , Endornaviridae , Hypoviridae, Megabirnaviridae , Partitiviridae , Picobirnaviridae , Reoviridae, or Totiviridae families, or other family of dsRNA virus. Exemplary dsRNA viruses and virus genera include, but are not limited to, Picobirnavirus, Rotavirus, Seadornavirus, Coltivirus, Orbivirus, and Orthoreovirus, or a subtype, species, or variant thereof.

In some embodiments, the virus is ssRNA virus, e.g., a positive- strand (sense) ssRNA virus, e.g., a Group IV virus. In some embodiments, expression of one or more PRRs (e.g., RIG-I, NOD2, STING) is induced through host-produced or viral-derived RNA. In some embodiments, the virus is a positive- strand (sense) ssRNA virus, and is a member of the Arteriviridae, Coronaviridae, Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Marnaviridae, Piconaviridae, Secoviridae, Alphaflexiviridae, Betaflexiviridae,

Gammaflexiviridae, Tymoviridae, Alphatetraviridae, Alvernaviridae, Astroviridae,

Barnaviridae, Bromoviridae, Caliciviridae, Carmotetraviridae, Closteroviridae, Flaviviridae, Leviviridae, Luteoviridae, Narnaviridae, Nodaviridae, Permutotetraviridae, Potyviridae, Togaviridae, or Virgaviridae families, or other family of positive- strand (sense) ssRNA virus. Exemplary positive-strand (sense) ssRNA viruses and virus genera include, but are not limited to, Yellow fever virus, West Nile virus, Hepatitis C virus, Dengue fever virus, Rubella virus, Ross River virus, Sindbis virus, Chikungya virus, Norwalk virus, Japanese encephalitis virus, Tick-borne encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, Kyasanur Forest disease virus (e.g., Monkey disease virus), Western Equine encephalitis virus, Eastern Equine encephalitis virus, Venezuelan Equine encephalitis virus, Sapporo virus, Norovirus, Sapovirus, Calicivirus, Parechovirus, Hepatitis A virus, Rhinovirus (e.g.,

Rhinovirus A, Rhinovirus B, and Rhinovirus C), Enterovirus (e.g., Enterovirus A, Enterovirus B, Enterovirus C (e.g., poliovirus), Enterovirus D, Enterovirus E, Enterovirus F, Enterovirus G, or Enterovirus H), Apthovirus (e.g., Foot and mouth disease virus), Nidovirales (e.g., Cavally virus, Nam Dinh virus, Middle East respiratory syndrome coronavirus (MERS-CoV),

Coronavirus HKU1, Coronavirus NL63, SARS-CoV, Coronavirus OC43, and Coronavirus 229E), Benyvirus, Blunevirus, Cilevirus, Hepevirus (e.g., Hepatitis E virus), Higrevirus, Idaeovirus, Negevirus, Ourmiavirus, Polemovirus, Sobemovirus, or Umbravirus, or a subtype, species, or variant thereof.

In some embodiments, the virus is ssRNA virus, e.g., a negative- strand (antisense) ssRNA virus, e.g., a Group V virus. In some embodiments, expression of one or more PRRs (e.g., RIG-I, NOD2, STING) is induced through host-produced or viral-derived RNA. In some embodiments, the virus is a negative-strand (antisense) ssRNA virus, and is a member of the Bornaviridae, Filoviridae, Paramyxoviridae , Rhabdoviridae , Nyamiviridae , Arenaviridae , Bunyaviridae, Ophioviridae, or Orthomyxoviridae families, or other family of negative-strand (antisense) ssRNA virus. Exemplary negative-strand (antisense) ssRNA viruses and virus genera include, but are not limited to, Brona disease virus, Ebola virus, Marburg virus, Measles virus, Mumps virus, Nipah virus, Hendra virus, Respiratory syncytial virus, Influenza and Parainfluenza viruses, Metapneumovirus, Newcastle disease virus, Deltavirus (e.g., Hepatitis D virus), Dichohavirus , Emaravirus, Nyavirus, Tenuivirus, Varicosavirus, or a subtype, species, or variant thereof.

In some embodiments, the virus is an ssRNA retrovirus (ssRNA RT virus), e.g., a Group VI virus. In some embodiments, expression of one or more PRRs (e.g., RIG-I, NOD2, STING) is induced through host-produced or viral-derived RNA. In some embodiments, the virus is an ssRNA RT virus and is a member of the Metaviridae, Pseudoviridae, or Retroviridae families, or other family of ssRNA RT virus. Exemplary ssRNA RT viruses and virus genera include, but are not limited to, Metavirus, Errantivirus, Alpharetrovirus (e.g., Avian leukosis virus, Rous sarcoma virus), Betaretrovirus (e.g., Mouse mammary tumor virus), Gammaretrovirus (e.g., Murine leukemia virus, Feline leukemia virus), Deltaretrovirus (e.g., human T- lymphotropic virus), Epsilonretrovirus (e.g., Walleye dermal sarcoma virus), Lentivirus (e.g., Human immunodeficiency virus 1 (HIV)), or a subtype, species, or variant thereof.

In some embodiments, the virus is a DNA virus, e.g., a dsDNA virus or an ssDNA virus. In some embodiments, the virus is a dsDNA virus, e.g., a Group I virus, and expression of one or more PRRs (e.g., RIG-I, NOD2, STING) is induced through host-produced or viral-derived RNA. In some embodiments, the virus is a dsDNA virus and is a member of the Myoviridae, Podoviridae, Siphoviridae , Alloherpesviridae, Herpesviridae, Malacoherpesviridae,

Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae , Ascoviridae, Asfarviridae, Baculoviridae, Bicaudaviridae , Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae, Marseilleviridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Polydnaviruses, Polymaviridae, Poxviridae,

Sphaerolipoviridae, Tectiviridae, or Turriviridae families, or other family of dsDNA virus. Exemplary dsDNA viruses and virus genera include, but are not limited to, Dinodnavirus, Nudivirus, smallpox, human herpes virus, Varicella Zoster virus, polyomavirus 6, polyomavirus 7, polyomavirus 9, polyomavirus 10, JC virus, BK virus, KI virus, WU virus, Merkel cell polyomavirus, Trichodysplasia spinulosa-associated polyomavirus, MX polyomavirus, Simian virus 40, or a subtype, species, or variant thereof.

