Compositions and methods for the treatment of viral infection using mitochondrial modulation

Abstract

Methods and compositions for the treatment of viral infections, such as SARS-CoV-2 infection, are disclosed herein which restore mitochondrial function, thereby inhibiting viral propagation.

Description

[0001] COMPOSITIONS AND METHODS FOR THE TREATMENT OF VIRAL INFECTION USING MITOCHONDRIAL MODULATION

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to US Provisional Application No. 63 / 656,959 filed June 6, 2024, which is incorporated herein by reference as though set forth in full.

[0004] GOVERNMENT INTEREST STATEMENT

[0005] This invention was made with government support under W81XWH-21-1-0128 awarded by the U.S. Department of Defense. The government has certain rights in the invention.

[0006] FIELD OF THE INVENTION

[0007] This invention relates to therapies for the treatment of viral infections including SARS- CoV-2. More specifically, the invention relates to methods and compositions for inhibiting viral biogenesis and restoring mitochondrial function to halt the advance of viral propagation.

[0008] BACKGROUND OF THE INVENTION

[0009] Beginning in 2019, the global coronavirus disease (CO VID-19) pandemic has presented a large-scale public health challenge, with the death toll in the United States exceeding 1 million (See the world wide web at covid.edc.gov / covid-data-tracker / #datatracker-home), and the global death count over 6 million as of July 2022 (world wide web at / / co vid 19. who .int / ) . COVID-19, which results from infection by the severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) virus (Zhu et at., 2020), is a disease primarily characterized by dry cough, fever, and fatigue. However, symptoms can also include sore throat, shortness of breath, loss of smell and / or taste, headache, chills, nausea, vomiting, and diarrhea (Guan et al., 2020; Song et al., 2021; Wang et al., 2020). Symptoms can also persist long after resolution of the initial infection, in some cases more than 14 months (Rays et al., 2022; de Miranda et al., 2022; Desai et al., 2022). Currently, the treatments for SARS-CoV-2 can be ineffective for ameliorating persistent symptoms.

[0010] Previous studies have shown that SARS-CoV-2 concertedly inhibits both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) mitochondrial gene expression. This is associated with activation of hypoxia inducible factor 1 subunit a (HIF-la) resulting in the induction of glycolysis and the further down regulation of mitochondria function. The inhibition of mitochondrial oxidative phosphorylation (OXPHOS) and the induction of glycolysis by SARS- CoV-2 redirects nutrients from oxidation by the mitochondria to the production of lipids and nucleic acids for the propagation of the virus.

[0011] Viral inhibition of mitochondrial function activates the mitochondrially-regulated innate immune system which is both important for SARS-CoV-2 dispersion along with the production of toxic cytokines. Perturbation of mitochondrial biogenesis also activates the 0MA1 mitochondrial protease which cleaves DAP3 binding cell death enhancer 1 (DELEI). Cleaved DELEI activates the elF2a kinase heme -regulated inhibitor (HRI kinase) which phosphorylates eIF2a, inhibiting cytosolic protein synthesis and activating the production of diffusible signaling factors including GDF-15 through Integrated Stress Response (ISR).

[0012] Initial nasopharyngeal infection causes the concerted impairment of mitochondrial gene expression resulting in lung pathology and the activation of the ISR with GDF-15 production. Diffusible factors such as GDF-15 may account for the perturbation of mitochondrial function in the hamster brain in the absence of viral entrance into the brain and thus may be the basis of the commonly observed “brain fog.” As the SARS-CoV-2 infection proceeds, viral titer declines until the autopsy tissues harbor no detectable viral RNA. While the absence of vims in the autopsy lung is associated with the restoration of nDNA and mtDNA gene expression, autopsy results reveal that heart, kidney and liver mitochondrial gene expression remains impaired.

[0013] Clearly, a need exists for leveraging these mechanisms in methods for treating persistent symptoms of viral infections, including, without limitation, SARS-CoV-2 infection.

[0014] SUMMARY OF THE INVENTION

[0015] In accordance with the present invention, methods of treating, inhibiting, or preventing a viral infection or viral propagation in a subject are provided. An exemplary method comprises administering a compound that inhibits reactive oxygen species (mROS) production to the subject. In certain embodiments, the compound is a mitochondria-targeted antioxidant (MnTBAP) or an inhibitory RNA molecule. In certain embodiments, the MnTBAP is selected from a mitochondrially-targeted catalase (mCAT) mimetic; vitamin C; vitamin E; vitamin KI; vitamin B; sodium pyruvate; a-lipoic acid; and a NAD precursor. In certain embodiments the MnTBAP is a mCAT mimetic selected from EUK8 and EUK134. In certain embodiments the compound enhances mitochondrially-targeted catalase (mCAT) expression. In certain embodiments the compound is a vector, such as an AAV or AAV6 vector, comprising a mCAT transgene.

[0016] In certain embodiments, the compound is a mitochondrial permeability transition pore (mtPTP) inhibitor, such as N-methyl-valine-cyclosporine and N-methyl-4-isoleucine- cyclosporine (NIM811), cyclosporine, such as Cyclosporine A, cyclophilin D, VDAC (voltage dependent anion channel) inhibitors, and adenine nuclease translocase (ANT) inhibitors.

[0017] In certain embodiments, at least two compounds listed above act synergistically to reduce virus infection symptoms.

[0018] In certain embodiments, the viral infection is caused by SARS-CoV-2 or a vims from the Hepadnaviridae family of viruses, hepatitis B, a retrovirus, an alpharetrovirus, Rous sarcoma vims, a coronavirus, a flavivirus, Tick-borne encephalitis vims, Dengue virus, Zika vims, a betaretrovims, simian retrovirus, a deltaretrovims, bovine leukemia virus, human T- lymphotrophic vims (HTLV), HTLV-1, HTLV-2, HTLV-3, a gammaretrovirus, murine leukemia vims, feline leukemia virus, a lentivims, human immunodeficiency virus (HIV), HIV-1, HIV-2, simian immunodeficiency vims, equine infectious anemia vims, bovine immunodeficiency vims, rabbit endogenous lentivirus type K (RELIK), or feline immunodeficiency virus.

[0019] In certain embodiments, the method reduces or suppresses at least one symptom of the viral infection. In certain embodiments, the symptom is selected from fatigue, feeling tired, weakness, brain fog (problems concentrating or thinking), headaches, tremor, rapid or pounding heartbeat, feeling of skipped heartbeats (palpitations), dizziness upon standing, symptoms that worsen after physical or mental activity (known as post-exertional malaise, PEM), gastrointestinal symptoms including stomach pain, diarrhea, and / or constipation, loss of or change in smell and / or taste, thirst (for instance, dry mouth), cough, changes in comfort or capacity for sex and / or desire for sex, chest pain, tightness, or pressure, hearing problems, including hearing loss or ringing in the ears (tinnitus), shortness of breath, muscle and / or joint pain, back pain, sleep apnea, fever, sweats, and / or chills, hair loss, sleep problems, including insomnia, bladder problems, including difficulty urinating or incontinence, vision problems, such as blurry vision, sensitivity to light, floaters, flashing lights, or difficulty reading or focusing eyes, depression, anxiety, swelling of the legs, problems with teeth, foot pain, skin rash, abnormal movements, skin color changes (for instance, skin that is red, white, or purple), and changes in menstrual cycle. In certain embodiments, the method prevents or treats symptoms of Long Co vid infection.

