WO2023230431A1 - Inhibitors of deoxyhypusine synthase for the treatment and prevention of respiratory virus infections - Google Patents

Inhibitors of deoxyhypusine synthase for the treatment and prevention of respiratory virus infections Download PDF

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WO2023230431A1
WO2023230431A1 PCT/US2023/067278 US2023067278W WO2023230431A1 WO 2023230431 A1 WO2023230431 A1 WO 2023230431A1 US 2023067278 W US2023067278 W US 2023067278W WO 2023230431 A1 WO2023230431 A1 WO 2023230431A1
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Prior art keywords
deoxyhypusine synthase
inhibitor
deoxyhypusine
seq
human
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PCT/US2023/067278
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French (fr)
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Istvan Bartha
Arthur CHASE
Johannes Grosse
Leah B. SORIAGA
Neil Robert Stokes
Amalio Telenti
Winston C. Tse
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Glaxo Wellcome Uk Limited
Vir Biotechnology, Inc.
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Publication of WO2023230431A1 publication Critical patent/WO2023230431A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system having sulfur as a ring hetero atom, e.g. ticlopidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses

Definitions

  • the present disclosure relates to the field of respiratory virus infections and modulation of deoxyhypusine synthase (DHPS) for the treatment or prevention of a respiratory virus infection.
  • DHPS deoxyhypusine synthase
  • the present disclosure also relates to compounds, compositions and systems for use in such methods and kits related thereto.
  • Respiratory infections are a major cause of morbidity and mortality for humans. A majority of these infections are caused by respiratory viruses such as coronaviruses, human rhinoviruses, influenza viruses, human parainfluenza viruses, respiratory syncytial viruses or human metapneumoviruses.
  • respiratory viruses such as coronaviruses, human rhinoviruses, influenza viruses, human parainfluenza viruses, respiratory syncytial viruses or human metapneumoviruses.
  • Coronaviruses contain an enveloped single- strand, positive-sense RNA genome of between 26 and 32 kilobases in size. They belong to the Coronaviridae family and are classified by phylogenetic similarity into four categories or genera: alpha, beta, gamma, and delta. Seven coronaviruses have been associated with human disease to date. These are the alphacoronaviruses 229E and NL63, and the betacoronaviruses OC43, HKU1, MERS-CoV, SARS-CoV, and SARS- CoV-2. People are frequently infected with human coronaviruses 229E, NL63, OC43 and HKU1.
  • Symptoms usually include mild-to-moderate upper-respiratory tract illnesses of short duration, such as runny nose, cough, sore throat, and fever which are symptoms consistent with a common cold.
  • MERS-CoV, SARS-CoV, and SARS-CoV-2 infection can cause severe human illness.
  • Coronaviruses utilize a membrane bound spike protein to bind to a host cell surface receptor in order to gain cellular entry. Following entry into the host cell, the RNA genome is translated into two large polypeptides by the host ribosomal machinery. The polypeptides are processed by two proteases, the coronavirus main protease (Mpro / 3CLpro) and a papain-like protease to generate the proteins required for viral replication and packaging.
  • Mpro / 3CLpro coronavirus main protease
  • papain-like protease to generate the proteins required for viral replication and packaging.
  • HRV Human Rhinovirus
  • Other viruses from this family that are important in the context of human health include Poliovirus, Coxsackievirus, Enterovirus D68, and Enterovirus A71.
  • HRV is classified into three species (A, B and C) comprising more than 100 genotypes.
  • HRV infection is a significant cause of the common cold, however it can also lead to more serious illness, particularly in individuals with underlying respiratory disease.
  • HRV has been identified as the most frequently isolated virus in exacerbations of asthma in adults and children, as well as in exacerbations of chronic obstructive pulmonary disease (COPD).
  • COPD chronic obstructive pulmonary disease
  • Influenza Virus is an enveloped, negative-sense, single- stranded RNA virus of the Orthomyxoviridae family. There are four types, namely A, B, C and D. Influenza A Virus and Influenza B Virus are associated with the seasonal human symptomatic respiratory illness influenza (“flu”). Influenza A viruses are divided into subtypes based on their hemagglutinin (H) and neuraminidase (N) surface proteins. The severity of influenza disease in infected individuals is highly variable, with children and older adults generally experiencing more severe outcomes. Disease burden is significant with median incidence among all age groups estimated to be 8.3% in the United States.
  • Influenza virus has also been associated with several pandemics, most notably the influenza pandemic of 1918 (“Spanish Flu”), which was caused by the H INI influenza A virus strain. Seasonal vaccines and direct-acting antiviral agents have been approved for the prevention and treatment of influenza virus infection. However, there is an ongoing need for the development of new therapeutics owing to antigenic changes in the virus and the emergence of resistant isolates, as well as for pandemic preparedness purposes.
  • PIV Human Parainfluenza Virus
  • RSV Human Respiratory Syncytial Virus
  • hMPV human metapneumo virus
  • RSV is a leading cause of respiratory tract infection in infants and the elderly. Over 33 million cases of RSV-associated acute lower respiratory tract infection in children under five years of age were estimated globally in 2015, resulting in three million hospitalizations and almost 60,000 in-hospital. There are no approved vaccines for RSV. Passive immunization is achievable via prophylactic administration of a humanized monoclonal antibody (palivizumab) directed to an epitope of the RSV Fusion protein.
  • a humanized monoclonal antibody palivizumab
  • RSV The only approved therapeutic for RSV is ribavirin, which is administered via aerosol treatment. Evidence for ribavirin’s efficacy is limited and it has an unfavorable safety profile.
  • Human metapneumovirus which was discovered in 2001, can cause upper and lower respiratory disease in people of all ages, especially among young children, older adults, and people with weakened immune systems.
  • hMPV is an enveloped, negative-sense, single-stranded RNA virus.
  • a and B Two major genotypes or lineages, termed A and B, have been identified, each comprising two sub-groups or sub-lineages, namely Al and A2, and Bl and B2.
  • One aspect of the disclosure is a method of treating or preventing a respiratory virus infection or one or more symptoms associated therewith or inhibiting or reducing viral protein synthesis, in a human subject in need thereof, comprising inhibiting activity of deoxyhypusine synthase within the human subject or decreasing an amount of deoxyhypusine synthase within the human subject in a manner to reduce the respiratory virus infection or one or more symptoms associated therewith.
  • composition comprising a deoxyhypusine synthase inhibitor and a pharmaceutically acceptable excipient.
  • Another aspect of the disclosure is use of a deoxyhypusine synthase inhibitor in the manufacture of a medicament for the treatment or prevention of a respiratory virus infection or one or more symptoms associated therewith or for reducing viral protein synthesis.
  • Another aspect of the disclosure is a deoxyhypusine synthase inhibitor for use in the treatment or prevention of a respiratory virus infection or one or more symptoms associated therewith or for use in inhibiting or reducing viral protein synthesis.
  • Fig. 1 is a schematic illustrating the process within a cell of synthesizing hypusine within precursor eIF5A to form mature eIF5A including hypusine.
  • Eukaryotic translation initiation factor 5A (“cIF5A”) is a protein associated with protein biosynthesis and peptide bond formation and, accordingly, protein synthesis within a eukaryotic cell that is important for cell viability and proliferation.
  • cIF5A Eukaryotic translation initiation factor 5A
  • two isoforms of eIF-5A have been described: eIF5A- 1 and eIF5A-2. Each are encoded by two distinct genes EIF5A and EIF5A2.
  • eIF5A is the only known protein in a eukaryotic cell that includes the uncommon amino acid hypusine.
  • Hypusination is the process of altering lysine to include a hypusine residue believed to be critical for e!F5A function. The region surrounding the hypusine residue is highly conserved and is believed essential to the function of eIF5A.
  • an eIF5A precursor includes a lysine (K50) that is hypusinated via the hypusination pathway to provide mature eIF5A.
  • Deoxyhypusine synthase uses spermidine as a substrate in the presence of NAD to transfer a 4-amino butyl moiety from spermidine to the terminal -NH2 of lysine to create an intermediate form of eIF5A with deoxyhypusine attached to the -NH2 of lysine.
  • the 4-amino butyl moiety attached to the terminal -NH2 of lysine is then hydroxylated by deoxyhypusine hydroxylase to result in mature eIF5A including hypusine.
  • Embodiments of the present disclosure are directed to methods, compounds, compositions, systems, medicaments and kits for the treatment or prevention of a respiratory infection associated with a virus (referred to herein as a “respiratory virus infection”), or one or more symptoms associated therewith, or to inhibit, modulate, reduce or otherwise interfere with deoxyhypusine synthase within a cell, such as a human cell within a subject, in need of such treatment or prevention.
  • a respiratory infection referred to herein as a “respiratory virus infection”
  • a respiratory virus infection referred to herein as a “respiratory virus infection”
  • inhibiting, modulating, reducing or otherwise interfering with the activity of deoxyhypusine synthase to add deoxyhypusine to the terminal -NH2 of lysine of eIF5A limits or reduces the ability of deoxyhypusine synthase to enable respiratory virus replication within a cell.
  • Embodiments of the present disclosure are directed to methods of treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof.
  • subject refers to an animal or human body.
  • activity within the subject of deoxyhypusine synthase is inhibited, modulated, reduced or otherwise interfered with in a manner to inhibit, modulate, reduce or otherwise interfere with the respiratory virus infection or one or more symptoms associated therewith.
  • activity within a cell, such as a human cell, of deoxyhypusine synthase is inhibited, modulated, reduced or otherwise interfered with by introducing to the cell one or more competitive inhibitor compounds to bind to an active site of deoxyhypusine synthase.
  • activity within a cell, such as a human cell, of deoxyhypusine synthase is inhibited, modulated, reduced or otherwise interfered with by introducing to the cell one or more allosteric inhibitor compounds to bind to deoxyhypusine synthase in a manner to alter an active site of deoxyhypusine synthase.
  • the cell may be in vitro, ex vivo or in vivo.
  • the method may include treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof.
  • activity within a cell, such as a human cell, of deoxyhypusine synthase is inhibited, modulated, reduced or otherwise interfered with by introducing to the cell one or more degradation-promoting compounds to promote deoxyhypusine synthase degradation through cellular processes such as proteolysis.
  • cellular concentration of deoxyhypusine synthase is reduced, resulting in reduced activity of deoxyhypusine synthase in the cell.
  • the cell may be in vitro, ex vivo or in vivo.
  • the method may include treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof.
  • activity within a cell, such as a human cell, of deoxyhypusine synthase is inhibited, modulated, reduced or otherwise interfered with by introducing to the cell one or more systems for inhibiting, modulating, reducing or otherwise interfering with transcription of a nucleic acid encoding deoxyhypusine synthase.
  • activity within a cell, such as a human cell, of deoxyhypusine synthase is inhibited, modulated, reduced or otherwise interfered with by introducing to the cell one or more systems for inhibiting, modulating, reducing or otherwise interfering with translation of messenger RNA encoding deoxyhypusine synthase.
  • the cell may be in vitro, ex vivo or in vivo.
  • the method may include treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof.
  • the present disclosure contemplates the use of deoxyhypusine synthase in the identification of active agents and systems and the manufacture of a medicament for the treatment or prevention of a respiratory virus infection.
  • active agents, systems and medicaments can be included in a kit, as further described herein.
  • one or more competitive inhibitory compounds may be used to inhibit, modulate, prevent, reduce or otherwise interfere with the ability of deoxyhypusine synthase to transfer the 4-amino butyl moiety from spermidine to the -NH2 of lysine of eIF5A, such as by competing with binding of spermidine or a compound having a structure similar to spermidine or sterically inhibiting the active site for binding to a substrate, such as a spermidine substrate, which would otherwise be subject to enzymatic activity.