In some embodiments, the virus is an ssDNA virus, e.g., a Group II virus, and expression of one or more PRRs (e.g., RIG-I, NOD2, STING) is induced through host-produced or viral-derived RNA. In some embodiments, the virus is an ssDNA virus and is a member of the Anelloviridae , Bacillariodnaviridiae, Bidnaviridae, Circoviridae, Geminiviridae ,

Inoviridae, Microviridae, Nanoviridae, Parvoviridae, or Spiraviridae families, or other family of ssDNA virus. Exemplary ssDNA viruses and virus genera include, but are not limited to, Torque teno virus, Torque teno midi virus, Torque teno mini virus, Gyrovirus, Circovirus, Parvovirus B 19, Bocaparvovirus, Dependoparvovirus, Erythroparvovirus, Protoparvovirus, Tetraparvovirus, Bombyx mori densovirus type 2, lymphoidal parvo-like virus,

Hepatopancreatic parvo-like virus, or a subtype, species, or variant thereof.

In some embodiments, the virus is a dsDNA reverse transcriptase (RT) virus, e.g., a Group VII virus, and expression of one or more PRRs (e.g., RIG-I, NOD2, STING) is induced through host-produced or viral-derived RNA. In some embodiments, the virus is a dsDNA RT virus and is a member of the Hepadnaviridae, or Caulimoviridae families, or other family of dsDNA RT virus. Exemplary dsDNA RT viruses and virus genera include, but are not limited to, Hepatitis B virus, or a subtype, species, or variant thereof.

In some embodiments, the virus (e.g., a virus described herein) is latent, e.g., within a cell. In some embodiments, the virus is an RNA virus (e.g., a double-stranded RNA (dsRNA) virus, a single-stranded RNA (ssRNA) virus (e.g., a positive- strand (sense) ssRNA virus or a negative-strand (antisense) ssRNA virus), or a ssRNA retrovirus) or a DNA virus (e.g., a dsDNA virus, ssDNA virus, or a dsDNA retrovirus) and is latent, e.g., within a cell. In some embodiments, the virus is a Group I, Group II, Group III, Group IV, Group V, Group VI, or Group VII class of virus, e.g., according to the Baltimore classification system, and is latent, e.g., within a cell.

In some embodiments, the virus is an RNA virus (e.g., an RNA virus described herein) and is latent, e.g., within a cell. In some embodiments, the virus is an ssRNA retrovirus (ssRNA RT virus), e.g., a Group VI virus, and is latent, e.g., within a cell. In some

embodiments, the virus is the human immunodeficiency virus 1 (HIV)), or a subtype, species, or variant thereof, and is latent, e.g., within a cell. In some embodiments, the methods of inducing expression of one or more PRRs (e.g., RIG-I, NOD2, STING) in a subject suffering from a viral infection disclosed herein result in an increase in PRR expression (e.g., RIG-I expression, NOD2 expression, STING expression). In some embodiments, expression of one or more PRRs (e.g., RIG-I, NOD2, STING) is induced by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more. In some embodiments, induction of expression of one or more PRRs (e.g., RIG-I, NOD2, STING) occurs within about 5 minutes of administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof. In some embodiments, induction of expression of one or more PRRs (e.g., RIG-I, NOD2, STING) occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof to a subject.

Treatment of Bacterial Infections

Recent studies have shown that PRRs (e.g., RIG-I, NOD2, STING) play a critical role in host recognition of bacterial infections stemming from a variety of species (Dixit, E. and Kagan, J.C. Adv Immunol (2013) 117:99-125). In some cases, bacteria may secrete nucleic acids during the exponential growth phase (e.g., Listeria monocytogenes; Abdullah, Z. et al, EMBO J (2012) 31:4153-4164), which in turn are detected by PRRs such as RIG-I and thus promote the induction of further PRR expression. In other cases, such as for Legionella pneumophila, bacterial DNA enters into the cytosol over the course of infection and is transcribed into an RNA ligand for RIG-I (Chiu, Y. H. et al, Cell (2009) 138:576-591), thus triggering downstream PRR-mediated signaling events. PRR expression (e.g., RIG-I expression, NOD2 expression, STING expression) may further be induced upon recognition of RNA released during phagocytotic uptake of bacteria. Additionally, bacterial cell wall components such as peptidoglycans (e.g., muramyl dipeptide, i.e., MDP) may serve as ligands for activation and induction of PRRs, namely NOD2, and bacterial-derived nucleic acids such as cyclic dinucleotides (e.g., cyclic di-GMP) may bind to and activate PRRs, in particular STING. In some embodiments, the expression of one or more PRRs may be induced through other means not explicitly recited herein

In some embodiments, the methods of inducing expression of one or more PRRs (e.g., RIG-I, NOD2, STING) disclosed herein comprise administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof to a subject infected with a microbial infection, e.g., a bacterial infection. In some embodiments, the bacterium is a Gram- negative bacterium or a Gram-positive bacterium. Exemplary bacteria include, but are not limited to, Listeria (e.g., Listeria monocytogenes), Francisella (e.g., Francisella tularensis), Mycobacteria (e.g., Mycobacteria tuberculosis), Brucella (e.g., Brucella abortis), Streptococcus (e.g., group B Streptococcus), Legionella (e.g., Legionella pneumophila), Escherichia (e.g., Escherichia coli), Pseudomonas (e.g., Psuedomonas aeruginosa), Salmonella (e.g., Salmonella typhi), Shigella (e.g., Shigella flexneri), Campylobacter (e.g., Campylobacter jejuni),

Clostridium (e.g., Clostrodium botulinum), Enterococcus (e.g., Enterococcus faecalis), Vibrio (e.g., Vibrio cholera), Yersinia (e.g., Yersinia pestis), Staphylococcus (e.g., Staphylococcus aureus), or other genera, species, subtypes, or variants thereof.

In some embodiments, the methods of inducing expression of one or more PRRs (e.g., RIG-I, NOD2, STING) in a subject suffering from a bacterial infection disclosed herein result in an increase in PRR expression (e.g., RIG-I expression, NOD2 expression, STING

expression). In some embodiments, expression of one or more PRRs (e.g., RIG-I, NOD2, STING) is induced by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more. In some embodiments, induction of expression of one or more PRRs (e.g., RIG-I, NOD2, STING) occurs within about 5 minutes of administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof. In some

embodiments, induction of expression of one or more PRRs (e.g., RIG-I, NOD2, STING) occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof.