[0020] In certain embodiments, the compound is administered by nasopharyngeal, enteral, parenteral, topical, or systemic administration.

[0021] In certain embodiments, the compound counteracts viral inhibition of oxidative phosphorylation. In certain embodiments, the compound directs nutrients away from viral biogenesis, reduces viral propagation and cytokine toxicity, and / or modulates a virally-induced cytokine storm. In certain embodiments, the method further comprises detecting inhibition of viral infection and / or propagation.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1: Graph Summary of the Progression of SARS-CoV-2 infection and modulation of mitochondrial function.

[0024] Figure 2: Creation of a Bi-Cistronic Lentiviral Vector for transduction of SARS-CoV-2 Viroporins E and Orf3a, designated “E3a”. “EV” = empty vector. Fura-Red measures cytosolic Ca++Rod-2 measures mitochondrial Ca++. FLIM permits quantification of the NAD+ / NADH ratio in living cells.

[0025] Figures 3A-3E: Expression of Orf3a (2-3a) elevates HIFla levels through mROS. 293T cells were infected with LV-EV or LV-3a. 24hours (hrs) post infection cells were cultured in the presence of Fig. 3A, 3C, 3D, 3E) 150mM of C0CI2 or Fig. 3B) 1% O2 for 24hrs then analyzed by immunoblot with antibodies against HIFla and FLAG-Tag to detect the FLAG-tagged 2-3a viroporins. GAPDH was a loading control. Fig. 3D) 293T cells were co-infected with LV- EV(mCAT) or LV-mCAT. Fig. 3C, 3D). At 24hpi cells were cultured in the presence of 150mM of C0CI2 for 24hr and levels of mROS were assessed using MitoSOX and MTDR fluorescence by confocal microscopy. MTDR fluorescence was used to normalize for mitochondrial content with mROS expressed as the ratio of MitoSOX / MTDR, by confocal microscopy. Error bars for Fig. 3A, 3B, 3C, 3E) represent SEM from at least 3 independent experiments; statistical data is indicated with asterisks (*).

[0026] Figures 4A-4D: The mtPTP is Essential for the Release of mtDNA into the Cytosol and the Activation of the Inflammasome. MnTBAP = Mitochondrially-targeted catalytic antioxidant. NIM811 binds cyclophilin D ad inhibits the mtPTP. P° cells lack mtDNA. Figures 5A-5G: The Mitochondrial Catalytic Antioxidant mCAT Transgene Protects Mice from SARS-CoV-2 Pathology. hACE2 = K18-hACE2 mice. DPI = days post infection. SARS-CoV2 N protein mRNA was quantified by qPCR. HIFla cells were quantified by immunofluorescence. * = <0.05, ** = < 0.01, *** = < 0.001.

[0027] Figures 6A-6B: Diminished Expression of Innate and Humoral Immunity Genes in SARS-CoV-2 Infected Mice Harboring the Systemic mCAT Gene.

[0028] Figure 7: Striking Reduction in mRNA Levels of Mitochondrial Bioenergetic and Biogenesis Genes as well as Inflammatory and Humoral Immunity Gene mRNAs in mCAT mice (Bottom Panels) Versus Control Mice (Top Panels).

[0029] Figures 8A-8D: Demonstration that Systemic Treatment with EUK8 Protects K18- hACE2 Mice from SARS-CoV-2 Pathogenicity. Fig. 8C: Pathology rating score system.

[0030] Figures 9A-9B: Demonstration that in K18-hACE2 Mice Systemic mCAT expression and EUK8 and EUK134 Treatment Markedly Reduce the Pathogenicity of SARS-CoV-2.

[0031] Figures 10A-10C: Confirmation that release of mtDNA and activation of the inflammasome are mediated by the mtDNA and activation can be mitigated by the mtPTP inhibitor NIM811 and in mice PPIF / _which lack the cyclophilin D NIM811 binding protein.

[0032] DETAILED DESCRIPTION OF THE INVENTION

[0033] Prolonged mitochondrial dysfunction is associated with the death of patients with severe Covid. In certain case, when less severe it appears to be the cause of Long COVID (Figure 1).

[0034] This 3l / 2 year study demonstrates that SARS-CoV-2 infection is associated with mitochondrial pathology and inhibition of mitochondrial function. This inhibition increases mitochondrial reactive oxygen species (mROS) production, thereby activating HIF-la which redirects nutrients from cellular mitochondrial OXPHOS to glycolysis and enhancing viral biogenesis. Inhibition of mitochondrial function also activates the innate immune system including the inflammasome, resulting in production of IL- 1 p and cGAS-STING pathway stimulation followed by production of Type I interferon. Hence, SARS-CoV-2 interference of mitochondrial function is central to both enhanced viral propagation and the activation of cytokine production which in extreme cases generates a cytokine storm (Figure 1).

[0035] The present claimed subject matter may be understood more readily by reference to the following detailed description which forms a part of this disclosure. It is to be understood that this invention is not limited to the specific products, methods, conditions or parameters described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention.

[0036] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. As employed above and throughout the disclosure, the following terms and abbreviations, unless otherwise indicated, shall be understood to have the following meanings.

[0037] In the present disclosure the singular forms "a," "an," and "the" include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to "a compound" is a reference to one or more of such compounds and equivalents thereof known to those skilled in the art, and so forth. The term "plurality", as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

[0038] The terms “about” or “approximately” in the context of numerical values and ranges refers to values or ranges that approximate or are close to the recited values or ranges such that the embodiment may perform as intended, such as having a desired amount of nucleic acids or polypeptides in a reaction mixture, as is apparent to the skilled person from the teachings contained herein. In some embodiments, about means plus or minus 10% of a numerical amount.

[0039] Furthermore, a compound "selected from the group consisting of" refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more of the compounds. According to the present invention, an isolated, or biologically pure molecule is a compound that has been removed from its natural milieu. As such, "isolated" and "biologically pure" do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthetic techniques or can be produced by any such chemical synthetic route. As used herein, the terms "component," "composition," "composition of compounds," "compound," "drug," "pharmacologically active agent," "active agent," "therapeutic," "therapy," "treatment," or "medicament" are used interchangeably herein to refer to a compound or compounds or composition of matter which, when administered to a subject (human or animal) induces a desired pharmacological and / or physiologic effect by local and / or systemic action.

[0040] Therapeutic Compounds

[0041] The inhibitors described herein may be used, alone or in combination, in methods for treating viral infections, such as SARS-CoV-2 infection.

[0042] The terms “inhibition” or “inhibit” refer to a decrease or cessation of any event (such as protein ligand binding) or to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and / or amount as compared to a reference. It is not necessary that the inhibition or reduction be complete. For example, in certain embodiments, “reduce” or “inhibit” refers to the ability to cause an overall decrease of 20% or greater. In another embodiment, “reduce” or “inhibit” refers to the ability to cause an overall decrease of 50% or greater. In yet another embodiment, “reduce” or “inhibit” refers to the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater. Inhibition can be a decrease in activity of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,

[0043] 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,

[0044] 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,

[0045] 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

[0046] An "inhibitor" (interchangeably termed "antagonist") of a polypeptide of interest is an agent that interferes with activation or function of the polypeptide of interest, e.g., partially or fully blocks, inhibits, or neutralizes a biological activity mediated by a polypeptide of interest. For example, an antagonist of polypeptide X may refers to any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity mediated by polypeptide X. Examples of inhibitors include antibodies; ligand antibodies; small molecule antagonists; antisense and inhibitory RNA (e.g., siRNA) molecules.