  • competitive inhibitor means a substance that competes with the binding of a substrate to the active site of deoxyhypusine synthase or otherwise binds to the active site of deoxyhypusine synthase and inhibits, modulates, prevents, reduces or otherwise interferes with the activity of deoxyhypusine synthase to enzymatically process a substrate for deoxyhypusine synthase, such as spermidine, and attach the 4-amino butyl moiety from spermidine to the -NH2 of lysine of eIF5A.
  • binding of the substrate is prevented, inhibited, reduced or weakened thereby reducing the enzymatic activity of deoxyhypusine synthase toward the substrate, such as spermidine.
  • Competitive inhibitors can be identified by screening compounds for binding efficiency to the active site of deoxyhypusine synthase using known assays or modifying known assays for this particular purpose.
  • One exemplary assay useful to assess inhibition of transfer of the 4-aminobutyl group from spermidine to eIF5A by deoxyhypusine synthase is carried out by using a reaction mixture including eIF5A, [ 3 H]- spermidine trihydrochloride, nicotinamide adenine dinucleotide (NAD + ), DTT, and various concentrations of deoxyhypusine synthase inhibitor.
  • such a reaction mixture may include 1 pM eIF5A, 2pM [ 3 H]- spermidine trihydrochloride (38.5 Ci/mmol), 14 pM nicotinamide adenine dinucleotide (NAD + ), 1 mM DTT, various concentrations of the inhibitor, and 24 nM DHPS in 50 mM Tris-HCL buffer, pH 8.0.
  • the enzyme reactions may be performed in buffer containing 1 M eIF5A, 2pM [ 3 H]- spermidine trihydrochloride, and 250 pM NAD + ).
  • Exemplary competitive inhibitors that target deoxyhypusine synthase include a small molecule, a protein, a peptide, a polypeptide, an antibody, a peptide aptamer, a nucleic acid, a nucleic acid aptamer, and the like.
  • a competitive inhibitor that targets deoxyhypusine synthase is a small molecule.
  • one or more allosteric inhibitory compounds may be used to inhibit, modulate, reduce or otherwise interfere with deoxyhypusine synthase by binding to a site different from the active site of deoxyhypusine synthase.
  • the binding allosterically changes the conformation of the active site of deoxyhypusine synthase so that binding of a substrate, such as spermidine, is prevented, inhibited, reduced or weakened or that binding of a co-factor, such as NAD, to deoxyhypusine synthase is prevented, inhibited, reduced or weakened, thereby reducing the enzymatic activity of deoxyhypusine synthase toward the substrate, such as spermidine.
  • allosteric inhibitor means a substance that binds deoxyhypusine synthase at an allosteric site which is different from the active site. Upon binding of the allosteric inhibitor to the allosteric site, deoxyhypusine synthase changes its three- dimensional shape or has a reduced ability to bind or otherwise interact with a co-factor, thereby slowing down its enzymatic activity or otherwise deactivating its enzymatic activity.
  • the allosteric inhibitor inhibits, modulates, prevents, reduces, or otherwise interferes with the activity of deoxyhypusine synthase to enzymatically process the substrate, such as spermidine, and attach the 4-amino butyl moiety from spermidine to the -NH2 of lysine of eIF5A.
  • Allosteric inhibitors can be identified by screening compounds for binding efficiency to deoxyhypusine synthase and determining the effect on active site binding using known assays or modifying known assays for this particular purpose.
  • One exemplary assay is described above with respect to competitive inhibitors.
  • Exemplary allosteric inhibitors that target dcoxyhypusinc synthase include a small molecule, a protein, a peptide, a polypeptide, an antibody, a peptide aptamer, a nucleic acid, a nucleic acid aptamer, and the like.
  • an allosteric inhibitor that targets deoxyhypusine synthase is a small molecule.
  • one or more degradation-promoting compounds may be used to inhibit, modulate, reduce or otherwise interfere with deoxyhypusine synthase by promoting deoxyhypusine synthase degradation through cellular processes such as proteolysis.
  • a degradation-promoting compound such as a proteolysis-targeting compound or “PROTAC”, which is a bifunctional degradation-promoting compound, can be used to increase deoxyhypusine synthase degradation by linking deoxyhypusine synthase to an ubiquitination enzyme, such as E3 ligase, or otherwise proximally locating deoxyhypusine synthase to an ubiquitination enzyme, such as E3 ligase, in order to promote degradation or proteolysis of deoxyhypusine synthase by a cellular proteosome, such as the 26S proteasome.
  • Such degradation-promoting compounds can be identified or designed, for example, by screening bifunctional compounds for binding efficiency to deoxyhypusine synthase, such as the competitive inhibitors and allosteric inhibitors described herein, and also binding efficiency to an ubiquitination enzyme, such as compounds known in the art to bind to the ubiquitination enzyme E3 ligase and for use in designing and making a PROTAC, and determining the degradation of deoxyhypusine synthase using known assays or modifying known assays for this particular purpose.
  • One exemplary assay useful to assess degradation of deoxyhypusine synthase using a degradation-promoting compound includes standard gel electrophoresis I antibody techniques such as Western blotting.
  • any assay designed to measure protein levels can be used to evaluate degradation of deoxyhypusine synthase.
  • exemplary degradation-promoting compounds that target deoxyhypusine synthase to promote degradation include a competitive or allosteric inhibitor described herein linked or otherwise bound to the E3 enzyme.
  • kits for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith are included in kits for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof.
  • a kit may include a pharmaceutically acceptable carrier.
  • a kit may also include instructions for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith.
  • a kit may also comprise, c.g., a buffering agent, a preservative, or a protein stabilizing agent.
  • a kit may also contain a control sample or a series of control samples which can be assayed and compared to a test sample. Other suitable components for including in a kit will be selected given the benefit of this disclosure.
  • one or more systems can be used to inhibit, modulate, reduce or otherwise interfere with cellular production of deoxyhypusine synthase, such as by interfering with transcription of the deoxyribonucleic acid encoding deoxyhypusine synthase or interfering with translation of messenger RNA encoding deoxyhypusine synthase.
  • Such systems are generally known and may utilize nucleic acid modifying agents or systems such as CRISPR-Cas systems, antisense molecules (e.g., interfering RNAs (e.g., small interfering RNAs, short hairpin RNAs), zinc finger nucleases, TALE systems, meganucleases, and ribozymes.
  • Such systems are generally designed to introduce nucleic acid constructs encoding the nucleic acid modifying agent or editor into a cell which is then expressed. The nucleic acid modifying agent or editor then targets a target nucleic acid.
  • Such systems include heterozygous gene knockout systems or homozygous gene knockout systems.
  • a gene is modified, such as by being cut or removed or replaced with a donor nucleic acid sequence through homologous recombination and the effect of the gene loss or inactivation is assayed.
  • Other systems include gene regulating systems using transcriptional repressors or translational repressors or compounds that otherwise repress promoters of a gene or otherwise block the ability of a cell’s transcription machinery to carry out transcription or the cell’s translation machinery to carry out translation. Such systems can be adapted for use after review of the present disclosure.
  • One exemplary system is a CRISPR/Cas system which can be used to target the nucleic acid encoding deoxyhypusine synthase and (1) cut the nucleic acid (in the case of a Cas enzyme) and interfere with expression of the nucleic acid, (2) nick the nucleic acid (in the case of a Cas nickase) and interfere with expression of the nucleic acid, (3) bind to the nucleic acid (in the case of a nuclease null Cas protein) and interfere with expression of the nucleic acid, or (4) bind to the nucleic acid (in the case of a nuclease null Cas protein having a transcriptional repressor attached thereto) and down-regulate expression of the nucleic acid encoding deoxyhypusine synthase.
  • CRISPR/Cas system which can be used to target the nucleic acid encoding deoxyhypusine synthase and (1) cut the nucleic acid (in the case of a Cas
  • a donor sequence can be inserted at the cut or nick site through homologous recombination, for example, to alter the target nucleic acid and interfere with expression of the target nucleic acid.
  • Nucleic acids encoding the Cas protein and/or one or more gRNAs are expressed by the cell and the Cas protein and one or more gRNAs then function to target a target nucleic acid.
  • Such CRISPR/Cas systems can be readily identified or designed after review of the present disclosure, for example by designing one or more gRNAs having a spacer sequence complementary to a protospacer sequence adjacent a protospacer adjacent motif (referred to as a “PAM” sequence) so as to form a colocalization complex with a Cas protein and a target nucleic acid sequence encoding deoxyhypusine synthase (and possibly also including a donor sequence) and then determine reduction of expression or production of deoxyhypusine synthase using known assays or modifying known assays for this particular purpose.
  • PAM protospacer adjacent motif
  • kits for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith are included in kits for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof.
  • a kit may include a pharmaceutically acceptable carrier.
  • a kit may also include instructions for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith.
  • a kit may also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent.
  • a kit may also contain a control sample or a series of control samples which can be assayed and compared to a test sample. Other suitable components for including in a kit can be selected, given the benefit of this disclosure.
  • Zinc-finger nucleases include DNA binding domains that can target the nucleic acid encoding deoxyhypusine synthase. Each zinc finger can recognize codons of the nucleic acid encoding deoxyhypusine synthase, and therefore can be modularly assembled to bind to the nucleic acid encoding deoxyhypusine synthase. These binding domains are coupled with a restriction endonuclease that can cause a double stranded break (DSB) in the nucleic acid encoding deoxyhypusine synthase.
  • DSB double stranded break
  • Repair processes such as nonhomologous end joining, may introduce mutations, such as insertions and/or deletions, that destroy functionality of the nucleic acid encoding deoxyhypusine synthase.
  • a nucleic acid encoding the zinc finger nuclease is expressed by the cell and the zinc finger nuclease then functions to target a target nucleic acid.
  • Such zinc finger nuclease systems can be readily identified or designed after review of the present disclosure, for example by designing a zinc finger nuclease that will bind to the nucleic acid encoding deoxyhypusine synthase and cut the nucleic acid encoding deoxyhypusine synthase and then determine reduction of expression or production of deoxyhypusine synthase using known assays or modifying known assays for this particular purpose.
  • Zinc finger nuclease system components such as a zinc finger nuclease or a nucleic acid encoding a zinc finger nuclease as described herein, are included in kits for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof.
  • a kit may include a pharmaceutically acceptable carrier.
  • a kit may also include instructions for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith.
  • a kit may also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent.
  • a kit may also contain a control sample or a series of control samples which can be assayed and compared to a test sample. Other suitable components for including in a kit can be selected, given the benefit of this disclosure.
  • TALEN transcription activator-like effector nuclease
  • a TALEN includes a DNA binding domain that can target the nucleic acid encoding deoxyhypusine synthase and a nuclease that can cleave the nucleic acid encoding deoxyhypusine synthase. Repair processes, such as nonhomologous end joining may introduce mutations, such as insertions and/or deletions, that destroy functionality of the nucleic acid encoding deoxyhypusine synthase.
  • a nucleic acid encoding the TALEN is expressed by the cell and the TALEN then functions to target a target nucleic acid.
  • Such TALEN systems can be readily identified or designed after review of the present disclosure, for example by designing a TALEN that will bind to the nucleic acid encoding deoxyhypusine synthase and cut the nucleic acid encoding deoxyhypusine synthase and then determine reduction of expression or production of deoxyhypusine synthase using known assays or modifying known assays for this particular purpose.
  • kits for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof may include a pharmaceutically acceptable carrier.
  • a kit may also include instructions for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith.
  • a kit may also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent.
  • a kit may also contain a control sample or a series of control samples which can be assayed and compared to a test sample. Other suitable components for including in a kit can be selected, given the benefit of this disclosure.
  • RNA interference system which can be used to interfere with messenger RNA to prevent transcription or translation.
  • RNA interference systems include those using small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, effective within the RNA interference (RNAi) pathway to degrade mRNA after transcription thereby preventing translation.
  • RNA interference systems include those using microRNA (miRNA), effective within the RNA interference (RNAi) pathway to inhibit translation of mRNA.