Treatment of Cancer

Recognition of RNA ligands by PRRs (e.g., RIG-I, NOD2, or STING) stimulates the production of type I interferons (e.g., IFN-a or IFN-β), thus triggering a series of downstream signaling events that may lead to apoptosis in susceptible cells. In recent years, a connection between the induction of RIG-I expression and a number of cancers has been discovered. For example, RIG-I expression has been shown to be significantly downregulated in hepatocellular carcinoma, and patients exhibiting low RIG-I expression in tumors had shorter survival and poorer responses to IFN-a therapy (Hou, J. et al, Cancer Cell (2014) 25:49-63). As such, it has been suggested that the level of RIG-I expression may be useful as a biomarker for prediction of prognosis and response to immunotherapy. In other cases, induction of RIG-I expression has been shown to induce immunogenic cell death of pancreatic cancer cells, prostate cancer cells, breast cancer cells, skin cancer cells, and lung cancer cells (Duewell, P. et al, Cell Death Differ (2014) 21: 1825-1837; Besch, R. et al, Clin Invest (2009) 119:2399-2411; Kaneda, Y.

Oncoimmunology (2013) 2:e23566; Li, X.Y. et al, Mol Cell Oncol (2014) l :e968016), highlighting a new approach in immune-mediated cancer treatment.

A role for STING in the stimulation of innate immunity in response to cancer has also been identified. Recent studies have revealed the presence of tumor-derived DNA in the cytosol of certain antigen-presenting cells, such as tumor-infiltrating dendritic cells, likely generated through tumor cell stress or cell death. This tumor-derived DNA has been shown to activate STING, resulting in production and sensing of associated type 1 interferons (Woo, S.R. et al, Immunity (2014) 41:830-842). Stimulation of STING and resulting downstream signaling pathways also likely contributes to effector T cell recruitment into the inflamed tumor microenvironment (Woo, S. R. Trends in Immunol (2015) 36:250-256).

In some embodiments, the methods of inducing expression of a PRR (e.g., a PRR described herein) comprise administration of a compound of Formula (I) or Formula (II) or a

pharmaceutically acceptable salt thereof to a subject suffering from cancer. In some

embodiments, the methods of inducing expression of RIG-I disclosed herein comprise administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof to a subject suffering from cancer. In some embodiments, the methods of inducing expression of NOD2 disclosed herein comprise administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof to a subject suffering from cancer. In some embodiments, the methods of inducing expression of STING disclosed herein comprise administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof to a subject suffering from cancer. In some embodiments, the cancer is selected from a cancer of the breast, bone, brain, cervix, colon, gastrointestinal tract, eye, gall bladder, lymph nodes, blood, lung, liver, skin, mouth, prostate, ovary, penis, pancreas, uterus, testicles, stomach, thymus, thyroid, or other part of the body. In some embodiments, the cancer comprises a solid tumor (e.g., a carcinoma, a sarcoma, or a lymphoma). In some embodiments, the cancer is a hepatocellular carcinoma or other cancer of the liver. In some embodiments, the cancer is a leukemia or other cancer of the blood. In some embodiments, the cancer comprises breast cancer, renal cell carcinoma, colon cancer, melanoma, ovarian cancer, head and neck squamous cell carcinoma, pancreatic cancer, prostate cancer, lung cancer, brain cancer, or gastrointestinal stromal cancer. In some embodiments, the cancer cells (e.g., tumor cells) comprise specific cancer-associated antigens that induce a T-cell response.

In some embodiments, the methods of inducing expression of one or more PRRs (e.g., RIG-I, NOD2, STING) in a subject suffering from a cancer disclosed herein result in an increase in PRR expression (e.g., RIG-I expression, NOD2 expression, STING expression). In some embodiments, expression of one or more PRRs (e.g., RIG-I, NOD2, STING) is induced by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more. In some embodiments, induction of expression of one or more PRRs (e.g., RIG-I, NOD2, STING) occurs within about 5 minutes of administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof. In some embodiments, induction of expression of one or more PRRs (e.g., RIG-I, NOD2, STING) occurs within about 5 minutes of

administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof. In some embodiments, induction of expression of one or more PRRs (e.g., RIG-I, NOD2, STING) occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof.

Pharmaceutical Compositions

The present invention features methods for inducing the expression of one or more PRRs (e.g., RIG-I, NOD2, STING) in a subject, the methods comprising administering a compound of Formula (I) or a prodrug thereof (e.g., a compound of Formula (II)), or a pharmaceutically acceptable salt thereof.

While it is possible for the compound of the present invention (e.g., a compound of Formula (I) or a prodrug thereof (e.g., a compound of Formula (II))) to be administered alone, it is preferable to administer said compound as a pharmaceutical composition or formulation, where the compounds are combined with one or more pharmaceutically acceptable diluents, excipients or carriers. The compounds according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine. In certain embodiments, the compounds included in the pharmaceutical preparation may be active itself, or may be a prodrug, e.g. , capable of being converted to an active compound in a physiological setting (e.g., a compound of Formula (II)). Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into a pharmaceutically acceptable dosage form such as described below or by other conventional methods known to those of skill in the art.

The amount and concentration of compounds of the present invention (e.g., a compound of Formula (I) or a prodrug thereof (e.g., a compound of Formula (II)) in the pharmaceutical compositions, as well as the quantity of the pharmaceutical composition administered to a subject, can be selected based on clinically relevant factors, such as medically relevant characteristics of the subject (e.g., age, weight, gender, other medical conditions, and the like), the solubility of compounds in the pharmaceutical compositions, the potency and activity of the compounds, and the manner of administration of the pharmaceutical compositions. For further information on Routes of Administration and Dosage Regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

Thus, another aspect of the present invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount or prophylactic ally effective amount of a compound described herein (e.g., a compound of Formula (I) or a prodrug thereof (e.g., a compound of Formula (II), formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical

compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for oral or parenteral administration, for example, by oral dosage, or by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension. However, in certain embodiments the subject compounds may be simply dissolved or suspended in sterile water. In certain embodiments, the pharmaceutical preparation is non-pyrogenic, i.e., does not elevate the body temperature of a patient.