[0047] In certain embodiments, the inhibitor is a small nucleic acid inhibitor. A "small nucleic acid inhibitor" refers to any sequence based nucleic acid molecule which, when introduced into a cell expressing the target nucleic acid, is capable of modulating expression of that target. siRNA, antisense, miRNA, shRNA and the like may be utilized in the methods of the invention.

[0048] The terms "miRNA" and "microRNA" refer to about 10-35 nt, preferably about 15-30 nt, and more preferably about 19-26 nt, non-coding RNAs derived from endogenous genes encoded in the genomes of plants and animals. They are processed from longer hairpin- like precursors termed pre-miRNAs that are often hundreds of nucleotides in length. MicroRNAs assemble in complexes termed miRNPs and recognize their targets by antisense complementarity. These highly conserved, endogenously expressed RNAs are believed to regulate the expression of genes by binding to the 3'-untranslated regions (3'-UTR) of specific mRNAs as well as other regions on targeted mRNAs. Without being bound by theory, a possible mechanism of action assumes that if the microRNAs match 100% their target, i.e. the complementarity is complete, the target mRNA is cleaved, and the miRNA acts like a siRNA. However, if the match is incomplete, i.e. the complementarity is partial, then the translation of the target mRNA is blocked. The manner by which a miRNA base-pairs with its mRNA target correlates with its function: if the complementarity between a mRNA and its target is extensive, the RNA target is cleaved; if the complementarity is partial, the stability of the target mRNA in not affected but its translation is repressed.

[0049] The term "RNA interference" or "RNAi" refers generally to a process or system in which a RNA molecule changes the expression of a nucleic acid sequence with which RNA molecule shares substantial or total homology. The term "RNAi agent" refers to an RNA sequence that elicits RNAi.

[0050] An "siRNA" refers to a molecule involved in the RNA interference process for a sequence-specific post-transcriptional gene silencing or gene knockdown by providing small interfering RNAs (siRNAs) that has homology with the sequence of the targeted gene. Small interfering RNAs (siRNAs) can be synthesized in vitro or generated by ribonuclease III cleavage from longer dsRNA and are the mediators of sequence-specific mRNA degradation. Preferably, the siRNA of the invention are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA / RNA synthesizer. The siRNA can be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions. Commercial suppliers of synthetic RNA molecules or synthesis reagents include Applied Biosystems (Foster City, Calif., USA), Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK).

[0051] The term “delivery” as used herein refers to the introduction of foreign molecule (i.e., salt taste receptor nucleotide containing nanoparticle) into cells. The term “administration” as used herein means the introduction of a foreign molecule into a cell. The term is intended to be synonymous with the term “delivery”.

[0052] A “vector” as used herein is a biological or chemical moiety comprising a nucleic acid sequence which can be introduced into an appropriate host cell for replication or expression of said nucleic acid sequence. Common vectors include non-viral vectors and viral vectors. As used herein, a non-viral system might be selected from nanoparticles, electroporation systems and novel biomaterials, naked DNA, phage, transposon, plasmids, cosmids (Phillip McClean, www.ndsu.edu / pubweb / ~mcclean / -plsc731 / cloning / cloning4.htm) and artificial chromoisomes (Gong, Shiaoching, et al. “A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.” Nature 425.6961 (2003): 917-925).

[0053] As used herein, an “expression cassette” refers to a nucleic acid molecule which comprises a biologically useful nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme or other useful gene product, mRNA, etc.) and regulatory sequences operably linked thereto which direct or modulate transcription, translation, and / or expression of the nucleic acid sequence and its gene product.

[0054] As used herein, “operably linked” sequences include both regulatory sequences that are contiguous with the nucleic acid sequence and regulatory sequences that act in trans or at a distance to control the sequence. Such regulatory sequences typically include, e.g., one or more of a promoter, an enhancer, an intron, a Kozak sequence, a polyadenylation sequence, and a TATA signal. The expression cassette may contain regulatory sequences upstream (5’ to) of the gene sequence, e.g., one or more of a promoter, an enhancer, an intron, etc., and one or more of an enhancer, or regulatory sequences downstream (3’ to) a gene sequence, e.g., 3’ untranslated region comprising a polyadenylation site, among other elements. In other embodiments, the term “transgene” refers to one or more DNA sequences from an exogenous source which are inserted into a target cell. Typically, such an expression cassette for generating a viral vector contains the coding sequence for the gene product described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein. In certain embodiments, a vector genome may contain two or more expression cassettes.

[0055] In addition to the coding sequence, in certain embodiments the vector includes regulatory sequences which direct expression in a host cell. In certain embodiments, the regulatory elements include a promoter.

[0056] In addition to a promoter, the vector may contain one or more appropriate “regulatory elements” or “regulatory sequences”, which comprise but are not limited to an enhancer; transcription factor; transcription terminator; efficient RNA processing signals such as splicing and polyadenylation signals (poly A); sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE); sequences that enhance translation efficiency (i.e. , Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. Examples of suitable polyA sequences include, e.g., SV40, bovine growth hormone (bGH), and TK polyA. Examples of suitable enhancers include, e.g., the alpha fetoprotein enhancer, the TTR minimal promoter / enhancer, LSP (TH-binding globulin promoter / alphal-microglobulin / bikunin enhancer), amongst others. These control sequences or the regulatory sequences are operably linked to the nuclease coding sequence or transgene coding sequence.

[0057] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in cells or target tissues. Such methods can be used to administer nucleic acids encoding inhibitory compounds to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bihm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994). Methods of non- viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91 / 17424; WO 91 / 16024. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).

[0058] The preparation of lipidmucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0059] The term “modulate” as used herein refers to the ability of a compound to change an activity in some measurable way as compared to an appropriate control. As a result of the presence of compounds in the assays, activities can increase or decrease as compared to controls in the absence of these compounds. Preferably, an increase in activity is at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound. Similarly, a decrease in activity is preferably at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound. A compound that increases a known activity is an “agonist”. One that decreases, or prevents, a known activity is an “antagonist”. Inhibition can be a change in activity of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

[0060] 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,

[0061] 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,

[0062] 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

[0063] In certain embodiments, the compounds described herein act to modulate mitochondrial function and are useful as therapeutic or prophylactic therapy when such modulation is desired, e.g., for the treatment of a viral infection, such as SARS-CoV-2. Unless otherwise indicated, when uses of the compounds of the present disclosure are described herein, it is to be understood that such compounds may be in the form of a composition (e.g., a pharmaceutical composition).

[0064] In certain embodiments, the compound inhibits mitochondrial reactive oxygen species (mROS) production. In certain embodiments, the compound is a mitochondria-targeted antioxidant (MnTBAP). The term “mitochondria-targeted antioxidant” or “MnTBAP” refers to compounds that have an antioxidant group linked to a moiety that targets mitochondria. In certain embodiments, the MnTBAP is selected from a mitochondrially-targeted catalase (mCAT) mimetic, such as EUK8 and EUK134; vitamin C; vitamin E; vitamin KI; vitamin B; sodium pyruvate and a-lipoic acid; and NAD precursors (for example, niacinamide).