  • miRNA microRNA
  • RNAi RNA interference
  • a nucleic acid encoding the RNA is expressed by the cell and the RNA then functions to target a target nucleic acid.
  • RNA interference systems can be readily identified or designed after review of the present disclosure, for example, by designing one or more interfering RNAs (iRNAs) whether siRNAs or miRNAs to interfere with either transcription of the nucleic acid encoding deoxyhypusine synthase or translation of the messenger RNA encoding deoxyhypusine synthase. Reduction of expression or production of deoxyhypusine synthase can be determined using known assays or modifying known assays for this particular purpose.
  • iRNAs interfering RNAs
  • kits for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith are included in kits for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof.
  • a kit may include a pharmaceutically acceptable carrier.
  • a kit may also include instructions for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith.
  • a kit may also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent.
  • a kit may also contain a control sample or a series of control samples which can be assayed and compared to a test sample. Other suitable components for including in a kit can be selected, given the benefit of this disclosure.
  • the present disclosure contemplates treating or preventing a respiratory virus infection in a subject, such as a human subject, in need thereof.
  • Respiratory viruses contemplated by the present disclosure include coronavirus, human rhinovirus, influenza virus, human parainfluenza virus, respiratory syncytial virus, metapneumo virus and the like.
  • the respiratory virus infection is a coronavirus infection.
  • Coronaviruses include, but are not limited to, alphacoronaviruses including human coronavirus 229E (HCoV-229E), and human coronavirus NL63 (HCoV-NL63), and betacoronaviruses including human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKUl), Middle East respiratory syndrome-related coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including variants thereof of the aforementioned alphacoronaviruses and betacoronaviruses. It is to be understood that the disclosure is not limited to particular genera, species, or variants of coronaviruses but includes any and all genera, species and variants of coronaviruses.
  • the respiratory virus infection is a human rhinovirus infection.
  • Rhinoviruses include, but are not limited to, human rhinovirus (HRV) species A, HRV species B and HRV species C, including any types, strains and variants thereof of the aforementioned species of HRV. It is to be understood that the disclosure is not limited to particular species, types, strains or variants of human rhinoviruses but includes any and all species, types, strains and variants of human rhinoviruses.
  • the respiratory virus infection is an influenza virus infection. Influenza viruses include, but arc not limited to, Influenza A virus (IAV) and Influenza B Virus (IBV), including any strains and variants thereof of the aforementioned types of influenza virus. It is to be understood that the disclosure is not limited to particular species, types, strains, or variants of influenza viruses but includes any and all species, types, strains and variants of influenza viruses.
  • the respiratory virus infection is a parainfluenza virus infection.
  • Parainfluenza viruses include, but are not limited to, human parainfluenza virus type 1 (PIV1), human parainfluenza type 2 (PIV2), human parainfluenza type 3 (PIV3), and human parainfluenza type 4 (PIV4), including any strains and variants thereof of the aforementioned types of parainfluenza viruses. It is to be understood that the disclosure is not limited to particular species, types, strains or variants of parainfluenza viruses but includes any and all species, types, strains and variants of parainfluenza viruses.
  • the respiratory virus infection is a respiratory syncytial virus (RSV) infection.
  • Respiratory syncytial viruses include, but are not limited to, RSV sub-types or groups A and B, including any strains and variants thereof of the aforementioned subtypes or groups of respiratory syncytial vims. It is to be understood that the disclosure is not limited to particular sub-types or groups, strains, or variants of respiratory syncytial viruses but includes any and all sub-types or groups, strains and variants of respiratory syncytial viruses.
  • the respiratory virus infection is a metapneumovirus infection.
  • Metapneumoviruses include, but are not limited to, human metapneumovirus genotype or lineage A and human metapneumovirus genotype or lineage B, and sub-groups or sub-lineages Al and A2, and Bl and B2 thereof, including any strains and variants of the aforementioned genotypes, lineages, sub-groups and sub-lineages of human metapneumovirus.
  • the disclosure is not limited to particular genotypes, lineages, sub-groups, sub-lineages, strains or variants of human metapneumoviruses but includes any and all genotypes, lineages, sub-groups, sub-lineages, strains and variants of human metapneumoviruses.
  • Treatment of one or more symptoms associated with respiratory virus infections is intended to include, but is not limited to, the use of a compound, composition, or system described herein to reduce or alleviate one or more symptoms of a respiratory virus infection.
  • the term “symptom associated with a respiratory virus infection” refers to the host’s response to infection by one or more respiratory viruses. Symptoms associated with respiratory virus infections are well known in the art.
  • Such responses include, but are not limited to, coughing, sneezing, runny nose, sore throat, fever, rapid breathing, trouble breathing, wheezing, congestion of nasal sinuses and/or lungs, body aches, fatigue, decrease in appetite, and exacerbation of other underlying respiratory conditions (e.g., asthma, COPD).
  • This list is exemplary only and is not exhaustive, as additional symptoms associated with respiratory virus infections based on the disclosure herein can be identified.
  • efficacy of the treatment and/or prevention methods described herein can be determined.
  • known assays or modified known assays can be used for this particular purpose.
  • common endpoints in the viral respiratory field can be assayed and used to determine whether a treatment is effective, including reduction in viral load (e.g., quantifying viral load in nasal swabs, throat swabs, bronchioalveolar lavage fluid, etc.), reduction in duration of illness, reduction in symptoms (either severity of symptoms or number of symptoms as determined from either a diagnostic point of view of a physician or patient experience/perception), decreased percentage of individuals requiring hospitalization, decreased percentage of individuals requiring supplemental oxygen, decreased number of deaths, decreased intensive care unit (ICU) admissions, reduced number of days in the hospital and/or ICU, etc., and the like.
  • ICU intensive care unit
  • DHPS deoxyhypusine synthase
  • deoxyhypusine synthase includes at least the amino acid sequence: EGSLEREAPAGALAAVLKHSSTLPPESTQVRGYDFNRGVNYRALLEAFGTTGFQATNFG RAVQQVNAMIEKKLEPLSQDEDQHADLTQSRRPLTSCTIFLGYTSNLISSGIRETIRYLVQ HNMVDVLVTTAGGVEEDLIKCLAPTYLGEFSLRGKELRENGINRIGNLLVPNENYCKFE DWLMPILDQMVMEQNTEGVKWTPSKMIARLGKEINNPESVYYWAQKNHIPVFSPALTD GSLGDMIFFHSYKNPGLVLDIVEAQEFDGSDSGARPDEAVSWGKIRVDAQPVKVYADA
  • SEQ ID NO:1 may include an NT-terminal amino acid M at the beginning of SEQ ID NO:1.
  • deoxyhypusine synthase includes at least the amino acid sequence: EGSLEREAPAGALAAVLKHSSTLPPESTQVRGYDFNRGVNYRALLEAFGTTGFQATNFG RAVQQVNAMIEKKLEPLSQDEDQHADLTQSRRPLTSCTIFLGYTSNLISSGIRETIRYLVQ HNMVDVLVTTAGGVEEDLIKCLAPTYLGEFSLRGKELRENGINRIGNLLVPNENYCKFE DWLMPILDQMVMEQNTEGVKWTPSKMIARLGKEINNPESVYYWAQKNHIPVFSPALTD GSLGDMIFFHSYKNPGLVLDIVEAQEFDGSDSGARPDEAVSWGKIRVDAQPVKVYADA SLVFPLLVAETFAQKMDAFMHEKNED. (SEQ ID NO:2).
  • SEQ ID NO:2 may include an N-terminal amino acid M at the beginning of SEQ ID NO:2.
  • deoxyhypusine synthase includes at least the amino acid sequence: PIIPAFWEAEAGGSREEEFETSLANMIEKKLEPLSQDEDQHADLTQSRRPLTSCTIFLGYT SNLISSGIRETIRYLVQHNMVDVLVTTAGGVEEDLIKCLAPTYLGEFSLRGKELRENGINR IGNLLVPNENYCKFEDWLMPILDQMVMEQNTEGVKWTPSKMIARLGKEINNPESVYYW AQKNHIPVFSPALTDGSLGDMIFFHSYKNPGLVLDIVEDLRLINTQAIFAKCTGMIILGGG VVKHHIANANLMRNGADYAVYINTAQEFDGSDSGARPDEAVSWGKIRVDAQPVKVYA DASLVFPLLVAETFAQKMDAFMHEKNED.
  • SEQ ID NO:3 may include an N-terminal amino acid M at the beginning of SEQ ID NO:3.
  • deoxyhypusine synthase includes at least the amino acid sequence: EGSLEREAPAGALAAVLKHSSTLPPESTQVRGYDFNRGVNYRALLEAFGTTGFQATNFG RAVQQVNAMIEKKLEPLSQDEDQHADLTQSRRPLTSCTIFLGYTSNLISSGIRETIRYLVQ HNMVDVLVTTAGGVEEDLIKCLAPTYLGEFSLRGKELRENGINRIGNLLVPNENYCKFE DWLMPILDQMVMEQNTEGVKWTPSKMIARLGKEINNPESVYYWAQKNHIPVFSPALTD GSLGDMTFFHSYKNPGLVLDIVEDLRLTNTQAIFAKCTGMITLGGGVVKHHIANANLMVP DQTRLSPGARSGWMHSPSRSMLTPPWSSPCLWLKPLPRRWMPSCMRRTRTERLRSQEG LTPSSIY. (SEQ ID N0:4).
  • SEQ ID NO:4 may include an N-terminal
  • deoxyhypusine synthase includes at least the amino acid sequence:
  • SEQ ID NO:5 may include an N-terminal amino acid M at the beginning of SEQ ID NO:5.
  • deoxyhypusine synthase includes at least the amino acid sequence: PILDQMVMEQNTEGVKWTPSKMIARLGKEINNPESVYYWAQKNHIPVFSPALTDGSLG DMIFFHSYKNPGLVLDIVEDLRLINTQAIFAKCTGMIILGGGVVKHHIANANLMRNGAD YAVYINTAQEFDGSDSGARPDEAVSWGKIRVDAQPVKVYADASLVFPLLVAETFAQKM DAFMHEKNED. (SEQ ID NO:6).
  • SEQ ID NO:6 may include an N- terminal amino acid M at the beginning of SEQ ID NO: 6.
  • small molecules may be used as competitive inhibitors.
  • An exemplary competitive inhibitor is a poly amine, such as a poly amine having structural similarity to spermidine.
  • Representative competitive inhibitors include N 1 - guanyl-l,7-diaminoheptane, N ’-guanyl- 1.8-diaminooctanc. N’-guanylcaldinc. N 1 - guanylspermidine, or N 8 -guanylspermidine and the like.
  • an exemplary competitive inhibitor is N,N’- bis [3 ,5 -bis [ 1 (aminoiminomethyl)hydrazonoethyl]phenyl] decanediamidetetrahydrochloride also known in the art as CNL1493, AXD455 or semapimod.
  • SMALL MOLECULE ALLOSTERIC INHIBITORS is N,N’- bis [3 ,5 -bis [ 1 (aminoiminomethyl)hydrazonoethyl]phenyl] decanediamidetetrahydrochloride also known in the art as CNL1493, AXD455 or semapimod.
  • small molecules may be used as allosteric inhibitors.
  • an exemplary allosteric inhibitor is
  • a deoxyhypusine synthase inhibitor is an antibody.
  • antibodies or antibody formats may be used as competitive inhibitors or allosteric inhibitors.
  • the term “antibody” is used herein in the broadest sense to refer to molecules with an immunoglobulin-like domain (for example IgG, IgM, IgA, IgD or IgE) and includes monoclonal, recombinant, synthetic, polyclonal, chimeric, human, humanized, multispccific antibodies, including bispccific antibodies, and hctcroconjugatc antibodies; a single variable domain, antigen binding antibody fragments (e.g., Fab, F(ab')2, Fv, disulphide linked Fv, single chain Fv, disulphide-linked scFv, diabodies, TANDAB, etc.) and modified versions of any of the foregoing.