The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of the compound other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" as used herein means a

pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, stabilizing agent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject antagonists from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) ascorbic acid; (17) pyrogen-free water; (18) isotonic saline; (19) Ringer's solution; (20) ethyl alcohol; (21) phosphate buffer solutions; (22) cyclodextrins such as Captisol®; and (23) other non-toxic compatible substances such as antioxidants and antimicrobial agents employed in pharmaceutical formulations.

As set out above, certain embodiments of the compounds described herein may contain a basic functional group, such as an amine, and are thus capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts" in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like (see, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19).

In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of the compound of the present invention (e.g., a compound of Formula (I) or a prodrug thereof (e.g., a compound of Formula (II))). These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like.

Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, for example, Berge et al., supra).

Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated

hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha- tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

The pharmaceutically acceptable carriers, as well as wetting agents, emulsifiers, lubricants, coloring agents, release agents, coating agents, sweetening, flavoring agents, perfuming agents, preservatives, antioxidants, and other additional components may be present in an amount between about 0.001% and 99% of the composition described herein. For example, said pharmaceutically acceptable carriers, as well as wetting agents, emulsifiers, lubricants, coloring agents, release agents, coating agents, sweetening, flavoring agents, perfuming agents, preservatives, antioxidants, and other additional components may be present from about 0.005%, about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1%, about 1.5%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 85%, about 90%, about 95%, or about 99% of the composition described herein.

Pharmaceutical compositions of the present invention may be in a form suitable for oral administration, e.g., a liquid or solid oral dosage form. In some embodiments, the liquid dosage form comprises a suspension, a solution, a linctus, an emulsion, a drink, an elixir, or a syrup. In some embodiments, the solid dosage form comprises a capsule, tablet, powder, dragee, or powder. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages. Pharmaceutical compositions may comprise, in addition to the compound described herein (e.g., a compound of Formula (I) or a prodrug thereof (e.g., a compound of Formula (II)),) or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and may optionally further comprise one or more pharmaceutically acceptable excipients, such as, for example, stabilizers (e.g., a binder, e.g., polymer, e.g., a precipitation inhibitor, diluents, binders, and lubricants.

In some embodiments, the composition described herein comprises a liquid dosage form for oral administration, e.g., a solution or suspension. In other embodiments, the composition described herein comprises a solid dosage form for oral administration capable of being directly compressed into a tablet. In addition, said tablet may include other medicinal or pharmaceutical agents, carriers, and or adjuvants. Exemplary pharmaceutical compositions include compressed tablets (e.g., directly compressed tablets), e.g., comprising a compound of the present invention (e.g., a compound of Formula (I) or a prodrug thereof (e.g., a compound of Formula (II)) or a pharmaceutically acceptable salt thereof.

Formulations of the present invention include those suitable for parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about 99 percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent. Pharmaceutical compositions of this invention suitable for parenteral administration comprise compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacterio stats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a compound of the present invention (e.g., a compound of Formula (I) or a prodrug thereof (e.g., a compound of Formula (II)) it may be desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered form of the compound of the present invention is accomplished by dissolving or suspending compound in an oil vehicle.

In some embodiments, it may be advantageous to administer the compound of the present invention (e.g., a compound of Formula (I) or a prodrug thereof (e.g., a compound of Formula (II)) in a sustained fashion. It will be appreciated that any formulation that provides a sustained absorption profile may be used. In certain embodiments, sustained absorption may be achieved by combining a compound of the present invention with other pharmaceutically acceptable ingredients, diluents, or carriers that slow its release properties into systemic circulation.

Routes of Administration

The compounds and compositions used in the methods described herein may be administered to a subject in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. Exemplary routes of administration of the compositions used in the methods described herein include topical, enteral, or parenteral applications. Topical applications include but are not limited to epicutaneous, inhalation, enema, eye drops, ear drops, and applications through mucous membranes in the body. Enteral applications include oral administration, rectal administration, vaginal administration, and gastric feeding tubes. Parenteral administration includes intravenous, intraarterial, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrastemal, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal, and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time. In certain embodiments of the invention, the compositions described herein comprising a compound of Formula (I) or a prodrug thereof (e.g., a compound of Formula (II)) is administered orally. In other embodiments of the invention, the compositions described herein comprising a compound of Formula (I) or a prodrug thereof (e.g., a compound of Formula (II)) is administered parenterally (e.g., intraperitoneally).

For intravenous, intraperitoneal, or intrathecal delivery or direct injection, the

composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

The choice of the route of administration will depend on whether a local or systemic effect is to be achieved. For example, for local effects, the composition can be formulated for topical administration and applied directly where its action is desired. For systemic, long term effects, the composition can be formulated for enteral administration and given via the digestive tract. For systemic, immediate and/or short term effects, the composition can be formulated for parenteral administration and given by routes other than through the digestive tract. Dosages

The compositions of the present invention are formulated into acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the compositions of the present invention (e.g., a compound of Formula (I) or a prodrug thereof (e.g., a compound of Formula (II)) may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of absorption of the particular agent being employed, the duration of the treatment, other drugs, substances, and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the composition required. For example, the physician or veterinarian can start doses of the substances of the invention employed in the composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the invention will be that amount of the substance which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Preferably, the effective daily dose of a therapeutic composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

Preferred therapeutic dosage levels are between about 0.1 mg/kg to about 1000 mg/kg (e.g. , about 0.2 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg) of the composition per day administered (e.g., orally or intraperitoneally) to a subject afflicted with the disorders described herein (e.g., HBV infection). Preferred prophylactic dosage levels are between about 0.1 mg/kg to about 1000 mg/kg (e.g. , about 0.2 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg) of the composition per day administered (e.g., orally or intraperitoneally) to a subject. The dose may also be titrated (e.g. , the dose may be escalated gradually until signs of toxicity appear, such as headache, diarrhea, or nausea).

The frequency of treatment may also vary. The subject can be treated one or more times per day (e.g. , once, twice, three, four or more times) or every so-many hours (e.g. , about every 2, 4, 6, 8, 12, or 24 hours). The composition can be administered 1 or 2 times per 24 hours. The time course of treatment may be of varying duration, e.g. , for two, three, four, five, six, seven, eight, nine, ten, or more days, two weeks, 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, or more than one year. For example, the treatment can be twice a day for three days, twice a day for seven days, twice a day for ten days. Treatment cycles can be repeated at intervals, for example weekly, bimonthly or monthly, which are separated by periods in which no treatment is given. The treatment can be a single treatment or can last as long as the life span of the subject (e.g. , many years).