[0065] In certain embodiments, the compound enhances mitochondrially-targeted catalase (mCAT) expression. In certain embodiments, the mCAT expression is enhanced via administration of a vector comprising a mCAT transgene. In certain embodiments, the mCAT transgene is an AAV vector. In certain embodiments, the AAV vector is an AAV6 serotype.

[0066] In certain embodiments, the compounds inhibit mitochondrial permeability transition pore (mtPTP). The “mitochondrial permeability transition pore” (mtPTP) is a protein within the inner membrane of the mitochondria that is permeable to molecules less than 1.5 kDa. The mPTP is usually closed, but may be opened under certain conditions including mitochondrial matrix Ca.sup.2+ accumulation, adenine nucleotide depletion, increased phosphate concentration, or oxidative stress. The opening of the mtPTP pore is associated with apoptosis. Cyclophilins (e.g., CypD) can regulate the opening and closing of the mPTP. Exemplary mtPTP inhibitors include N-methyl-valine-cyclosporine and N-methyl-4-isoleucine-cyclosporine (NIM811), cyclosporine, such as Cyclosporine A, cyclophilin D, VDAC (voltage dependent anion channel) inhibitors, and adenine nuclease translocase (ANT) inhibitors.

[0067] Methods of Treatment and Administration

[0068] In certain embodiments, the compounds described above are used in methods of treating, preventing, and / or inhibiting viral infection and / or propagation. In certain embodiments, the compounds are used in methods of treating, preventing, and / or SARS-CoV-2 infection and / or propagation. The term “preventing” as used herein refers to administering a compound prior to the onset of clinical symptoms of a disease or conditions so as to prevent a physical manifestation of aberrations associated with the disease or condition.

[0069] The term “in need of treatment” as used herein refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals) that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a care giver's expertise, but that includes the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the disclosed compounds.

[0070] As used herein, “subject” includes, but is not limited to, animals, plants, bacteria, viruses, parasites and any other organism or entity. The subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian. The subject can be an invertebrate, more specifically an arthropod (e.g., insects and crustaceans). The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.

[0071] By “treatment” and “treating” is meant the medical management of a subject with the intent to cure, ameliorate, or stabilize, a pathological condition or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. It is understood that treatment, while intended to cure, ameliorate, or stabilize, a disease, pathological condition, or disorder, need not actually result in the cure, ameliorization, or stabilization. The effects of treatment can be measured or assessed as described herein and as known in the art as is suitable for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and / or quantitative terms. Thus, for example, characteristics or features of a disease, pathological condition, or disorder and / or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount.

[0072] As used herein, the term “viral infection” or “disease caused by virus” describes a diseased state in which a virus invades healthy cells, uses the cell's reproductive machinery to multiply or replicate and ultimately lyse the cell resulting in cell death, release of viral particles and the infection of other cells by the newly produced progeny viruses. Latent infection by certain viruses is also a possible result of viral infection.

[0073] Viral infections that can be treated or prevented by the compounds of the present disclosure can be any infection caused by a virus, e.g., a vims from the Hepadnaviridae family of viruses, e.g., hepatitis B; or any retrovirus, e.g., an alpharetrovirus, such as Rous sarcoma virus;a coronavirus, such as SARS-CoV-2; a flavivirus, such as Tick-borne encephalitis virus, Dengue vims or Zika virus; a betaretrovims, such as simian retrovirus; a deltaretrovirus, such as bovine leukemia virus or human T-lymphotrophic vims (HTLV) including HTLV-1, HTLV-2, and HTLV-3; a gammaretrovirus, such as murine leukemia virus or feline leukemia virus; or a lentivims, such as human immunodeficiency vims (HIV) including HIV-1 and HIV-2, simian immunodeficiency vims, equine infectious anemia virus, bovine immunodeficiency virus, rabbit endogenous lentivims type K (RELIK), or feline immunodeficiency virus. In preferred embodiments, the viral infection is a SARS-CoV-2 infection.

[0074] SARS-COV-2 is a betacoronavims with 79% genetic homology with SARS-COV, and 98% homology to the bat coronavirus RaTG13 (Zhou et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin, Nature, 2020;579 (7798):270-3). It is spread in respiratory droplets and aerosols and infects nasal, bronchial and alveolar epithelial cells by binding of the viral spike protein to its cellular receptor, ACE2 (Walls et al. Stmcture, Function, and Antigenicity of the SARS-COV-2 Spike Glycoprotein. Cell. 2020; 181(2):281-92 e6). Owing to the error prone nature of the viral replication process, RNA vimses such as SARS- COV-2 accumulate mutations resulting in some sequence diversity. Nevertheless, different strains of SARS-COV-2 can be recognized by sequencing and phylogenetic sequence trees. Exemplification of such phylogenetic tree analysis with rapid sequencing of isolates is reported for example by Meredith et al. Rapid Implementation of SARS-COV-2 sequencing to investigate cases of health-care associated COVID-19: a prospective genomic surveillance study in The Lancet, published on-line 14th July 2020. Sequences of amplified SARS-COV-2 vims genome can be compared with the NCBI Reference sequence NC_045512.2 or equivalent GenBank reference MN908947.3 for SARS-COV-2 corresponding to the SARS-COV-2 isolate Wuhan- Hu-1 complete genome (Wu et al. Nature 579, 265-269). SARS-COV-2 variant strains can thus be recognized and can be expected to have high level of sequence homology to the reference genome, e.g. at least 90%, at least 95%, at least 98% or at least 99%. Such sequencing surveillance can equally enable any new SARS-COV-2 virus infecting humans to be identified. The use of compounds of the present invention may therefore be used for treating humans infected with any SARS-COV virus, especially any SARS-COV-2 viral strain (which can include variants thereof).

[0075] SARS-CoV-2 can cause a variety of symptoms, from fever and cough to respiratory distress (Respiratory Distress Syndrome — RDS), renal failure and even death in infected individuals. With certain SARS-CoV-2 infections, the patient may develop “Long CO VID”, also known as “Post-COVID Conditions”. Long COVID refers to a wide range of symptoms and conditions that some people experience four or more weeks after an initial infection by SARS- CoV-2. The symptoms and conditions, which may last for weeks, months, or years, can be persistent (meaning they developed during an acute CO VID- 19 illness and haven’t gone away), recurrent (meaning they may go away after the initial illness then return), or new (meaning they were not present initially but developed later). Some people may experience only one of these (or other) symptoms, while others may have two or more. Symptoms can vary greatly from one person to the next. Symptoms of Long COVID include, without limitation, fatigue, feeling tired, weakness, brain fog (problems concentrating or thinking), headaches, tremor, rapid or pounding heartbeat, feeling of skipped heartbeats (palpitations), dizziness upon standing, symptoms that worsen after physical or mental activity (known as post-exertional malaise, PEM), gastrointestinal symptoms including stomach pain, diarrhea, and / or constipation, loss of or change in smell and / or taste, thirst (for instance, dry mouth), cough, changes in comfort or capacity for sex and / or desire for sex, chest pain, tightness, or pressure, hearing problems, including hearing loss or ringing in the ears (tinnitus), shortness of breath, muscle and / or joint pain, back pain, sleep apnea, fever, sweats, and / or chills, hair loss, sleep problems, including insomnia, bladder problems, including difficulty urinating or incontinence, vision problems, such as blurry vision, sensitivity to light, floaters, flashing lights, or difficulty reading or focusing eyes, depression, anxiety, swelling of the legs, problems with teeth, foot pain, skin rash, abnormal movements, skin color changes (for instance, skin that is red, white, or purple), and changes in menstrual cycle. COVID- 19 can damage multiple organs throughout the body, including the brain, heart, lungs, liver, and kidneys, among others. When this happens, it can increase the risk of the development of new medical conditions.