  • Fab fragment antigen binding antibody fragments
  • domain refers to a folded protein structure which retains its tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.
  • single variable domain refers to a folded polypeptide domain comprising sequences characteristic of antibody variable domains.
  • a single variable domain that is capable of binding an antigen or epitope independently of a different variable region or domain may be referred to as a “domain antibody” or “dAB.”
  • a single variable domain may be a human single variable domain, but also includes single variable domains from other species such as rodent, nurse shark and Camelid VHH dABs.
  • Camelid VHH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Such VHH domains may be humanized according to standard techniques available in the art, and such domains are considered to be “single variable domains.” As used herein VH includes camelid VHH domains.
  • “Alternative antibody formats” are those where the CDRs are arranged onto a suitable non-immunoglobulin protein scaffold or skeleton.
  • the non-immunoglobulin scaffold may be a derived from the group consisting of CTLA-4, lipocalin, Protein A derived molecules such as Z-domain of Protein A (Affibody, SpA), A-domain (Avimer/Maxibody); heat shock proteins such as GroEl and GroES; transferrin (trans-body); ankyrin repeat protein (DARPin); peptide aptamer; C-type lectin domain (Tetranectin); human y-cry stallin and human ubiquitin (affilins); PDZ domains; LDL receptor class A domains; EGF domains; scorpion toxin kunitz type domains of human protease inhibitors; and fibronectin/adnectin.
  • a deoxyhypusine synthase inhibitor is an aptamer.
  • Aptamers can block activity of a protein by mechanisms such as hydrogen bonding, electrostatic complementarity, hydrophobic contacts, and steric exclusion.
  • An aptamer may be a peptide aptamer or a nucleic acid aptamer.
  • a dcoxyhypusinc synthase inhibitor is a peptide aptamer.
  • a “peptide aptamer” as used herein is a small combinatorial polypeptide, about 5-20 amino acid residues long, containing one or more short variable peptide domains. Peptide aptamers typically are embedded as a loop within a protein scaffold. Methods of generating, selecting, synthesizing, and administering peptide aptamers are known.
  • a deoxyhypusine synthase inhibitor is a nucleic acid aptamer.
  • a “nucleic acid aptamer” as used herein is a single- stranded nucleic acid molecule, typically about 20-100 bases in length, that folds into a three-dimensional structure that selectively binds to a molecular target (e.g., a small molecule, a protein, a nucleic acid, a cell).
  • Nucleic acid aptamers can be RNA aptamers, DNA aptamers, or XNA aptamers (synthetic xeno nucleic acids, having a different sugar backbone than naturally occurring DNA and RNA). Methods of generating, selecting, synthesizing, and administering nucleic acid aptamers are known.
  • a deoxyhypusine synthase inhibitor is a CRISPR- Cas system.
  • a CRISPR-Cas system may be used as a nucleic acid modifying agent.
  • a “CRISPR-Cas system” as used herein uses a guide RNA (gRNA) complexed with a Cas protein to cleave a nucleic acid molecule encoding deoxyhypusine synthase at specific sites.
  • gRNA guide RNA
  • the Cas protein creates a double-strand break.
  • the Cas protein creates a single-strand break.
  • Cas proteins typically interact with gRNAs via at least one RNA binding or recognition domain.
  • a Cas protein also comprises one or more of a nuclease domain (e.g., RNase or DNase), DNA binding domain, helicase domain, protein-protein interaction domain, or dimerization domain.
  • Cas proteins include a wild-type Cas9 protein, a wild -type Cpfl protein (e.g., FnCpfl), Casl, CaslB, Cast, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, CasF, CasG, CasH, Csy 1 , Csy2, Csy3, Csel (CasA), Cse2 (Cas6), Cse3 (CasE), Cse4 (CasC), Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, C
  • a Cas protein and at least one gRNA form a colocalization complex with a gRNA recognition sequence (also known as a protospacer sequence) in a nucleic acid molecule encoding deoxyhypusine synthase.
  • gRNAs can be designed and synthesized using methods known in the art.
  • a gRNA spacer sequence complementary to a target protospacer sequence includes from about 17 to about 23 nucleotides, from about 18 to about 22 nucleotides, or from about 19 to about 21 nucleotides.
  • a gRNA spacer sequence includes 20 nucleotides.
  • a gRNA recognizes a site in a deoxyhypusine synthase genomic nucleic acid molecule that encodes a deoxyhypusine synthase protein having the amino acid sequence SEQ ID NO: 1. In some embodiments, a gRNA recognizes a site in a deoxyhypusine synthase genomic nucleic acid molecule that encodes a deoxyhypusine synthase protein having the amino acid sequence SEQ ID NO:2. In some embodiments, a gRNA recognizes a site in a deoxyhypusine synthase genomic nucleic acid molecule that encodes a deoxyhypusine synthase protein having the amino acid sequence SEQ ID NO:3.
  • a gRNA recognizes a site in a deoxyhypusine synthase genomic nucleic acid molecule that encodes a deoxyhypusine synthase protein having the amino acid sequence SEQ ID NO:4. In some embodiments, a gRNA recognizes a site in a deoxyhypusine synthase genomic nucleic acid molecule that encodes a deoxyhypusine synthase protein having the amino acid sequence SEQ ID NO:5. In some embodiments, a gRNA recognizes a site in a deoxyhypusine synthase genomic nucleic acid molecule that encodes a deoxyhypusine synthase protein having the amino acid sequence SEQ ID NO:6.
  • cleavage occurs on one or both strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the nucleic acid sequence present in the deoxyhypusine synthase genomic nucleic acid molecule to which a DNA-targeting segment of a gRNA binds.
  • a dcoxyhypusinc synthase inhibitor is a zinc finger nuclease.
  • a zinc finger nuclease or system may be used as a nucleic acid modifying agent in which arrays of zinc finger (ZF) modules, each targeting three DNA bases, are assembled into a zinc finger protein (ZFP). Examples of using ZFPs to target genes are known.
  • a deoxyhypusine synthase inhibitor is a TALE system.
  • TALEs Transcription Activator- Like Effector proteins
  • They contain a nucleic acid binding domain composed of tandem repeats of highly conserved monomer polypeptides, typically 33, 34, or 35 amino acids long, that differ from each other at amino acids 12 and 13 (“repeat variable di-residues” or RVD).
  • RVD refpeat variable di-residues
  • the nucleotide binding affinity of a TALE monomer is determined by the amino acids in its RVD.
  • TALE proteins can be fused to a nuclease domain (e.g., FokI) to generate a TALE system, or TALEN system, for gene editing applications. Examples of using TALE systems to effect gene modifications are known.
  • a deoxyhypusine synthase inhibitor is a meganuclease, i.e., an endodeoxyribonuclease characterized by a large recognition site (e.g., double-stranded DNA sequences of 12-40 base pairs). Examples of using meganucleases are known.
  • a deoxyhypusine synthase inhibitor is an antisense molecule.
  • Antisense molecules are oligonucleotide molecules containing sequence complementarity to target RNA molecules that bind to the target RNA and inhibit its function. Antisense molecules can be used to reduce expression of deoxyhypusine synthase mRNA, thereby reducing levels of deoxyhypusine synthase protein. Any region of deoxyhypusine synthase mRNA can be used as the target region.
  • Antisense molecules include, but are not limited to, single-stranded DNA molecules (antisense oligodcoxyribonuclcotidcs), small interfering RNA (siRNA), ribozymes, and DNAzymes.
  • siRNA small interfering RNA
  • a deoxyhypusine synthase inhibitor is an siRNA.
  • An siRNA (“short interfering RNA”) is a double-stranded RNA that can induce RNAi, thereby inhibiting the activity of a gene, in this case deoxyhypusine synthase.
  • An siRNA can be obtained by chemical synthesis, biochemical synthesis, or biosynthesis. Methods of making and using siRNAs are known.
  • a deoxyhypusine synthase inhibitor is an antisense oligodeoxyribonucleotide.
  • An “antisense oligodeoxyribonucleotide” or “antisense oligonucleotide” is an oligonucleotide in which at least a portion of the nucleotide sequence is complementary to a target nucleic acid and hybridizes to the target nucleic acid. Methods of making and using antisense oligonucleotides are known.
  • a deoxyhypusine synthase inhibitor is a DNAzyme.
  • a “DNAzyme,” also called a DNA enzyme, deoxyribozyme, or catalytic DNA, are non-naturally occurring DNA oligonucleotides that can perform a specific chemical reaction and are often, but not always, catalytic. Methods of making and using DNAzymes are known.
  • a deoxyhypusine synthase inhibitor is a ribozyme.
  • ribozyme also known as an RNA enzyme or catalytic RNA, is an RNA molecule that participates in RNA processing reactions, such as RNA splicing and transfer RNA biosynthesis. Examples of ribozymes include hammerhead ribozymes, hairpin ribozymes, hepatitis delta virus ribozymes, Group I intron ribozymes, RNAse P ribozymes, VS ribozymes, and Leadzymes. Methods of using ribozymes are known. THERAPEUTIC USES
  • the present disclosure provides a use of, or a method of administering, an inhibitor of human deoxyhypusine synthase to treat or prevent respiratory virus infections, such as coronavirus, human rhinovirus, influenza virus, human parainfluenza virus, respiratory syncytial virus, and metapneumo virus infections.
  • respiratory virus infections such as coronavirus, human rhinovirus, influenza virus, human parainfluenza virus, respiratory syncytial virus, and metapneumo virus infections.
  • the present disclosure provides for both prophylactic and therapeutic methods of treating a subject for a respiratory virus infection or symptoms associated therewith.
  • the invention provides a method for preventing in a subject, a respiratory virus infection or symptom associated with a respiratory virus infection by administering to the subject one or more inhibitory compounds, compositions, or systems as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms, such that a respiratory virus infection or symptom associated therewith is prevented or, alternatively, delayed in its progression.
  • prevent includes preventing the severity or frequency of a symptom of a viral respiratory infection, such as coughing, sneezing, runny nose, sore throat, fever, trouble breathing, congestion of nasal sinuses and/or lungs, body aches, and fatigue or preventing the onset of the respiratory virus infection itself.
  • a subject who is at risk of being infected with a respiratory virus infection such as an individual in an at-risk population, such as immunocompromised individuals or individuals having underlying conditions, is provided with a deoxyhypusine synthase inhibitor or composition described herein.
  • a subject who has been exposed to a respiratory virus infection is provided with a deoxyhypusine synthase inhibitor or composition described herein for post-exposure prophylaxis to prevent the severity or frequency of a symptom of a viral respiratory infection, or to prevent the onset of the respiratory virus infection itself.
  • the invention provides a method for treating in a subject, a respiratory virus infection or symptom associated with a respiratory virus infection by administering to the subject one or more inhibitory compounds such as a deoxyhypusine synthase inhibitor, compositions, or systems as described herein.
  • Administration of a treatment agent can occur during or after the manifestation of symptoms, such that a respiratory virus infection or symptom associated therewith is reduced or, alternatively, delayed in its progression.
  • treatment or “treating” refers to ameliorating or stabilizing the specified condition, reducing or eliminating the symptoms of the condition, slowing or eliminating the progression of the condition, and preventing or delaying reoccurrence of the condition in a previously afflicted patient or subject.
  • Treatment or “treating” as used herein includes reducing the severity or frequency of a symptom of a viral respiratory infection, such as coughing, sneezing, runny nose, sore throat, fever, trouble breathing, congestion of nasal sinuses and/or lungs, body aches, and fatigue and/or reducing the respiratory virus infection itself.
  • a symptom of a viral respiratory infection such as coughing, sneezing, runny nose, sore throat, fever, trouble breathing, congestion of nasal sinuses and/or lungs, body aches, and fatigue and/or reducing the respiratory virus infection itself.