Patient Selection and Monitoring

The methods of the present invention described herein entail administration of a compound of Formula (I) or a prodrug thereof (e.g., a compound of Formula (II)) or a pharmaceutically acceptable salt thereof to a subject to induce expression of one or more PRRs (e.g., RIG-I, NOD2, STING). In some embodiments, the subject is suffering from or is diagnosed with a condition, e.g., a microbial infection or a cancer. Accordingly, a patient and/or subject can be selected for treatment using a compound of Formula (I) or a prodrug thereof (e.g., a compound of Formula (II), or a pharmaceutically acceptable salt thereof by first evaluating the patient and/or subject to determine whether the subject is infected with a microbial infection (e.g., a viral infection or bacterial infection) or a cancer. A subject can be evaluated as infected with a microbial infection (e.g., a viral infection or bacterial infection) or a cancer using methods known in the art. The subject can also be monitored, for example, subsequent to administration of a compound described herein (e.g., a compound of Formula (I) or a prodrug thereof (e.g., a compound of Formula (II)) or a pharmaceutically acceptable salt thereof.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is an adult. In some embodiments, the subject is suffering from a microbial infection (e.g., a viral infection, a bacterial infection, a fungal infection, or a parasitic infection). In some embodiments, the subject is suffering from a viral infection (e.g., an infection caused by an RNA virus or a DNA virus). In some embodiments, the subject is suffering from a bacterial infection. In some embodiments, the subject is suffering from a cancer.

In some embodiments, the subject is infected with a virus. In some embodiments, the subject is infected with a virus, and the virus is in a latent stage. In some embodiments, the subject is infected with an RNA virus (e.g., a double- stranded RNA (dsRNA) virus, a single- stranded RNA (ssRNA) virus (e.g., a positive- strand (sense) ssRNA virus or a negative- strand (antisense) ssRNA virus), or a ssRNA retrovirus) or a DNA virus (e.g., a dsDNA virus, ssDNA virus, or a dsDNA retrovirus) and the virus is in a latent stage. In some embodiments, the subject is infected with a Group I, Group II, Group III, Group IV, Group V, Group VI, or Group VII class of virus, e.g., according to the Baltimore classification system, and the virus is in a latent stage. In some embodiments, the subject is infected with an RNA virus (e.g., an RNA virus described herein), and the virus is in a latent stage. In some embodiments, the virus is an ssRNA retrovirus (ssRNA RT virus), e.g., a Group VI virus, and is latent, e.g., within a cell. In some embodiments, the virus is the human immunodeficiency virus 1 (HIV)), or a subtype, species, or variant thereof, and is latent, e.g., within a cell.

In some embodiments, the subject is infected with a virus and is symptomatic. In some embodiments, the subject is infected with a virus and is asymptomatic. In some embodiments, the subject is infected with an ssRNA retrovirus (ssRNA RT virus), e.g., a Group VI virus, and is asymptomatic. In some embodiments, the subject is infected with the HIV virus and is asymptomatic.

In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a woodchuck, e.g., the eastern woodchuck. The eastern woodchuck (Marmota monax) is naturally infected with the woodchuck hepatitis virus (WHV), a hepadnavirus which is genetically closely related to human HBV. Neonatal infection of woodchucks with WHV parallels the main route of human (vertical) transmission for chronic HBV infection and displays a disease course similar to that in HBV-infected patients. Thus, chronic WHV infection in woodchucks is a fully immunocompetent model for studying CHB and HBV-induced HCC, and chronic WHV carriers have extensively been used to evaluate efficacy and safety of current and new HBV therapeutics. The recent comparison of hepatic transcriptional profiles in woodchucks and humans with acute self-limiting and chronic hepadnaviral infections identified important parallels in the antiviral immune responses and demonstrated molecular similarities in HCC induced by WHV and HBV. As these studies have established the translational value of this animal model for CHB, woodchucks with chronic WHV infection may used to evaluate antiviral efficacy, safety and pharmacodynamics associated with treatment.

Combination Therapies

In some embodiments, additional therapeutic agents may be administered with compositions of the present invention for the treatment of a microbial infection (e.g., a viral infection, a bacterial infection, a fungal infection, or a parasitic infection), a cancer, or any symptom or associated condition thereof. When combination therapy is employed, the additional therapeutic agent(s) can be administered as a separate formulation or may be combined with any of the compositions described herein.

For example, any of the methods described herein may further comprise the

administration of a therapeutically effective amount of an additional agent. In some

embodiments, the additional agent is an antiviral agent, an antibacterial agent, or an anticancer agent. In some embodiments, the antiviral agent comprises an interferon, a nucleoside analog, a non-nucleoside antiviral, or an immune enhancer (e.g., a non-interferon immune enhancer or a small molecule immune enhancer). In some embodiments, the antiviral agent is a capsid inhibitor, an entry inhibitor, a secretion inhibitor, a microRNA, an antisense RNA agent, an RNAi agent, or other agent designed to inhibit viral RNA or DNA. In some embodiments, the antiviral agent is selected from entecavir, lamuvidine, adefovir, darunavir, sofosbuvir, telaprevir, tenofovir, zidovudine, and ribavirin. In some embodiments, the antibacterial agent is selected from gentamicin, kanamycin, streptomycin, chloramphenicol, ceftobiprole, amoxicillin, penicillin, bacitracin, tetracycline, rifabutin, tigecycline, and vancomycin. In some embodiments, the anticancer agent is selected from methotrexate, 5-fluorouracil, doxorubicin, vincristine, bleomycin, vinblastine, dacarbazine, toposide, cisplatin, epirubicin, and sorafenib tosylate.

EXAMPLES

Example 1. Induction of RIG-I expression in the Woodchuck model of HBV infection.

Chronic WHV carrier woodchucks were used in this study. Formula (II) was administered orally at two doses (15 mg/kg/day and 30 mg/kg/day) to two groups of 5 woodchucks daily for 12 weeks. After the end of dosing, monitoring of the animals was continued for an additional 4 weeks.

At each time point in the study (0, 6, 12, and 20 weeks), liver biopsy samples were obtained under general anesthesia using 16-gauge disposable biopsy needles directed by ultrasound imaging according to the study schedule. The needle was inserted at a site near the ventral midline caudal to the xiphoid cartilage and directed dorsolaterally and cranially into the left lateral lobe of the liver. The biopsy specimen was processed for histopathological examination using standard conditions. Histological sections were stained by

immunohisochemistry (IHC) for RIG-I induction using a commercially available anti-RIG-I antibody under standard protocols, and the results of this analysis is summarized below in Table 1.