[0076] The term "drug response" as used herein, means any biological response in an organism that is the result of exposure to the drug. Drug responses can be favorable, such as when a patient's disease is eradicated by treatment with the drug, or unfavorable, such as when a patient enters a coma upon treatment with a drug.

[0077] The term “synergy” or “synergistic” refers to the interaction or cooperation of two or more substances, or other agents to produce a combined effect greater than the sum of their separate effects.

[0078] In certain embodiments, treatment comprises the use of at two or more of the compounds described herein, the combination improving the therapy using either one of the substances alone, by maximizing efficacy, reducing toxicity, and addressing interpatient variability, as well as delaying and / or overcoming innate or acquired resistance.

[0079] Provided herein are methods of treatment of viral infections including a SARS-CoV-2 infection. The methods include administration of an effective amount of at least one compound that inhibits mitochondrial reactive oxygen species (mROS) production to a subject in need thereof. In certain embodiments the compound is a mitochondria-targeted antioxidant (MnTBAP. In certain embodiments, the MnTBAP is selected from a mitochondrially-targeted catalase (mCAT) mimetic, such as EUK8 and EUK134; vitamin C; vitamin E; vitamin KI; vitamin B; sodium pyruvate and a-lipoic acid; and NAD precursors (for example, niacinamide).

[0080] In certain embodiments, the compound enhances mitochondrially-targeted catalase (mCAT) expression. In certain embodiments, the mCAT expression is enhanced via administration of a vector comprising a mCAT transgene. In certain embodiments, the mCAT transgene is an AAV vector. In certain embodiments, the AAV vector is an AAV6 serotype.

[0081] In certain embodiments, the compounds inhibit mitochondrial permeability transition pore (mtPTP) activity. The “mitochondrial permeability transition pore” (mtPTP) is a protein within the inner membrane of the mitochondria that is permeable to molecules less than 1.5 kDa. The mPTP is usually closed, but may be opened under certain conditions including mitochondrial matrix Ca2+accumulation, adenine nucleotide depletion, increased phosphate concentration, or oxidative stress. The opening of the mtPTP pore is associated with apoptosis. Cyclophilins (e.g., CypD) can regulate the opening and closing of the mPTP. Exemplary mtPTP inhibitors include N-methyl-valine-cyclosporine and N-methyl-4-isoleucine-cyclosporine (NIM811), cyclosporine, such as Cyclosporine A, cyclophilin D, VDAC (voltage dependent anion channel) inhibitors, and adenine nuclease translocase (ANT) inhibitors

[0082] In certain embodiments, the method of treatment effectively suppresses symptoms associated one or more symptoms associated with a viral infection, such as SARS-CoV-2 infection.

[0083] Symptoms vary according to the location and type of viral infection being treated. In certain embodiments, symptoms of viral infection include, fever, chills, head and body aches, fatigue, sore throat, cough, sneezing, runny or stuffy nose, nausea, vomiting, diarrhea, Rashes, sores, blisters, warts, weakness, night sweats, mouth ulcers, swelling, swollen lymph nodes and no symptoms at all. In certain embodiments, the treatment ameliorates symptoms of long Covid infection.

[0084] The compounds described herein can be formulated for nasopharyngeal, enteral, parenteral, topical, or systemic administration. The compounds can be combined with one or more pharmaceutically acceptable carriers and / or excipients that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. The carrier is all components present in the pharmaceutical formulation other than the active ingredient or ingredients. Typical carriers and conventional methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the compounds are known by those skilled in the art. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

[0085] The compounds described herein can be formulated for nasopharyngeal administration. For example, administration may include introduction of the compounds as vapors or sprays through the nasopharyngeal space.

[0086] The compounds described herein can be formulated for parenteral administration. For example, parenteral administration may include administration to a patient intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intravitreally, intratumor ally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, intrapericardially, intraumbilically, by injection, and by infusion.

[0087] Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Typically, such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and microemulsions thereof, liposomes, or emulsomes.

[0088] For intravenous administration, the compositions may be packaged in solutions of sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent. The components of the composition are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or concentrated solution in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent. If the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water or saline can be provided so that the ingredients may be mixed prior to injection.

[0089] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and / or by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

[0090] Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, viscosity modifying agents, and combination thereof. Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface-active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions.

[0091] The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent(s).

[0092] The formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

[0093] Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above.

[0094] The compounds described herein can be administered in an effective amount to a subject that is in need of alleviation or amelioration from one or more symptoms associated with a viral infection, such as SARS-CoV-2 infection.

[0095] The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact “effective amount.” However, an appropriate effective amount can be determined by one of ordinary skill in the art using only routine experimentation. The dosages or amounts of the compounds described herein are large enough to produce the desired effect in the method by which delivery occurs. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the subject and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician based on the clinical condition of the subject involved. The dose, schedule of doses and route of administration can be varied. The compositions are administered in an effective amount and for a period of time effect to reduce one or more symptoms associated with the disease to be treated. It should be understood that the “effective amount” for a composition having anti-viral proliferation properties may vary. In one embodiment an effective amount includes without limitation about 0.001 to about 25 mg / kg subject body weight. In one embodiment, the range of effective amount is 0.001 to 0.01 mg / kg body weight. In another embodiment, the range of effective amount is 0.001 to 0.1 mg / kg body weight. In another embodiment, the range of effective amount is 0.001 to 1 mg / kg body weight. In another embodiment, the range of effective amount is 0.001 to 10 mg / kg body weight. In another embodiment, the range of effective amount is 0.001 to 20 mg / kg body weight. In another embodiment, the range of effective amount is 0.01 to 25 mg / kg body weight. In another embodiment, the range of effective amount is 0.01 to 0.1 mg / kg body weight. In another embodiment, the range of effective amount is 0.01 to 1 mg / kg body weight. In another embodiment, the range of effective amount is 0.01 to 10 mg / kg body weight. In another embodiment, the range of effective amount is 0.01 to 20 mg / kg body weight. In another embodiment, the range of effective amount is 0.1 to 25 mg / kg body weight. In another embodiment, the range of effective amount is 0.1 to 1 mg / kg body weight. In another embodiment, the range of effective amount is 0.1 to 10 mg / kg body weight. In another embodiment, the range of effective amount is 0.1 to 20 mg / kg body weight. In another embodiment, the range of effective amount is 1 to 25 mg / kg body weight. In another embodiment, the range of effective amount is 1 to 5 mg / kg body weight. In another embodiment, the range of effective amount is 1 to 10 mg / kg body weight. In another embodiment, the range of effective amount is 10 to 20 mg / kg body weight. In another embodiment, the range of effective amount is 20 to 30 mg / kg body weight. In another embodiment, the range of effective amount is 30 to 40 mg / kg body weight. In another embodiment, the range of effective amount is 40 to 50 mg / kg body weight. In another embodiment, the range of effective amount is 1 to 50 mg / kg body weight. Still other doses falling within these ranges are expected to be useful.