  • treatment refers to one or more of reducing viral load (e.g., quantifying viral load in nasal swabs, throat swabs, bronchioalveolar lavage fluid, etc.), reducing duration of illness, reducing symptoms (either severity of symptoms or number of symptoms as determined from either a diagnostic point of view of a physician or patient experience/perception), decreasing percentage of individuals requiring hospitalization, decreasing percentage of individuals requiring supplemental oxygen, decreasing number of deaths, decreasing ICU admissions, reducing number of days in the hospital and/or ICU, etc., and the like.
  • reducing viral load e.g., quantifying viral load in nasal swabs, throat swabs, bronchioalveolar lavage fluid, etc.
  • reducing duration of illness e.g., quantifying viral load in nasal swabs, throat swabs, bronchioalveolar lavage fluid, etc.
  • reducing symptoms either severity of symptoms or number of symptoms as determined from either a diagnostic point of view of a physician or patient
  • the inhibitor of human deoxyhypusine synthase is administered in combination with at least one other (i.e., at least a second) therapeutic agent used to treat symptoms of a respiratory virus infection.
  • agents include, but are not limited to, acetaminophen, nonsteroidal anti-inflammatory drugs, antihistamines, nasal ipratropium, antitussives, steroids, decongestants, and combinations thereof.
  • the inhibitor of human deoxyhypusine synthase is administered in combination with at least one other (i.e., at least a second) therapeutic agent used to treat a respiratory virus infection, as opposed to symptoms of the respiratory virus infection, such as antivirals (such as nucleos(t)ide inhibitors, protease inhibitors, etc.)
  • the inhibitor of human deoxyhypusine synthase and the other therapeutic agent(s) may be administered together or separately and, when administered separately, administration may occur simultaneously or sequentially, in any order. Simultaneous administration may be achieved by administration of (1) a unitary pharmaceutical composition including the therapeutic agents; or (2) simultaneous administration of separate pharmaceutical compositions each including one of the therapeutic agents. Alternatively, the combination may be administered separately in a sequential manner wherein one treatment agent is administered first and the other second or vice versa. Such sequential administration may be close in time or remote in time.
  • An inhibitor of deoxyhypusine synthase may be administered by any convenient route.
  • the inhibitor of deoxyhypusine synthase may be administered orally, parenterally, intranasally, or by inhalation.
  • the inhibitor of deoxyhypusine synthase is administered in a pharmaceutical composition.
  • the inhibitor of deoxyhypusine synthase is formulated in a pharmaceutical composition adapted for oral or parenteral administration, or for administration intranasally or by inhalation.
  • the invention provides a pharmaceutical composition comprising an inhibitor of deoxyhypusine synthase and a pharmaceutically acceptable excipient.
  • the invention provides a process for the preparation of a pharmaceutical composition comprising admixing an inhibitor of deoxyhypusine synthase with a pharmaceutically acceptable excipient.
  • compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.
  • compositions adapted for nasal administration can comprise a coarse powder having a particle size for example in the range 20 to 500 microns that is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the inhibitor of deoxyhypusine synthase.
  • Fine particle dusts or mists which may be generated by means of various types of metered, dose pressurized aerosols, nebulizers or insufflators, all of which are well known in the art.
  • compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain anti-oxidants, buffers, l ' l bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets using known techniques.
  • formulations described herein may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
  • a deoxyhypusine synthase inhibitor and one or more other therapeutic agent(s) may be administered together.
  • the dose of each therapeutic agent may differ from the dose of that therapeutic agent when used alone.
  • a deoxyhypusine synthase inhibitor may be administered by an effective route, according to the type of inhibitor.
  • the deoxyhypusine synthase inhibitor may be administered by an injection for example subcutaneously or intravenously.
  • a pharmaceutical composition comprising a deoxyhypusine synthase inhibitor and a pharmaceutically acceptable excipient.
  • the present disclosure provides a process for the preparation of a pharmaceutical composition comprising admixing a deoxyhypusine synthase inhibitor with a pharmaceutically acceptable excipient.
  • compositions may be presented as units such as solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.
  • the inhibitor is present in an aqueous solution.
  • compositions adapted for injection include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example scaled ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • the present disclosure also provides unitary pharmaceutical compositions in which a deoxyhypusine synthase inhibitor and one or more other therapeutic agent(s) disclosed herein may be administered in combination, concurrently or sequentially.
  • a method of treating or preventing a respiratory virus infection or symptom associated therewith includes the step of administering a therapeutically and/or prophylactically effective amount of a compound or system to a subject, such as a human subject, in need of such treatment or prevention.
  • a subject is administered a therapeutically and/or prophylactically effective amount that is effective to treat or prevent a respiratory virus infection or symptom associated therewith.
  • therapeutically effective amount refers to the quantity of a compound or a pharmaceutically acceptable salt thereof, which will elicit the desired biological response in an animal or human body. It may vary depending on the compound, the disease and its severity and the age and weight of the subject to be treated.
  • Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the respiratory virus infection or symptom associated therewith, previous treatments, the general health and/or age of the subject, and other diseases present.
  • Treatment of a subject with a therapeutically and/or prophylactically effective amount of a compound or system can include a single treatment or can include a series of treatments. It will also be appreciated that the effective dosage used for treatment or prevention may increase or decrease over the course of a particular treatment.
  • MRC-5 human epithelial cells were seeded at a density of 5 x 10 3 per well in 96-well tissue culture plates and allowed to adhere overnight. The following day, six different dilutions of Compound 1, Compound 2, and Compound 3 ranging from about 10 nM to 500 pM, i.e., 50 pM, 15.8 pM, 5 pM, 1.58 pM, 0.5 pM and 0.16 pM were added for a pre-treatment period of three hours at 37 °C and 5 % CO2.
  • Human coronavirus strain 229E HCV-229E
  • diluted to a pre-determined titre to yield 85 % to 95 % killing at six days post-infection was added to the plates.
  • Cell viability was measured by XTT tetrazolium dye staining following six days of incubation at 37 °C and 5 % CO2, with optical density measured spectrophotometrically at 450 nm and 650 nm using SOFTMAX Pro 4.6 software. Cellular toxicity due to compound treatment alone was measured in parallel with uninfected cells. The concentrations of compound that reduced the CPE by 50% (EC 50) and uninfected cell viability by 50% (CC50) were calculated using parameter curve fit analysis, and are provided in Table 1 below for HCoV-229E.
  • Hl-HeLa human epithelial cells were seeded at a density of 5 x 10 per well in 96-well tissue culture plates and allowed to adhere overnight. The following day, six different dilutions of Compound 1, Compound 2, and Compound 3 ranging from about 10 nM to 500 pM, i.e., 50 pM, 15.8 pM, 5 pM, 1.58 pM, 0.5 pM and 0.16 pM were added for a pre-treatment period of three hours at 37 °C and 5 % CO2. Human rhinovirus type 16 strain 11757 (HRV 16), diluted to a pre-determined titre to yield 85 % to 95 % killing at six days post-infection, was added to the plates.
  • HRV 16 Human rhinovirus type 16 strain 11757
  • RPMI2650 cells were seeded at a density of 5 x 10 3 per well in 96-well tissue culture plates and allowed to adhere overnight. The following day, the cells were washed and six different dilutions of Compound 1, Compound 2, and Compound 3 ranging from about 10 nM to 500 pM, i.e., 50 pM, 15.8 pM, 5 pM, 1.58 pM, 0.5 pM and 0.16 pM were added for a pretreatment period of three hours at 37 °C and 5 % CO2.
  • Human lung epithelial A549 cells were harvested, counted, and resuspended in phenol red-free DMEM medium containing 10% FBS, HEPES, glutamine and pyruvate.
  • PIV3-GFP virus was applied to the cells at a target multiplicity- of-infection (MOI) to yield 40-60% infection of cells by GFP fluorescence two days after infection.
  • MOI target multiplicity- of-infection
  • the cells were fixed in 2% paraformaldehyde containing 10 pM Hoechst dye. After one-hour incubation, plates were washed three times with PBS and imaged at 4x using blue and green fluorescence channels on a PerkinElmer ENSIGHT imager. Hoechst-stained nuclei were identified using a segmentation algorithm. GFP-positive nuclei were quantified using a minimum fluorescence threshold determined from positive controls. The maximum and minimum GFP positive nuclei were calculated from positive and negative controls and used to normalize data for putative inhibitors against PIV3. EC50 values were calculated by fitting a sigmoidal variable slope nonlinear regression model to the data.
  • nuclei counts were normalized to positive controls for EC50 calculation.
  • the EC50 data and CC50 data are provided in Table 4 below for PIV3-GFP. indicates that a reduction in CPE by 50% was not achieved over the compound concentration range tested.
  • HEp-2 human epithelial cells were seeded at a density of 5 x 10 3 per well in 96-well tissue culture plates and allowed to adhere overnight. The following day, six different dilutions of Compound 1, Compound 2, and Compound 3 ranging from about 10 nM to 500 pM, i.e., 50 pM, 15.8 pM, 5 pM, 1.58 pM, 0.5 pM and 0.16 pM were added for a pre-treatment period of three hours at 37 °C and 5 % CCh.
  • Human RSV strain A2 (RSV A2), diluted to a pre -determined titre to yield 85 % to 95 % killing at six days post-infection, was added to the plates.
  • LLC-MK2 American Type Culture Collection CCL-7 cells are seeded at an appropriate density per well in 96-well tissue culture plates and allowed to adhere overnight. The following day, dilutions of Compound 1, Compound 2, and Compound 3 ranging from about 10 nM to 500 p M are added for a pre-treatment period of three hours at 37 °C and 5 % CO2. Human mctapncumovirus, diluted to an appropriate prc-dctcrmincd titre, is added to the plates.
  • Cell viability is measured by XTT tetrazolium dye staining following four to seven days of incubation at 37 °C and 5 % CO2, with optical density measured spectrophotometrically at 450 nm and 650 nm using SOFTMAX Pro 4.6 software. Cellular toxicity due to compound treatment alone is measured in parallel with uninfected cells. The concentrations of compound that reduced the CPE by 50% (EC50) and uninfected cell viability by 50% (CC50) are calculated using parameter curve fit analysis, and the EC50 data and CC50 data are collected.
  • qPCR Quantitative PCR
  • ACTB housekeeping gene beta-actin
  • Ct Threshold cycle
  • a CRISPR-Cas system was used to genetically knock out deoxyhypusine synthase in cells and the effect of deoxyhypusine synthase genetic knock out on viral infection/replication of respiratory viruses was determined.
  • Genome-wide CRISPR knockout (KO) screens were performed in cellular infection models for human rhinovirus (HRV), parainfluenza virus (PIV), respiratory syncytial virus (RSV) and Influenza A vims (IAV).
  • CRISPR library generation was performed as previously described. Briefly, CRISPR KO sgRNA libraries were introduced into cell lines via lentivirus transduction. Cas9/sgRNA expressing cells were selected via antibiotic resistance and expanded. For each screening experiment, cells were infected with virus and enriched for a specific phenotype of interest (susceptibility/resi stance to virus infection-dependent gene expression and/or virus- induced cytopathic effect.) Details of each cellular infection model are described in Table 7 below.
  • NGS Next-generation-sequencing
  • Deoxyhypusine synthase was a statistically significant gene-level hit (MAGeCK score p-value ⁇ 0.001) in the genome-wide and/or focused custom CRISPR KO screens for each virus tested.
  • the CRISPR screen results are summarized in Table 8 and Table 9 below. “X” denotes that the gene was a statistically significant screen hit (p-value ⁇ 0.001).
  • CRISPR editing of deoxyhypusine synthase was performed by transducing cells with Cas9/sgRNA expressing lentivirus.
  • Non-targeting (NT) sgRNA sequences were used where indicated as negative controls.
  • a list of sgRNA sequences is provided in Table 10 below.