In the table, the intensity of the IHC staining is described on a scale from 0-3, where 0 represents no staining, 1 represents weak staining, 2 represents intermediate or moderate staining, and 3 represents strong or intense staining. Similarly, the distribution of the staining throughout the biopsy specimen is described on a scale from 0-5, where 0 represents 0%, 1 represents 1-20%, 2 represents 21-40%, 3 represents 41-60%, and 5 represents 81-100%. The sum of the intensity and distribution columns are summarized in the column entitled "Total."

Table 1. Summary of IHC data from RIG-I induction study.

2 1 3

3 2 5

1

2 5 7

3 3 6

3 4 7

2F

3 5 8

1 5 6

Example 2. siRNA-mediated silencing of NOD2 and RIG-I in human lung epithelial (HLE) cells.

In this experiment, human lung epithelial (HLE) cells were transfected with 20 pmol of either control siRNA (con siRNA), NOD2 siRNA, or RIG-I siRNA using standard protocols. At 24 hours post-transfection, the expression of NOD2 and RIG-I were determined through RT-PCR analysis. GAPDH expression served as a loading control. As shown in FIGS. 8A- 8B, diminished expression of NOD2 (FIG. 8A) and RIG-I (FIG. 8B) was observed after transfection of NOD2 and RIG-I siRNAs, respectively.

Example 3. NOD2 and RIG-I expression is required for induction of IFN-β by Formula (Π).

In order to investigate the connection between Formula (II) administration and IFN induction, HLE cells were transfected with 20 pmol of either control siRNA (con siRNA), NOD2 siRNA, or RIG-I siRNA as described in Example 2. At 24 hours post-transfecrtion, the cells were treated with either water (control) or Formula (II). After 12 hours, the cells were pelleted and the supernatant was collected to analyze IFN-β levels through ELISA analysis. The results of this analysis are depicted in FIG. 9, wherein the ELISA levels represent the mean standard deviation, and * indicates p < 0.05 using the student's t test. As shown in FIG. 9, a significant reduction in IFN-β induction and production was observed in Formula (II)- treated cells transfected with RIG-I and NOD2 siRNAs. IFN-β induction or production was not observed in cells treated with water (data not shown). These results indicate that both RIG-I and NOD2 are required for IFN-β induction upon administration of Formula (II).

Example 4. Dose-dependent binding of Formula (I) to RIG-I and STING.

An analysis of Formula (I) binding to PRRs was carried out using a sandwich ELISA assay. Anti-DDK antibody was immobilized on a microplate and used to capture either RIG-I or STING, followed by incubation with varying concentrations of 5'-biotinylated Formula (I) (20 μg/mL, 10 μg/mL, 5 μg/mL, 2.5 μg/mL, 1.3 μg/mL, 0.6 μg/mL, 0.3 μg/mL, and 0 μg/mL). Detection of biotinylated Formula (I) bound to the microplate was carried out with horseradish peroxidase (HRP)-conjugated streptavidin. Upon substrate development, the absorbance at 450 nm (OD 450 nm) was measured and quantified (FIGS. 10A-10B). In these graphs, the y- axis represents the fold change in absorbance compared to undosed samples, and the error bars represent standard deviation in multiple wells. In the case of both RIG-I (FIG. 10A) and STING (FIG. 10B), increasing concentration of Formula (I) led to a concomitant increase in absorbance at 450 nm, indicating the dose-dependent binding of Formula (I) to both PRRs.

EQUIVALENTS

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. While this disclosure has been described with reference to specific aspects, it is apparent that other aspects and variations may be devised by others skilled in the art without departing from the true spirit and scope of the disclosure. The appended claims are intended to be construed to include all such aspects and equivalent variations. Any patent, publication, or other disclosure material, in whole or in part, that is said to be incorporated by reference herein is incorporated herein only to the extent that the incorporated material does not conflict with existing definitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference.

While this disclosure has been particularly shown and described with references 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 disclosure encompassed by the appended claims.

Claims

What is claimed is:

1. A method of inducing the expression of a pattern recognition receptor (PRR) in a subject, the method comprising administering to the subject a compound of Formula (I), wherein the compound is selected from:

Formula (la) Formula (lb) Formula (Ic) or a prodrug or pharmaceutically acceptable salt thereof to thereby treat the subject.

2 A method of inducing the expression of a pattern recognition receptor in a subject suffering from a microbial infection or a cancer, the method comprising administering to the subject a compound of Formula (I), wherein the compound is selected from:

3. A method of activating a pattern recognition receptor (PRR) in a subject, the method comprising administering to the subject a compound of Formula (I), wherein the compound is selected from:

4. The method of claims 1-3, wherein the prodrug of Formula (I) is a compound of Formula (II), wherein the compound is selected from:

Formula (Ila) Formula (lib)

Formula (lie) or a pharmaceutically acceptable salt thereof.

5. The method of claim 4, wherein the PRR comprises a RIG-I-like receptor or a NOD-like receptor.

6. The method of claim 5, wherein the PRR comprises RIG-I, NOD2, MDA5, LPG2, or STING (e.g., RIG-I).

7. The method of claim 6, wherein the PRR comprises RIG-I.

8. The method of claim 6, wherein the PRR comprises NOD2.

9. The method of claim 6, wherein the PRR comprises STING.

10. The method of any one of claims 1-3, wherein the subject is administered a composition comprising a mixture of compounds of Formula (I), e.g., Formula (lb) and Formula (Ic).

11. The method of claim 10, wherein the composition comprises Formula (lb) and comprises less than about 5% of Formula (Ic) (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1% of Formula (Ic)), or is substantially free of Formula (Ic).

12. The method of claim 10, wherein the composition comprises Formula (Ic) and comprises less than about 5% of Formula (lb) (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1% of Formula (lb), or is substantially free of Formula (lb)).

13. The method of claim 4, wherein the subject is administered a composition comprising a mixture of compounds of Formula (II), e.g., Formula (lib) and Formula (lie).

14. The method of claim 13, wherein the composition comprises Formula (lib) and comprises less than about 5% of Formula (lie) (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1% of Formula (lie), or is substantially free of Formula (lie)).