[0096] In another embodiment, the range of effective amount is O.OOlmg to 10g. In another embodiment, the range of effective amount is 0.01 mg to 1 g. In another embodiment, the range of effective amount is 0.01 mg to 100 mg. In another embodiment, the range of effective amount is 0.1 mg to 100 mg. In another embodiment, the range of effective amount is 0.1 mg to 500 mg. In another embodiment, the range of effective amount is 1 mg to 100 mg. In another embodiment, the range of effective amount is 10 mg to 500 mg. In another embodiment, the range of effective amount is 10 mg to 750 mg. In another embodiment, the range of effective amount is 0.01 mg to 100 mg. In another embodiment, the range of effective amount is 1 mg to 500 mg.

[0097] In certain embodiments, the compositions described herein is administered via intraperitoneal administration. In these embodiments, the effective amount of the compositions described herein may be between 2-20mg / kg, 0-20mg / kg, about Img / kg, about 2mg / kg, about 3mg / kg, about 4mg / kg, about 5mg / kg, about 6mg / kg, about 7mg / kg, about 8mg / kg, about 9mg / kg, about lOmg / kg, about l lmg / kg, about 12mg / kg, about 13mg / kg, about 14mg / kg, about 15mg / kg, about 16mg / kg, about 17mg / kg, about 18mg / kg, about 19mg / kg, or about 20mg / kg.

[0098] In certain embodiments, the compositions described herein is administered via systemic or intravenous administration. In these embodiments, the effective amount of the compositions described herein may be between 2-10mg / kg, 2-20mg / kg, 0-20mg / kg, 14-50mg / kg, 12.5- lOOmg / kg, or at least about Img / kg, about 2mg / kg, about 3mg / kg, about 4mg / kg, about 5mg / kg, about 6mg / kg, about 7mg / kg, about 8mg / kg, about 9mg / kg, about lOmg / kg, about l lmg / kg, about 12mg / kg, about 13mg / kg, about 14mg / kg, about 15mg / kg, about 16mg / kg, about 17mg / kg, about 18mg / kg, about 19mg / kg, about 20mg / kg, about 21mg / kg, about 22mg / kg, about 23mg / kg, about 24mg / kg, about 25mg / kg, about 26mg / kg, about 27mg / kg, about 28mg / kg, about 29mg / kg, about 30mg / kg, about 3 Img / kg, about 32mg / kg, about 33mg / kg, about 34mg / kg, about 35mg / kg, about 36mg / kg, about 37mg / kg, about 38mg / kg, about 39mg / kg, about 40mg / kg, about 41mg / kg, about 42mg / kg, about 43mg / kg, about 44mg / kg, about 45mg / kg, about 46mg / kg, about 47mg / kg, about 48mg / kg, about 49mg / kg, about 50mg / kg, about 5 Img / kg, about 52mg / kg, about 53mg / kg, about 54mg / kg, about 55mg / kg, about 56mg / kg, about 57mg / kg, about 58mg / kg, about 59mg / kg, about 60mg / kg, about 6 Img / kg, about 62mg / kg, about 63mg / kg, about 64mg / kg, about 65mg / kg, about 66mg / kg, about 67mg / kg, about 68mg / kg, about 69mg / kg, about 70mg / kg, about 7 Img / kg, about 72mg / kg, about 73mg / kg, about 74mg / kg, about 75mg / kg, about 76mg / kg, about 77mg / kg, about 78mg / kg, about 79mg / kg, about 80mg / kg, about 8 Img / kg, about 82mg / kg, about 83mg / kg, about 84mg / kg, about 85mg / kg, about 86mg / kg, about 87mg / kg, about 88mg / kg, about 89mg / kg, about 90mg / kg, about 9 Img / kg, about 92mg / kg, about 93mg / kg, about 94mg / kg, about 95mg / kg, about 96mg / kg, about 97mg / kg, about 98mg / kg, about 99mg / kg, or about lOOmg / kg.

[0099] In certain embodiments, the compositions described herein are administered via oral administration. In these embodiments, the effective amount of the compositions described herein may be between 2-10mg / kg, 2-20mg / kg, 0-20mg / kg, 0-40mg / kg, 2-40mg / kg, 10-40mg / kg, 10- 20mg / kg, 20-40mg / kg, about Img / kg, about 2mg / kg, about 3mg / kg, about 4mg / kg, about 5mg / kg, about 6mg / kg, about 7mg / kg, about 8mg / kg, about 9mg / kg, about lOmg / kg, about llmg / kg, about 12mg / kg, about 13mg / kg, about 14mg / kg, about 15mg / kg, about 16mg / kg, about 17mg / kg, about 18mg / kg, about 19mg / kg, or about 20mg / kg, about 21mg / kg, about 22mg / kg, about 23mg / kg, about 24mg / kg, about 25mg / kg, about 26mg / kg, about 27mg / kg, about 28mg / kg, about 29mg / kg, about 30mg / kg, about 31mg / kg, about 32mg / kg, about 33mg / kg, about 34mg / kg, about 35mg / kg, about 36mg / kg, about 37mg / kg, about 38mg / kg, about 39mg / kg, or about 40mg / kg.

[0100] Kits and Articles of Manufacture

[0101] Any of the aforementioned products can be incorporated into a kit which may contain at least one of the inhibitors described herein, a pharmaceutically acceptable carrier, instructions for use, a container, a vessel for administration, or any combination thereof.

[0102] Any patent, patent application publication, or scientific publication, cited herein, is incorporated by reference herein in its entirety.

[0103] The examples are presented in order to more fully illustrate embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

[0104] EXAMPLE I

[0105] ELIMINATION OF MTDNA AND MITOCHONDRIAL FUNCTION ENHANCES VIRAL REPLICATION AND BIOGENESIS

[0106] To assess the importance of the mitochondrion and mtDNA in SARS-CoV-2 replication, human lung A549 cells expressing the human ACE2 receptor (A549-ACE2 p+) were treated with with ethidium bromide resulting in A549-ACE2 p° cells with reduced or lacking mtDNA.

[0107] When infected with the SARS-CoV-2 WAI (Washington 1 strain), the A549-ACE2 p° cells produced 100 times more infectious than the A549-ACE2 p+cells as quantified by plaque forming units (PFUs). Restoration of the A549-ACE2 p° cell’s mtDNA by adding the mtDNA back using transmitochondrial cybrid fusions reverted the viral production back to that of the A549-ACE2 p+cells. SARS-CoV-2 replication is also enhanced 5- to 10- times by treating A549-ACE2 p+cells with the mitochondrial protein synthesis inhibitor, chloramphenicol (CAP), and by treating A549-ACE2 p+cells with OXPHOS inhibitors. The enhanced viral propagation in the A549- ACE2 p° cells demonstrates that viral replication is maximized when the competition for nutrients between mitochondrial OXPHOS and glycolysis is eliminated permitting the total nutrient flow to go through glycolysis to enhance viral propagation. Thus, inhibition of mitochondrial function is critical to SARS-CoV-2 replication and that restoration of normal mitochondrial function inhibits vims production. Hence, therapies to enhance mitochondrial function will diminish viral propagation.