  • Hl-HeLa cells were plated in 96- well plates and infected with HRV at various multiplicities of infection (MOI). Viral infection was measured by determining the level of virus-induced cytopathic effect using the CELLTITER-GLO (CTG) viability assay (Promega) and measuring luminescence signal in infected wells relative to uninfected controls. The impact of deoxyhypusine synthase gene perturbation on viral infection was measured by determining relative survival in deoxyhypusine synthase KO cells compared to NT controls.
  • CCG CELLTITER-GLO
  • influenza virus A549 cells were plated in 96-well plates and infected with IAV. The impact of deoxyhypusine synthase gene perturbation on IAV infection was measured by determining the percentage of cells that were positive for the influenza viral protein nucleoprotein (NP) in deoxyhypusine synthase KO cells compared to NT controls.
  • NP influenza viral protein nucleoprotein
  • CRISPR knockout experiments cells were infected in suspension whereas in the knockdown experiments, cells were infected 48 hours after siRNA transfection.
  • A549 cells were plated and infected with PIV-GFP at various MOI.
  • the impact of deoxyhypusine synthase gene perturbation on viral infection was measured by determining the percentage of cells that were positive for GFP expression in deoxyhypusine synthase KO cells compared to NT controls.
  • HEp-2 cells were plated in 96-well plates and infected with RSV-GFP.
  • the impact of deoxyhypusine synthase gene editing on RSV infection was measured either by calculating the percentage of cells that were positive for GFP or relative survival (using the CTG viability assay) in deoxyhypusine synthase KO cells compared to NT controls.
  • Results of the individual genetic perturbation experiments are summarized in Tabic 11 below.
  • Deoxyhypusine synthase CRISPR knockout cell lines displayed resistance to virus infection validating deoxyhypusine synthase as a target for the treatment or prevention of a respiratory virus infection.
  • Results in CRISPR KO cells for rhinovirus and parainfluenza virus are shown in Fig. 2 and Fig. 3.
  • Fig. 2 is directed to evaluating HRV infection (human rhinovirus 16) in CRISPR-mediated deoxyhypusine synthase knockout cell lines.
  • Deoxyhypusine synthase knockout cells compared to NT (non-target) controls show increased resistance to human rhinovirus-induced cytopathic effect.
  • Fig. 3 is directed to evaluating PIV infection (Parainfluenza virus 3 strain JS) in CRISPR-mediated deoxyhypusine synthase knockout cell lines.
  • Deoxyhypusine synthase knockout cells show decreased percentage of Parainfluenza Virus Type 3 (PIV)-GFP positive cells compared to NT controls.
  • sgRNAs targeting SLC35A1 were used as a positive control against PIV (SLC35A1) to evaluate the effect of known pro-pathogen genes in this infection model.
  • EXAMPLE 9 Embodiments
  • the present disclosure provides a method of treating or preventing a respiratory virus infection or one or more symptoms associated therewith or inhibiting or reducing viral protein synthesis, in a human subject in need thereof, which includes inhibiting activity of deoxyhypusine synthase within the human subject or decreasing an amount of deoxyhypusine synthase within the human subject in a manner to reduce the respiratory virus infection or one or more symptoms associated therewith.
  • inhibiting activity of deoxyhypusine synthase within the human subject is carried out by administering to the human subject an effective amount of a competitive inhibitor that binds to an active site of deoxyhypusine synthase and inhibits enzymatic activity of deoxyhypusine synthase.
  • the competitive inhibitor is a molecule that competes for binding with a molecule having structural similarity to spermidine and that binds to the active site of deoxyhypusine synthase and inhibits enzymatic activity of deoxyhypusine synthase.
  • inhibiting activity of deoxyhypusine synthase within the human subject is carried out by administering to the human subject an effective amount of an allosteric inhibitor that binds to deoxyhypusine synthase and inhibits enzymatic activity of deoxyhypusine synthase.
  • reducing the amount of deoxyhypusine synthase within the human subject is carried out by administering an effective amount of a degradation-promoting compound to the human subject that binds to deoxyhypusine synthase and promotes degradation of deoxyhypusine synthase.
  • the deoxyhypusine synthase includes an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO: 6.
  • the respiratory virus infection is caused by a coronavirus, a human rhinovirus, an influenza virus, a human parainfluenza virus, a respiratory syncytial virus or a human metapneumovirus.
  • reducing the respiratory virus infection is carried out by reducing viral load.
  • the competitive inhibitor is administered by inhalation, intranasally, transdermally, orally, rectally, transmucosally, intestinally, parenterally, intramuscularly, subcutaneously or intravenously.
  • the allosteric inhibitor is administered by inhalation, intranasally, transdermally, orally, rectally, transmucosally, intestinally, parenterally, intramuscularly, subcutaneously or intravenously.
  • the degradation-promoting compound is administered by inhalation, intranasally, transdermally, orally, rectally, transmucosally, intestinally, parenterally, intramuscularly, subcutaneously or intravenously.
  • the present disclosure provides a composition including at least a deoxyhypusine synthase inhibitor and a pharmaceutically acceptable excipient.
  • the deoxyhypusine synthase inhibitor is a competitive inhibitor, an allosteric inhibitor or a degradation-promoting compound.
  • the composition is provided as a single dose unit including an inhibitory effective amount of the deoxyhypusine synthase inhibitor.
  • the present disclosure provides use of a deoxyhypusine synthase inhibitor in the manufacture of a medicament for the treatment or prevention of a respiratory virus infection or one or more symptoms associated therewith or for reducing viral protein synthesis.
  • the deoxyhypusine synthase inhibitor is a competitive inhibitor, an allosteric inhibitor or a degradation-promoting compound.
  • the medicament is provided as a single dose unit including an inhibitory effective amount of the deoxyhypusine synthase inhibitor.
  • the present disclosure provides a deoxyhypusine synthase inhibitor for use in the treatment or prevention of a respiratory virus infection or one or more symptoms associated therewith or for use in inhibiting or reducing viral protein synthesis.
  • the treatment or prevention includes inhibiting activity of deoxyhypusine synthase or decreasing an amount of deoxyhypusine synthase in a manner to reduce the respiratory virus infection or one or more symptoms associated therewith.
  • the deoxyhypusine synthase inhibitor is a competitive inhibitor that competes for binding with a molecule having structural similarity to spermidine and binds to an active site of deoxyhypusine synthase and inhibits enzymatic activity of deoxyhypusine synthase.
  • the deoxyhypusine synthase inhibitor is an allosteric inhibitor that binds to deoxyhypusine synthase and inhibits enzymatic activity of deoxyhypusine synthase.
  • the deoxyhypusine synthase inhibitor is a degradation-promoting compound that binds to deoxyhypusine synthase and promotes degradation of deoxyhypusine synthase.
  • the deoxyhypusine synthase inhibitor is a competitive inhibitor, an allosteric inhibitor or a degradation-promoting compound.
  • the deoxyhypusine synthase includes an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.
  • the respiratory virus infection is caused by a coronavirus, a human rhinovirus, an influenza virus, a human parainfluenza virus, a respiratory syncytial virus or a human mctapncumovirus.
  • the treatment or prevention includes reducing the respiratory virus infection by reducing viral load.
  • the deoxyhypusine synthase inhibitor is administered by inhalation, intranasally, transdermally, orally, rectally, transmucosally, intestinally, parenterally, intramuscularly, subcutaneously or intravenously.

Abstract

The disclosure relates to deoxyhypusine synthase (DHPS) inhibitors for use in treating or preventing respiratory virus infections, such as infection with coronaviruses, human rhinoviruses, influenza viruses, human parainfluenza viruses, respiratory syncytial viruses or human metapneumoviruses.

Description

INHIBITORS OF DEOXYHYPUSINE SYNTHASE FOR THE TREATMENT AND PREVENTION OF RESPIRATORY VIRUS INFECTIONS
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The subject application claims priority to U.S. Patent Application No. 63/345,008 filed on May 23, 2022, the entirety of which is incorporated herein by reference.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 16, 2023, is named 054624-09-5045- WO_Sequence_Listing.xml and is 16,457 bytes in size.
FIELD OF THE DISCLOSURE
[0003] The present disclosure relates to the field of respiratory virus infections and modulation of deoxyhypusine synthase (DHPS) for the treatment or prevention of a respiratory virus infection. The present disclosure also relates to compounds, compositions and systems for use in such methods and kits related thereto.
BACKGROUND OF THE DISCLOSURE
[0004] Respiratory infections are a major cause of morbidity and mortality for humans. A majority of these infections are caused by respiratory viruses such as coronaviruses, human rhinoviruses, influenza viruses, human parainfluenza viruses, respiratory syncytial viruses or human metapneumoviruses.
[0005] Coronaviruses contain an enveloped single- strand, positive-sense RNA genome of between 26 and 32 kilobases in size. They belong to the Coronaviridae family and are classified by phylogenetic similarity into four categories or genera: alpha, beta, gamma, and delta. Seven coronaviruses have been associated with human disease to date. These are the alphacoronaviruses 229E and NL63, and the betacoronaviruses OC43, HKU1, MERS-CoV, SARS-CoV, and SARS- CoV-2. People are frequently infected with human coronaviruses 229E, NL63, OC43 and HKU1. Symptoms usually include mild-to-moderate upper-respiratory tract illnesses of short duration, such as runny nose, cough, sore throat, and fever which are symptoms consistent with a common cold. MERS-CoV, SARS-CoV, and SARS-CoV-2 infection can cause severe human illness. Coronaviruses utilize a membrane bound spike protein to bind to a host cell surface receptor in order to gain cellular entry. Following entry into the host cell, the RNA genome is translated into two large polypeptides by the host ribosomal machinery. The polypeptides are processed by two proteases, the coronavirus main protease (Mpro / 3CLpro) and a papain-like protease to generate the proteins required for viral replication and packaging. Although vaccines and therapeutics have been developed for selected coronaviruses, notably for SARS-CoV-2, the emergence of novel variants with altered susceptibility to these medicines necessitates the discovery of new prophylaxis and treatment options.
[0006] Human Rhinovirus (HRV) is a positive-strand RNA virus from the Enterovirus genus of the Picornaviridae family. Other viruses from this family that are important in the context of human health include Poliovirus, Coxsackievirus, Enterovirus D68, and Enterovirus A71. HRV is classified into three species (A, B and C) comprising more than 100 genotypes. HRV infection is a significant cause of the common cold, however it can also lead to more serious illness, particularly in individuals with underlying respiratory disease. HRV has been identified as the most frequently isolated virus in exacerbations of asthma in adults and children, as well as in exacerbations of chronic obstructive pulmonary disease (COPD). Currently there are no vaccines or medicines available for the specific prevention and treatment of HRV infection. In light of this, there is a need for new therapeutics for the prophylaxis and treatment of infection by HRV and other enteroviruses.
[0007] Influenza Virus is an enveloped, negative-sense, single- stranded RNA virus of the Orthomyxoviridae family. There are four types, namely A, B, C and D. Influenza A Virus and Influenza B Virus are associated with the seasonal human symptomatic respiratory illness influenza (“flu”). Influenza A viruses are divided into subtypes based on their hemagglutinin (H) and neuraminidase (N) surface proteins. The severity of influenza disease in infected individuals is highly variable, with children and older adults generally experiencing more severe outcomes. Disease burden is significant with median incidence among all age groups estimated to be 8.3% in the United States. Influenza virus has also been associated with several pandemics, most notably the influenza pandemic of 1918 (“Spanish Flu”), which was caused by the H INI influenza A virus strain. Seasonal vaccines and direct-acting antiviral agents have been approved for the prevention and treatment of influenza virus infection. However, there is an ongoing need for the development of new therapeutics owing to antigenic changes in the virus and the emergence of resistant isolates, as well as for pandemic preparedness purposes.
[0008] Human Parainfluenza Virus (PIV) is a negative-sense, single- stranded RNA virus of the family Paramyxoviridae . PIV is a generally common infection. In the United States, approximately one-half of children aged one year and almost all children aged six years have been infected with at least one type of PIV. PIV s are the second most common cause of acute respiratory illness leading to hospitalization in children under five years of age. No specific treatment is available for PIV infection.