15. The method of claim 13, wherein the composition comprises Formula (lie) and comprises less than about 5% of Formula (lib) (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1% of Formula (lib), or is substantially free of Formula (lib)).

16. The method of claim 4, wherein the induction of PRR expression in the subject comprises an increase, enhancement, or initiation of expression of RIG-I or NOD2 (e.g., in the liver).

17. The method of claim 16, wherein the increase or enhancement of expression of RIG-I or NOD2 (e.g., in the liver) is between about 5% and about 95% compared with a reference standard or reference treatment (e.g., between about 10% and about 90%).

18. The method of claim 16, wherein the expression of RIG-I or NOD2 is induced by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more relative to a reference standard.

19. The method of claim 16, wherein the induction of expression of RIG-I or NOD2 occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a compound of Formula (I) or Formula (II), or a

pharmaceutically acceptable salt thereof to a subject.

20. The method of claim 19, wherein the induction of expression of RIG-I or NOD2 continues after administration of a compound of Formula (I) or Formula (II) has ended.

21. The method of claim 4, wherein the induction of PRR expression in the subject comprises an increase, enhancement, or initiation of expression of STING.

22. The method of claim 21, wherein the induction of STING expression occurs in the microenvironment of cancer cells (e.g., in the tumor microenvironment).

23. The method of claim 22, wherein the induction of STING expression occurs in antigen- presenting cells (e.g., dendritic cells) in the microenvironment of cancer cells (e.g., the tumor microenvironment) .

24. The method of claim 21, wherein the increase or enhancement of expression of STING (e.g., in the tumor microenvironment) is between about 5% and about 95% compared with a reference standard or reference treatment (e.g., between about 10% and about 90%).

25. The method of claim 21, wherein the expression of STING is induced by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more relative to a reference standard.

26. The method of claim 21, wherein the induction of expression of STING occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a compound of Formula (I) or Formula (II) or a

pharmaceutically acceptable salt thereof to a subject.

27. The method of claim 26, wherein the induction of expression of STING continues after administration of a compound of Formula (I) or Formula (II) has ended.

28. The method of claim 4, wherein the subject is a mammal.

29. The method of claim 28, wherein the subject is a human.

30. The method of claim 4, wherein the subject is suffering from a microbial infection (e.g., a viral infection, a bacterial infection, a fungal infection, or a parasitic infection) or a cancer.

31. The method of claim 30, wherein the cancer is selected from a cancer of the breast, bone, brain, cervix, colon, gastrointestinal tract, eye, gall bladder, lymph nodes, blood, lung, liver, skin, mouth, prostate, ovary, penis, pancreas, uterus, testicles, stomach, thymus, thyroid, or other part of the body.

32. The method of claim 30, wherein the cancer comprises a solid tumor (e.g., a carcinoma, a sarcoma, or a lymphoma).

33. The method of claim 32, wherein the cancer comprises hepatocellular carcinoma.

34. The method of claim 33, wherein the cancer comprises breast cancer, renal cell carcinoma, colon cancer, melanoma, ovarian cancer, head and neck squamous cell carcinoma, pancreatic cancer, prostate cancer, lung cancer, brain cancer, or gastrointestinal stromal cancer.

35. The method of claim 30, wherein the microbial infection (e.g., viral infection) comprises infection with an RNA virus or a DNA virus.

36. The method of claim 35, wherein the RNA virus or DNA virus comprises a single- stranded virus (e.g., positive single-stranded or negative single- stranded) or a double- stranded virus.

37. The method of claim 35, wherein the RNA virus or DNA virus comprises a virus in Group I, Group II, Group III, Group IV, Group V, Group VI, or Group VII class of virus.

38. The method of claim 35, wherein the RNA virus comprises a dsRNA virus, an ssRNA virus (e.g., a positive-strand ssRNA virus or a negative-strand ssRNA virus), or an ssRNA RT virus.

39. The method of claim 38, wherein the dsRNA virus is a member of the Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae, Megabirnaviridae, Partitiviridae, Picobirnaviridae , Reoviridae, or Totiviridae families, or other family of dsRNA virus.

40. The method of claim 38, wherein the positive-strand ssRNA virus is a member of the Arteriviridae, Coronaviridae, Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae,

Marnaviridae, Piconaviridae, Secoviridae, Alphaflexiviridae, Betaflexiviridae,

Gammaflexiviridae, Tymoviridae, Alphatetraviridae, Alvernaviridae, Astroviridae, Barnaviridae, Bromoviridae, Caliciviridae, Carmotetraviridae, Closteroviridae, Flaviviridae, Leviviridae, Luteoviridae, Narnaviridae, Nodaviridae, Permutotetraviridae, Potyviridae, Togaviridae, or Virgaviridae families, or other family of positive-strand (sense) ssRNA virus.

41. The method of claim 38, wherein the negative- strand ssRNA virus is a member of the Bornaviridae , Filoviridae, Paramyxoviridae , Rhabdoviridae, Nyamiviridae , Arenaviridae , Bunyaviridae, Ophioviridae, or Orthomyxoviridae families, or other family of negative-strand (antisense) ssRNA virus.

42. The method of claim 38, wherein the ssRNA RT virus is a member of the Metaviridae, Pseudoviridae, or Retroviridae families, or other family of ssRNA RT virus.

43. The method of claim 38, wherein the ssRNA RT virus is a member of the Lentivirus genus (e.g., human immunodeficiency virus 1 (HIV), or a subtype, species, or variant thereof).

44. The method of claim 43, wherein the ssRNA RT virus is HIV or a subtype, species, or variant thereof.

45. The method of claim 35, wherein the DNA virus comprises a dsDNA virus, an ssDNA virus, or a dsDNA RT virus.

46. The method of claim 45, wherein the dsDNA virus is a member of the Myoviridae, Podoviridae, Siphoviridae , Alloherpesviridae, Herpesviridae, Malacoherpesviridae,

Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae , Ascoviridae, Asfarviridae, Baculoviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae, Marseilleviridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Polydnaviruses, Polymaviridae, Poxviridae,

Sphaerolipoviridae, Tectiviridae, or Turriviridae families, or other family of dsDNA virus.

47. The method of claim 45, wherein the ssDNA virus is a member of the Anelloviridae, Bacillariodnaviridiae, Bidnaviridae, Circoviridae, Geminiviridae, Inoviridae, Microviridae, Nanoviridae, Parvoviridae, or Spiraviridae families, or other family of ssDNA virus.