[0108] EXAMPLE 2 MITOCHONDRIAL MROS AND MTPTP INHIBITORS MODULATE HIFla AND INFLAMMASOME ACTIVATION IN CELL MODELS

[0109] Since SARS-CoV-2 and other viruses cannot eliminate mitochondrial OXPHOS, they developed systems to impair mitochondrial function, redirect nutrients toward glycolysis and enhance viral replication. Therefore, elucidating the mechanisms by which SARS-CoV-2 impairs mitochondrial function can elucidate other mitochondrial drug targets to impair vims production.

[0110] To determine the underlying pathophysiology of viral inhibition of mitochondrial function, we created a novel system in which we co-expressed SARS-CoV-2 proteins in cultured human kidney cells (HEK293T) cells and determine the viral effects on mitochondrial function.

[0111] Cell Model of SARS-CoV-2 Protein Toxicity Delineates Steps in Mitochondrial Modulation and Reveals Drug Targets for Limiting Viral Propagation and Virus Induced Hyperinflammation

[0112] To directly interrogate the physiological basis of SARS-CoV-2 toxicity to the mitochondrion, we created a bi-cistronic expression vector of the SARS-CoV-2 envelope (E) and open reading frame (Orf) 3a proteins. The E and Orf3a proteins are “viroporins” which introduce Ca++channels in the plasma and endoplasmic reticulum (ER) membranes. Since mitochondria are the primary Ca++modulators in non-muscle and heart cells, we surmised that the increased cytosolic Ca++would be taken up by the mitochondrion where it would activate the TCA cycle dehydrogenases, generating excess NADH, which would overload the electron transport chain (ETC) of OXPHOS resulting in increased mROS (Figure 2). The increased mROS generated by E & Orf3a expression stabilizes and activates HIF-la. This effect could be eliminated by the transduction of the mitochondrially-targeted catalase (mCAT) (3) which removes mitochondrial H2O2. mCAT transduction completely blocked the viroporin stabilization of HIF-la (Figure 3)(2). Hence, removal of mROS should block the redirection of metabolites from the mitochondrion to glycolysis thus inhibiting SARS-CoV-2 replication.

[0113] In addition, increased mROS damages mitochondrial and mtDNA leading to the activation of the innate immune system by the release of damaged mtDNA from the mitochondrion. The resulting cytosolic mtDNA binds to and activates the inflammasome resulting in the processing of pro-inflammatory cytokines such as pro-IL-ip to active cytokines, specifically IL- Ip. In fact, cells lacking mtDNA were unable to activate inflammasome maturation of IL- 1 P (Figure 4D). mtDNA released into the cytosol was inhibited by expression of mCAT and by the treatment with the mitochondrially-targeted catalytic antioxidant (MnTBAP), a non-therapeutic drug (Figure 4B and 4C).

[0114] Most importantly, however, NIM811, an activation inhibitor of the mitochondrial permeability transition pore (mtPTP), binds to cyclophilin D in the mtPTP complex (6). Through this binding it is as effective as cells with no mtDNA at blocking the viroporin activation of the inflammasome and maturation of pro-IL-ip (Figure 4B). Hence, we have shown for the first time that the mitochondrial activation of inflammation is exclusively mediated by the release of the mtDNA through the mtPTP. This mechanism makes the mtPTP a drug target for blocking SARS-CoV-2 induced inflammation (Figure 4A).

[0115] Using mice expressing the human ACE2 receptor (K18-hACE2 mice) into which we bred our constitutive mCAT transgene (3), we tested how the removal of mitochondrial H2O2 effects the pathogenicity and propagation of SARS-CoV-2 WAI following nasal infection. We first analyzed the effect of a viral infection dose of 2.5X104PFUs on the outcome of variable weight loss to assess the severity of viral pathogenicity in the mouse (Figure 5). Expression of mCAT in the K18-hACE2 mice significantly reduced the progression of weight loss in (Figure 5A), the overall health status of (Figure 5B), and the six-day mortality of (Figure 5C) the infected mice. This was associated with a significant reduction in SARS-CoV-2 N protein at 2 days post infection (DPI) (Figure 5D) and a striking reduction in both the number of cells with activated HIF-la and the overall level of HIF-la (Figure 5E, 5F). A similar result was found using 1X103PFU inoculum (Figure 5G). mCAT expression resulted in a striking decrease in the expression of the full range of inflammatory and Interferon pathway genes as assessed by qPCR of mouse lung tissue (Figure 6). Further, mCAT expression strongly mitigated virally induced genes of mitochondrial function (TCA cycle, OXPHOS, Folate metabolism nucleotide synthesis and pentose phosphate pathway) as well as innate immune pathways (mitochondrial innate response, interferon, ISR, adaptive immune response (Figure 7, Top versus Bottom).

[0116] To assess if virally-induced mROS expression was druggable, we employed the mitochondrially targeted catalytic antioxidant drugs, EUK8 and EUK134 (Figure 8). Using weight loss as the outcome variable, we infected K18-hACE2 mice with 2.5X104PFU of the SARS-CoV-2 WAI strain. We found the same degree of reduction in pathogenicity by EUK8 as seen with systemically expressed mCAT (Figure 5). The beneficial effect of EUK8 is also seen in a pathology index rating (Figure 8B, 8C) and in the number of mice surviving at eight DPI (Figure 8D).

[0117] To extend the relevance of EUK8 and EUK134 on the Omicron strain of SARS-CoV-2, we infected KI 8- hACE2 mice with and without mCAT with 2.5X104PFU virus. While Omicron reduced the weight of the wild type mice, the presence of mCAT almost completely blocked the negative effects of the virus on mouse weight, and the mCAT mice continued to gain weight (Figure 9A). Similarly, in mice infected 2.5X104PFU, both EUK8 and EUK134 protected the mice from weight loss (Figure 9B).

[0118] From these data, we conclude that mitochondrially-targeted catalytic antioxidant drugs should significantly reduce SARS-CoV-2 viral replication and pathogenicity. Hence, EUK8 and EUK134 should exhibit efficacy as drugs for treating SARS-CoV-2 and other viruses that impair mitochondrial function.

[0119] EXAMPLE 3

[0120] INACTIVATION OF THE MTPTP COMPONENT CYCLOPHILIN D IN PPIF7MUTANT MICE CONFIRMS THAT INHIBITION OF THE MTPTP IMPAIRS SARS- COV-2 PATHOLOGY.

[0121] The mitochondrially-targeted catalytic antioxidants are generally useful to treating viral infection. Additionally, the anti-mtPTP drugs such as NIM811 are very important in mitigating the toxicity of induced cytokine storms. NIM811 inhibition of the mtPTP markedly reduced the release of mtDNA from the infected cell (Figure 10A) as well as reduced the production of the inflammatory cytokine, IL- 1 P, by 50% (Figure 10B). To confirm the relevance of mtPTP inhibition in protecting from SARS-CoV-2 pathogenicity, we infected mice deficient in the cyclophilin D (PPIF_ / ), the regulatory subunit of the mtPTP and the binding site of NIM811, with 5.0X104PFU of 30-passage mouse adapted virus (Perlman strain). Absence of cyclophilin D resulted in consistent reduction in weight loss of the infected mice (Figure 10C). Hence, inhibition of activation of the mtPTP with drugs such as NIM811 can have a significant beneficial effect on the pathogenicity of SARS-CoV-2 and presumably other viruses that impair mitochondrial function.