[0009] Human Respiratory Syncytial Virus (RSV) is a negative-sense, single- stranded RNA virus of the family Pneumoviridae. Other members of this family are also important mammalian pathogens, such as human metapneumo virus (hMPV). RSV is a leading cause of respiratory tract infection in infants and the elderly. Over 33 million cases of RSV-associated acute lower respiratory tract infection in children under five years of age were estimated globally in 2015, resulting in three million hospitalizations and almost 60,000 in-hospital. There are no approved vaccines for RSV. Passive immunization is achievable via prophylactic administration of a humanized monoclonal antibody (palivizumab) directed to an epitope of the RSV Fusion protein. The only approved therapeutic for RSV is ribavirin, which is administered via aerosol treatment. Evidence for ribavirin’s efficacy is limited and it has an unfavorable safety profile. Human metapneumovirus, which was discovered in 2001, can cause upper and lower respiratory disease in people of all ages, especially among young children, older adults, and people with weakened immune systems. Like RSV, hMPV is an enveloped, negative-sense, single-stranded RNA virus. Two major genotypes or lineages, termed A and B, have been identified, each comprising two sub-groups or sub-lineages, namely Al and A2, and Bl and B2. There is currently no specific antiviral therapy or vaccine for hMPV. Given the burden of disease and lack of adequate treatment options, there is a need for new therapeutics for the prophylaxis and treatment of infection by RSV and other pneumoviruses, such as human metapneumovirus.
SUMMARY OF THE DISCLOSURE
[0010] One aspect of the disclosure is a method of treating or preventing a respiratory virus infection or one or more symptoms associated therewith or inhibiting or reducing viral protein synthesis, in a human subject in need thereof, comprising inhibiting activity of deoxyhypusine synthase within the human subject or decreasing an amount of deoxyhypusine synthase within the human subject in a manner to reduce the respiratory virus infection or one or more symptoms associated therewith.
[0011] Another aspect of the disclosure is a composition comprising a deoxyhypusine synthase inhibitor and a pharmaceutically acceptable excipient.
[0012] Another aspect of the disclosure is use of a deoxyhypusine synthase inhibitor in the manufacture of a medicament for the treatment or prevention of a respiratory virus infection or one or more symptoms associated therewith or for reducing viral protein synthesis.
[0013] Another aspect of the disclosure is a deoxyhypusine synthase inhibitor for use in the treatment or prevention of a respiratory virus infection or one or more symptoms associated therewith or for use in inhibiting or reducing viral protein synthesis.
[0014] These and other aspects, objects, features, and advantages of the example embodiments will become apparent upon consideration of the following detailed description of example embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The foregoing and other features and advantages of the present disclosure will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.
[0016] Fig. 1 is a schematic illustrating the process within a cell of synthesizing hypusine within precursor eIF5A to form mature eIF5A including hypusine.
[0017] Fig. 2 is a graph showing that deoxyhypusine synthase knockout cells compared to NT (non-target) controls show reduced susceptibility to human rhinovirus (HRV)- induced cytopathic effects. Results are presented as the mean values of four independent experiments. Error bars represent standard error of the mean (SEM). * = p-value <0.05 compared to NT1 and NT2 as determined by one-way ANOVA with Tukey comparison.
[0018] Fig. 3 is a graph showing that deoxyhypusine synthase knockout cells show decreased percentage of Parainfluenza Virus Type 3 (PIV)-GFP positive cells compared to NT controls. Results are presented as the mean values of four independent experiments. Error bars represent standard error of the mean (SEM). * = p-value <0.05 compared to NT1 and NT2 as determined by one-way ANOVA with Tukey comparison. DETAILED DESCRIPTION OF THE DISCLOSURE
[00019] Eukaryotic translation initiation factor 5A (“cIF5A”) is a protein associated with protein biosynthesis and peptide bond formation and, accordingly, protein synthesis within a eukaryotic cell that is important for cell viability and proliferation. In humans, two isoforms of eIF-5A have been described: eIF5A- 1 and eIF5A-2. Each are encoded by two distinct genes EIF5A and EIF5A2. eIF5A is the only known protein in a eukaryotic cell that includes the uncommon amino acid hypusine. Hypusination is the process of altering lysine to include a hypusine residue believed to be critical for e!F5A function. The region surrounding the hypusine residue is highly conserved and is believed essential to the function of eIF5A.
[00020] The enzyme deoxyhypusine synthase is involved in the hypusination pathway. With reference to Fig. 1, an eIF5A precursor includes a lysine (K50) that is hypusinated via the hypusination pathway to provide mature eIF5A. Deoxyhypusine synthase uses spermidine as a substrate in the presence of NAD to transfer a 4-amino butyl moiety from spermidine to the terminal -NH2 of lysine to create an intermediate form of eIF5A with deoxyhypusine attached to the -NH2 of lysine. The 4-amino butyl moiety attached to the terminal -NH2 of lysine is then hydroxylated by deoxyhypusine hydroxylase to result in mature eIF5A including hypusine.
[00021] Embodiments of the present disclosure are directed to methods, compounds, compositions, systems, medicaments and kits for the treatment or prevention of a respiratory infection associated with a virus (referred to herein as a “respiratory virus infection”), or one or more symptoms associated therewith, or to inhibit, modulate, reduce or otherwise interfere with deoxyhypusine synthase within a cell, such as a human cell within a subject, in need of such treatment or prevention. Aspects of the present disclosure include inhibiting, modulating, reducing or otherwise interfering with the activity of deoxyhypusine synthase to add deoxyhypusine to the terminal -NH2 of lysine of eIF5A using spermidine as a substrate. Without wishing to be bound by scientific theory, inhibiting, modulating, reducing or otherwise interfering with the activity of deoxyhypusine synthase to add deoxyhypusine to the terminal -NH2 of lysine of eIF5A limits or reduces the ability of deoxyhypusine synthase to enable respiratory virus replication within a cell.
[00022] Embodiments of the present disclosure are directed to methods of treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof. The term "subject" refers to an animal or human body. According to one aspect and as further described herein, activity within the subject of deoxyhypusine synthase is inhibited, modulated, reduced or otherwise interfered with in a manner to inhibit, modulate, reduce or otherwise interfere with the respiratory virus infection or one or more symptoms associated therewith.
[00023] According to one aspect and as further described herein, activity within a cell, such as a human cell, of deoxyhypusine synthase is inhibited, modulated, reduced or otherwise interfered with by introducing to the cell one or more competitive inhibitor compounds to bind to an active site of deoxyhypusine synthase. According to one aspect and as further described herein, activity within a cell, such as a human cell, of deoxyhypusine synthase is inhibited, modulated, reduced or otherwise interfered with by introducing to the cell one or more allosteric inhibitor compounds to bind to deoxyhypusine synthase in a manner to alter an active site of deoxyhypusine synthase. According to these aspects, the cell may be in vitro, ex vivo or in vivo. To the extent the cell is in vivo, the method may include treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof.
[00024] According to one aspect and as further described herein, activity within a cell, such as a human cell, of deoxyhypusine synthase is inhibited, modulated, reduced or otherwise interfered with by introducing to the cell one or more degradation-promoting compounds to promote deoxyhypusine synthase degradation through cellular processes such as proteolysis. According to this aspect, cellular concentration of deoxyhypusine synthase is reduced, resulting in reduced activity of deoxyhypusine synthase in the cell. According to this aspect, the cell may be in vitro, ex vivo or in vivo. To the extent the cell is in vivo, the method may include treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof.
[00025] According to one aspect and as further described herein, activity within a cell, such as a human cell, of deoxyhypusine synthase is inhibited, modulated, reduced or otherwise interfered with by introducing to the cell one or more systems for inhibiting, modulating, reducing or otherwise interfering with transcription of a nucleic acid encoding deoxyhypusine synthase. According to one aspect and as further described herein, activity within a cell, such as a human cell, of deoxyhypusine synthase is inhibited, modulated, reduced or otherwise interfered with by introducing to the cell one or more systems for inhibiting, modulating, reducing or otherwise interfering with translation of messenger RNA encoding deoxyhypusine synthase. According to these aspects, cellular production of deoxyhypusine synthase is inhibited, modulated, reduced or otherwise interfered with. According to these aspects, the cell may be in vitro, ex vivo or in vivo. To the extent the cell is in vivo, the method may include treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof.
[00026] According to certain aspects, the present disclosure contemplates the use of deoxyhypusine synthase in the identification of active agents and systems and the manufacture of a medicament for the treatment or prevention of a respiratory virus infection. Such active agents, systems and medicaments can be included in a kit, as further described herein.
[00027] According to one aspect, one or more competitive inhibitory compounds may be used to inhibit, modulate, prevent, reduce or otherwise interfere with the ability of deoxyhypusine synthase to transfer the 4-amino butyl moiety from spermidine to the -NH2 of lysine of eIF5A, such as by competing with binding of spermidine or a compound having a structure similar to spermidine or sterically inhibiting the active site for binding to a substrate, such as a spermidine substrate, which would otherwise be subject to enzymatic activity. The term “competitive inhibitor” as used herein means a substance that competes with the binding of a substrate to the active site of deoxyhypusine synthase or otherwise binds to the active site of deoxyhypusine synthase and inhibits, modulates, prevents, reduces or otherwise interferes with the activity of deoxyhypusine synthase to enzymatically process a substrate for deoxyhypusine synthase, such as spermidine, and attach the 4-amino butyl moiety from spermidine to the -NH2 of lysine of eIF5A.
[00028] According to this aspect, binding of the substrate, such as a spermidine substrate, is prevented, inhibited, reduced or weakened thereby reducing the enzymatic activity of deoxyhypusine synthase toward the substrate, such as spermidine. Competitive inhibitors can be identified by screening compounds for binding efficiency to the active site of deoxyhypusine synthase using known assays or modifying known assays for this particular purpose. One exemplary assay useful to assess inhibition of transfer of the 4-aminobutyl group from spermidine to eIF5A by deoxyhypusine synthase is carried out by using a reaction mixture including eIF5A, [3H]- spermidine trihydrochloride, nicotinamide adenine dinucleotide (NAD+), DTT, and various concentrations of deoxyhypusine synthase inhibitor. For example, such a reaction mixture may include 1 pM eIF5A, 2pM [3H]- spermidine trihydrochloride (38.5 Ci/mmol), 14 pM nicotinamide adenine dinucleotide (NAD+), 1 mM DTT, various concentrations of the inhibitor, and 24 nM DHPS in 50 mM Tris-HCL buffer, pH 8.0. For analysis of higher concentrations of NAD+, the enzyme reactions may be performed in buffer containing 1 M eIF5A, 2pM [3H]- spermidine trihydrochloride, and 250 pM NAD+). After a 120-minute incubation at room temperature, incorporation of radio-labeled aminobutylidene is terminated by the addition of 20 pL of 300 pM GC-7 ; 25 pL of the stopped reaction is transferred to a streptavidin (SA) plate (NeutrAvidin Coated Plates, Thermo Scientific), and is incubated for 90-120 minutes at room temperature to allow complete binding of all proteins to the plates. Plates are washed three times with PBS, and 50pL of scintillation cocktail is added (OptiPhase SUPERMIX from PerkinElmer). The plate is measured with TPO count (PerkinElmer). Exemplary competitive inhibitors that target deoxyhypusine synthase include a small molecule, a protein, a peptide, a polypeptide, an antibody, a peptide aptamer, a nucleic acid, a nucleic acid aptamer, and the like. In one embodiment, a competitive inhibitor that targets deoxyhypusine synthase is a small molecule.