48. The method of claim 35, wherein the dsDNA RT virus is a member of the

Hepadnaviridae, or Caulimoviridae families, or other family of dsDNA RT virus.

49. The method of claim 35, wherein the virus is in a latent stage.

50. The method claim 35, wherein the virus is an ssRNA retrovirus (ssRNA RT virus), e.g., a Group VI virus, and is latent, e.g., within a cell.

51. The method of claim 50, wherein the virus is the Human immunodeficiency virus 1 (HIV)), or a subtype, species, or variant thereof, and is latent, e.g., within a cell.

52. The method of claim 51, wherein the subject is infected with the HIV virus and is asymptomatic.

53. The method of claim 30, wherein the bacterial infection comprises infection with a Gram- negative bacterium or a Gram-positive bacterium.

54. The method of claim 53, wherein the bacterial infection comprises a bacterium selected from Listeria (e.g., Listeria monocytogenes), Francisella (e.g., Francisella tularensis),

Mycobacteria (e.g., Mycobacteria tuberculosis), Brucella (e.g., Brucella abortis), Streptococcus (e.g., group B Streptococcus), Legionella (e.g., Legionella pneumophila), Escherichia (e.g., Escherichia coli), Pseudomonas (e.g., Psuedomonas aeruginosa), Salmonella (e.g., Salmonella typhi), Shigella (e.g., Shigella flexneri), Campylobacter (e.g., Campylobacter jejuni),

Clostridium (e.g., Clostrodium botulinum), Enterococcus (e.g., Enterococcus faecalis), Vibrio (e.g., Vibrio cholera), Yersinia (e.g., Yersinia pestis), and Staphylococcus (e.g., Staphylococcus aureus), or other genera, species, subtypes, or variants thereof.

55. The method of claim 4, further comprising analyzing or receiving analysis of a biopsy specimen or biological tissue sample from the subject at least once prior to the end of treatment.

56. The method of claim 55, wherein the biopsy specimen is a liver biopsy specimen.

57. The method of claim 55, wherein the liver biopsy specimen is analyzed by

immunohistochemical staining.

58. The method of claim 57, wherein the liver biopsy specimen is analyzed by immunohistochemical staining for the extent of induction of a PRR (e.g., RIG-I or NOD2).

59. The method of claim 57, wherein the immunohistochemical staining indicates an increase in the distribution of PRR expression (e.g., RIG-I expression or NOD2 expression) in the liver over the course of treatment of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof.

60. The method of claim 59, wherein the increase in the distribution of expression of a PRR (e.g., RIG-I or NOD2) in the liver is from the onset of treatment to the end of treatment is between about 5% and about 95%.

61. The method of claim 55, wherein the liver biopsy specimen is analyzed for the levels of one or more markers of microbial infection, e.g., viral DNA, viral RNA, viral antigens, cccDNA, bacterial load.

62. The method of claim 56, wherein the liver biopsy specimen is analyzed for the expression level of interferon (e.g., interferon alfa or interferon beta), an interferon stimulating protein (e.g., ISG15, CXCL10, OAS 1), or other cytokines.

63. The method claim 56, wherein the liver biopsy specimen is analyzed for the reduction of liver inflammation, necrosis, steatosis, or fibrosis.

64. The method of claim 4, wherein the subject is treatment naive.

65. The method of claim 4, wherein the subject has previously been treated for a microbial infection (e.g., a viral infection, a bacterial infection, a fungal infection, or a parasitic infection) or cancer.

66. The method of claim 65, wherein the subject has previously been treated for HBV infection or HCV infection.

67. The method of claim 65, wherein the subject has been previously treated for a bacterial infection or cancer.

68. The method of claim 65, wherein the subject has been previously treated for a viral infection and is asymptomatic.

69. The method of claim 4, wherein the compound of Formula (I) or Formula (II) is administered orally (e.g., the compound of Formula (II) is administered orally).

70. The method of claim 4, wherein the compound of Formula (I) or Formula (II) is administered parenterally (e.g., the compound of Formula (II) is administered parenterally).

71. The method of claim 4, wherein the method comprises daily administration of said compound.

72. The method of claim 4, wherein the administration is once daily.

73. The method of claim 4, wherein the method comprises administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof for a duration between about 1 week and 20 weeks.

74. The method of claim 4, wherein the method comprises administration of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof for a duration between about 1 week and 12 weeks.

75. The method of claim 4, wherein the dosage of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof comprises about 0.5 mg/kg to about 100 mg/kg.

76. The method of claim 4, wherein the dosage of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof comprises 0.5 mg/kg to about 50 mg/kg.

77. The method of claim 4, wherein the dosage of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof comprises 5 mg/kg to about 50 mg/kg.

78. The method claim 4, wherein the dosage comprises a liquid or a solid dosage form.

79. The method of claim 78, wherein the liquid dosage form comprises a suspension, a solution, a linctus, an emulsion, a drink, an elixir, or a syrup.

80. The method of claim 78, wherein the solid dosage form comprises a capsule, tablet, powder, dragee, or a microencapsulated dose form.

81. The method of claim 4, further comprising the administration of a therapeutically effective amount of an additional agent.

82. The method of claim 81, wherein the additional agent is an antiviral agent, an

antibacterial agent, or an anticancer agent.

83. The method of claim 82, wherein the antiviral agent comprises an interferon, a nucleoside analog, a non-nucleoside antiviral, or a small-molecule immune enhancer.

84. The method claim 82, wherein the antiviral agent comprises a capsid inhibitor, an entry inhibitor, a secretion inhibitor, a microRNA, an antisense RNA agent, an RNAi agent, or other agent designed to inhibit viral RNA or DNA.

85. The method of claim 82, wherein the antiviral agent comprises entecavir, lamuvidine, adefovir, darunavir, sofosbuvir, telaprevir, tenofovir, zidovudine, and ribavirin.

86. The method of claim 82, wherein the antibacterial agent comprises gentamicin, kanamycin, streptomycin, chloramphenicol, ceftobiprole, amoxicillin, penicillin, bacitracin, tetracycline, rifabutin, tigecycline, and vancomycin.

87. The method of claim 82, wherein the anticancer agent is selected from methotrexate, 5- fluorouracil, doxorubicin, vincristine, bleomycin, vinblastine, dacarbazine, toposide, cisplatin, epirubicin, and sorafenib tosylate.