[0122] References

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[0124] 2. J. W. Guamieri et al., SARS-CoV-2 viroporins activate the NLRP3-inflammasome by the mitochondrial permeability transition pore. Frontiers in Immunology (in press), (2023).

[0125] 3. S. E. Schriner et al., Extension of murine life span by overexpression of catalase targeted to mitochondria. Science 308, 1909-1911 (2005).

[0126] 4. B. J. Day, Catalytic antioxidants: a radical approach to new therapeutics. Drug Discovery Today 9, 557-566 (2007).

[0127] 5. J. Tong, S. E. Schriner, D. McCleary, B. J. Day, D. C. Wallace, Life extension through neurofibromin mitochondrial regulation and antioxidant therapy for Neurofibromatosis- 1 in Drosophila melanogaster. Nat. Genet. 39, 476-485 (2007).

[0128] 6. A. Angelin et al., Mitochondrial dysfunction in the pathogenesis of Ullrich congenital muscular dystrophy and prospective therapy with cyclosporins. Proc. Natl. Acad. Sci. USA 104, 991-996 (2007).

[0129] 7. S. Melov et al., Lifespan extension and rescue of spongiform encephalopathy in superoxide dismutase 2 nullizygous mice treated with superoxide dismutase-catalase mimetics. Journal of Neuroscience 21, 8348-8353 (2001).

[0130] 8. J. Li et al., Association of mitochondrial biogenesis with variable penetrance of schizophrenia. JAMA Psychiatry 78, 911-921 (2021).

[0131] EXAMPLE 5: CLINICAL EXAMPLE

[0132] The information herein above can be applied clinically to patients for therapeutic intervention. A preferred embodiment of the invention comprises clinical application of the information described herein to a patient. This can occur after a patient arrives in the clinic and presents with symptoms of a viral infection, e.g, symptoms of SARS-CoV-2 infection . A nonlimiting example of an effective dose range for a therapeutic compound described herein is from about 0.1 and 5,000 mg / kg of body weight / per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0133] The therapeutic compounds described herein have been shown to be well tolerated and the symptoms were assessed using clinical scores criteria. The treatment protocol can also optionally include administration of effective amounts of one or more of therapeutic agents that treat or inhibit a viral infection, such as SARS-CoV-2 infection. The treatment protocol can also optionally include administration of effective amounts of one or more of therapeutic agents that treat or inhibit viral infection. Such agents, include without limitation, compounds that inhibit mROS production, mnTBAPs, compounds that enhance mCAT expression, mCAT mimetics, and mtPTP inhibitors. The treatment protocol can also optionally include additional therapeutic compounds.

[0134] While certain features of the invention have been described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

What is claimed is:

1. A method for treating, inhibiting, or preventing a viral infection or viral propagation in a subject, the method comprising administrating a compound that inhibits reactive oxygen species (mROS) production to the subject.

2. The method of claim 1, wherein the compound is a mitochondria-targeted antioxidant (MnTBAP) or an inhibitory RNA molecule.

3. The method of claim 2, wherein the MnTBAP is selected from a mitochondrially- targeted catalase (mCAT) mimetic; vitamin C; vitamin E; vitamin KI; vitamin B; sodium pyruvate; a-lipoic acid; and a NAD precursor.

4. The method of claim 3, wherein the MnTBAP is a mCAT mimetic selected from EUK8 and EUK 134 and inhibits viral propagation.

5. The method of claim 1, wherein the compound enhances mitochondrially-targeted catalase (mCAT) expression.

6. The method of claim 5, wherein the compound is a vector comprising a mCAT transgene.

7. The method of claim 6, wherein the vector is an AAV6 vector.

8. The method of claim 1, wherein the compound is a mitochondrial permeability transition pore (mtPTP) inhibitor.

9. The method of claim 8, wherein the mtPTP inhibitor is selected from N-methyl-valine- cyclosporine and N-methyl-4-isoleucine-cyclosporine (NIM811), cyclosporine, such as Cyclosporine A, cyclophilin D, VDAC (voltage dependent anion channel) inhibitors, and adenine nuclease translocase (ANT) inhibitors.

10. The method of claim 9, wherein the mtPTP inhibitor is NIM811.

11. The method of any one of the preceding claims, wherein the viral infection is caused by SARS-CoV-2 or a virus from the Hepadnaviridae family of viruses, hepatitis B, a retrovirus, an alpharetrovirus, Rous sarcoma virus, a coronavirus, a flavivirus, Tick-bome encephalitis virus, Dengue virus, Zika vims, a betaretrovirus, simian retrovirus, a deltaretrovirus, bovineleukemia virus, human T-lympho trophic virus (HTLV), HTLV-1, HTLV-2, HTLV-3, a gammaretrovirus, murine leukemia virus, feline leukemia virus, a lentivirus, human immunodeficiency virus (HIV), HIV-1, HIV-2, simian immunodeficiency virus, equine infectious anemia virus, bovine immunodeficiency vims, rabbit endogenous lentivirus type K (RELIK), or feline immunodeficiency vims.

12. The method of claim 11, wherein the viral infection is caused by SARS-CoV-2.

13. The method of any one of the preceding claims, wherein the treatment reduces or suppresses at least one symptom of the viral infection.

14. The method of claim 13, wherein the symptom is selected from fatigue, feeling tired, weakness, brain fog (problems concentrating or thinking), headaches, tremor, rapid or pounding heartbeat, feeling of skipped heartbeats (palpitations), dizziness upon standing, symptoms that worsen after physical or mental activity (known as post-exertional malaise, PEM), gastrointestinal symptoms including stomach pain, diarrhea, and / or constipation, loss of or change in smell and / or taste, thirst (for instance, dry mouth), cough, changes in comfort or capacity for sex and / or desire for sex, chest pain, tightness, or pressure, hearing problems, including hearing loss or ringing in the ears (tinnitus), shortness of breath, muscle and / or joint pain, back pain, sleep apnea, fever, sweats, and / or chills, hair loss, sleep problems, including insomnia, bladder problems, including difficulty urinating or incontinence, vision problems, such as blurry vision, sensitivity to light, floaters, flashing lights, or difficulty reading or focusing eyes, depression, anxiety, swelling of the legs, problems with teeth, foot pain, skin rash, abnormal movements, skin color changes (for instance, skin that is red, white, or purple), and changes in menstrual cycle.

15. The method of any one of the preceding claims, wherein the method prevents or treats Long Co vid infection.

16. The method of any one of the preceding claims, wherein the compound is administered by nasopharyngeal, enteral, parenteral, topical, or systemic administration.

17. The method of claim 16, wherein the compound is administered by nasopharyngeal administration.

18. The method of any one of the preceding claims, wherein the compound counteracts viral inhibition of oxidative phosphorylation.

19. The method of claim 18, wherein the compound directs nutrients away from viral biogenesis.

20. The method of any one of the preceding claims, wherein the compound reduces viral propagation and cytokine toxicity.

21. The method of any one of the preceding claims, wherein the compound modulates a virally-induced cytokine storm.

22. The method of any one of the preceding claims, further comprising detecting inhibition of viral infection and / or propagation.

23. The method of any one of claims 2, 3, 4, 8, 9 or 11, where at least two of the compounds act synergistically to reduce symptoms of viral infection.

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