[00029] According to one aspect, one or more allosteric inhibitory compounds may be used to inhibit, modulate, reduce or otherwise interfere with deoxyhypusine synthase by binding to a site different from the active site of deoxyhypusine synthase. According to one aspect, the binding allosterically changes the conformation of the active site of deoxyhypusine synthase so that binding of a substrate, such as spermidine, is prevented, inhibited, reduced or weakened or that binding of a co-factor, such as NAD, to deoxyhypusine synthase is prevented, inhibited, reduced or weakened, thereby reducing the enzymatic activity of deoxyhypusine synthase toward the substrate, such as spermidine. The term “allosteric inhibitor” as used herein means a substance that binds deoxyhypusine synthase at an allosteric site which is different from the active site. Upon binding of the allosteric inhibitor to the allosteric site, deoxyhypusine synthase changes its three- dimensional shape or has a reduced ability to bind or otherwise interact with a co-factor, thereby slowing down its enzymatic activity or otherwise deactivating its enzymatic activity. In this manner, the allosteric inhibitor inhibits, modulates, prevents, reduces, or otherwise interferes with the activity of deoxyhypusine synthase to enzymatically process the substrate, such as spermidine, and attach the 4-amino butyl moiety from spermidine to the -NH2 of lysine of eIF5A.
[00030] Allosteric inhibitors can be identified by screening compounds for binding efficiency to deoxyhypusine synthase and determining the effect on active site binding using known assays or modifying known assays for this particular purpose. One exemplary assay is described above with respect to competitive inhibitors. Exemplary allosteric inhibitors that target dcoxyhypusinc synthase include a small molecule, a protein, a peptide, a polypeptide, an antibody, a peptide aptamer, a nucleic acid, a nucleic acid aptamer, and the like. In one embodiment, an allosteric inhibitor that targets deoxyhypusine synthase is a small molecule.
[00031] According to one aspect, one or more degradation-promoting compounds may be used to inhibit, modulate, reduce or otherwise interfere with deoxyhypusine synthase by promoting deoxyhypusine synthase degradation through cellular processes such as proteolysis. According to this aspect, a degradation-promoting compound, such as a proteolysis-targeting compound or “PROTAC”, which is a bifunctional degradation-promoting compound, can be used to increase deoxyhypusine synthase degradation by linking deoxyhypusine synthase to an ubiquitination enzyme, such as E3 ligase, or otherwise proximally locating deoxyhypusine synthase to an ubiquitination enzyme, such as E3 ligase, in order to promote degradation or proteolysis of deoxyhypusine synthase by a cellular proteosome, such as the 26S proteasome. Such degradation-promoting compounds can be identified or designed, for example, by screening bifunctional compounds for binding efficiency to deoxyhypusine synthase, such as the competitive inhibitors and allosteric inhibitors described herein, and also binding efficiency to an ubiquitination enzyme, such as compounds known in the art to bind to the ubiquitination enzyme E3 ligase and for use in designing and making a PROTAC, and determining the degradation of deoxyhypusine synthase using known assays or modifying known assays for this particular purpose. One exemplary assay useful to assess degradation of deoxyhypusine synthase using a degradation-promoting compound includes standard gel electrophoresis I antibody techniques such as Western blotting. In principle, any assay designed to measure protein levels can be used to evaluate degradation of deoxyhypusine synthase. Exemplary degradation-promoting compounds that target deoxyhypusine synthase to promote degradation include a competitive or allosteric inhibitor described herein linked or otherwise bound to the E3 enzyme.
[00032] Inhibitory compounds, whether competitive inhibitors or allosteric inhibitors, and compositions as described herein and degradation-promoting compounds and compositions as described herein, are included in kits for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof. A kit may include a pharmaceutically acceptable carrier. In an additional example, a kit may also include instructions for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith. Tn some examples, a kit may also comprise, c.g., a buffering agent, a preservative, or a protein stabilizing agent. In other examples, a kit may also contain a control sample or a series of control samples which can be assayed and compared to a test sample. Other suitable components for including in a kit will be selected given the benefit of this disclosure.
[00033] According to one aspect, one or more systems can be used to inhibit, modulate, reduce or otherwise interfere with cellular production of deoxyhypusine synthase, such as by interfering with transcription of the deoxyribonucleic acid encoding deoxyhypusine synthase or interfering with translation of messenger RNA encoding deoxyhypusine synthase. Such systems are generally known and may utilize nucleic acid modifying agents or systems such as CRISPR-Cas systems, antisense molecules (e.g., interfering RNAs (e.g., small interfering RNAs, short hairpin RNAs), zinc finger nucleases, TALE systems, meganucleases, and ribozymes. Such systems are generally designed to introduce nucleic acid constructs encoding the nucleic acid modifying agent or editor into a cell which is then expressed. The nucleic acid modifying agent or editor then targets a target nucleic acid.
[00034] Such systems include heterozygous gene knockout systems or homozygous gene knockout systems. In general, a gene is modified, such as by being cut or removed or replaced with a donor nucleic acid sequence through homologous recombination and the effect of the gene loss or inactivation is assayed. Other systems include gene regulating systems using transcriptional repressors or translational repressors or compounds that otherwise repress promoters of a gene or otherwise block the ability of a cell’s transcription machinery to carry out transcription or the cell’s translation machinery to carry out translation. Such systems can be adapted for use after review of the present disclosure.
[00035] One exemplary system is a CRISPR/Cas system which can be used to target the nucleic acid encoding deoxyhypusine synthase and (1) cut the nucleic acid (in the case of a Cas enzyme) and interfere with expression of the nucleic acid, (2) nick the nucleic acid (in the case of a Cas nickase) and interfere with expression of the nucleic acid, (3) bind to the nucleic acid (in the case of a nuclease null Cas protein) and interfere with expression of the nucleic acid, or (4) bind to the nucleic acid (in the case of a nuclease null Cas protein having a transcriptional repressor attached thereto) and down-regulate expression of the nucleic acid encoding deoxyhypusine synthase. In the case where a nucleic acid is cut or nicked, a donor sequence can be inserted at the cut or nick site through homologous recombination, for example, to alter the target nucleic acid and interfere with expression of the target nucleic acid. Nucleic acids encoding the Cas protein and/or one or more gRNAs are expressed by the cell and the Cas protein and one or more gRNAs then function to target a target nucleic acid. Such CRISPR/Cas systems can be readily identified or designed after review of the present disclosure, for example by designing one or more gRNAs having a spacer sequence complementary to a protospacer sequence adjacent a protospacer adjacent motif (referred to as a “PAM” sequence) so as to form a colocalization complex with a Cas protein and a target nucleic acid sequence encoding deoxyhypusine synthase (and possibly also including a donor sequence) and then determine reduction of expression or production of deoxyhypusine synthase using known assays or modifying known assays for this particular purpose.
[00036] CRISPR/Cas system components such as a Cas protein or a nucleic acid encoding a Cas protein, and one or more guide RNAs or a nucleic acid encoding one or more gRNAs as described herein, are included in kits for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof. A kit may include a pharmaceutically acceptable carrier. In an additional example, a kit may also include instructions for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith. In some examples, a kit may also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. In other examples, a kit may also contain a control sample or a series of control samples which can be assayed and compared to a test sample. Other suitable components for including in a kit can be selected, given the benefit of this disclosure.
[00037] One exemplary system is a zinc finger nuclease system which can be used to target the nucleic acid encoding deoxyhypusine synthase and cut the nucleic acid. Zinc-finger nucleases include DNA binding domains that can target the nucleic acid encoding deoxyhypusine synthase. Each zinc finger can recognize codons of the nucleic acid encoding deoxyhypusine synthase, and therefore can be modularly assembled to bind to the nucleic acid encoding deoxyhypusine synthase. These binding domains are coupled with a restriction endonuclease that can cause a double stranded break (DSB) in the nucleic acid encoding deoxyhypusine synthase. Repair processes, such as nonhomologous end joining, may introduce mutations, such as insertions and/or deletions, that destroy functionality of the nucleic acid encoding deoxyhypusine synthase. A nucleic acid encoding the zinc finger nuclease is expressed by the cell and the zinc finger nuclease then functions to target a target nucleic acid. Such zinc finger nuclease systems can be readily identified or designed after review of the present disclosure, for example by designing a zinc finger nuclease that will bind to the nucleic acid encoding deoxyhypusine synthase and cut the nucleic acid encoding deoxyhypusine synthase and then determine reduction of expression or production of deoxyhypusine synthase using known assays or modifying known assays for this particular purpose.
[00038] Zinc finger nuclease system components such as a zinc finger nuclease or a nucleic acid encoding a zinc finger nuclease as described herein, are included in kits for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof. A kit may include a pharmaceutically acceptable carrier. In an additional example, a kit may also include instructions for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith. In some examples, a kit may also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. In other examples, a kit may also contain a control sample or a series of control samples which can be assayed and compared to a test sample. Other suitable components for including in a kit can be selected, given the benefit of this disclosure.
[00039] One exemplary system is a TALEN (“transcription activator-like effector nuclease”) system which can be used to target the nucleic acid encoding deoxyhypusine synthase and cut the nucleic acid. A TALEN includes a DNA binding domain that can target the nucleic acid encoding deoxyhypusine synthase and a nuclease that can cleave the nucleic acid encoding deoxyhypusine synthase. Repair processes, such as nonhomologous end joining may introduce mutations, such as insertions and/or deletions, that destroy functionality of the nucleic acid encoding deoxyhypusine synthase. A nucleic acid encoding the TALEN is expressed by the cell and the TALEN then functions to target a target nucleic acid. Such TALEN systems can be readily identified or designed after review of the present disclosure, for example by designing a TALEN that will bind to the nucleic acid encoding deoxyhypusine synthase and cut the nucleic acid encoding deoxyhypusine synthase and then determine reduction of expression or production of deoxyhypusine synthase using known assays or modifying known assays for this particular purpose. [00040] TALEN system components such as a TALEN or a nucleic acid encoding a TALEN as described herein, arc included in kits for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof. A kit may include a pharmaceutically acceptable carrier. In an additional example, a kit may also include instructions for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith. In some examples, a kit may also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. In other examples, a kit may also contain a control sample or a series of control samples which can be assayed and compared to a test sample. Other suitable components for including in a kit can be selected, given the benefit of this disclosure.
[00041] One exemplary system is an RNA interference system which can be used to interfere with messenger RNA to prevent transcription or translation. Such RNA interference systems include those using small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, effective within the RNA interference (RNAi) pathway to degrade mRNA after transcription thereby preventing translation. Such RNA interference systems include those using microRNA (miRNA), effective within the RNA interference (RNAi) pathway to inhibit translation of mRNA. A nucleic acid encoding the RNA is expressed by the cell and the RNA then functions to target a target nucleic acid. Such RNA interference systems can be readily identified or designed after review of the present disclosure, for example, by designing one or more interfering RNAs (iRNAs) whether siRNAs or miRNAs to interfere with either transcription of the nucleic acid encoding deoxyhypusine synthase or translation of the messenger RNA encoding deoxyhypusine synthase. Reduction of expression or production of deoxyhypusine synthase can be determined using known assays or modifying known assays for this particular purpose.
[00042] RNA interference system components such as a double stranded interference RNA as described herein, are included in kits for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith, in a subject, such as a human subject, in need thereof. A kit may include a pharmaceutically acceptable carrier. In an additional example, a kit may also include instructions for treating or preventing a respiratory virus infection or treating or preventing one or more symptoms associated therewith. In some examples, a kit may also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. In other examples, a kit may also contain a control sample or a series of control samples which can be assayed and compared to a test sample. Other suitable components for including in a kit can be selected, given the benefit of this disclosure.
[00043] It will be recognized that the compounds, compositions, methods, systems and kits disclosed herein provide significant advantages over prior technology. Compounds, compositions, methods, systems and kits can be designed or selected to treat or prevent a respiratory virus infection and/or relieve and/or alleviate symptoms in a patient suffering from a respiratory virus infection. These and other aspects and examples are described below.
RESPIRATORY VIRUSES AND RESPIRATORY VIRUS INFECTION SYMPTOMS
[00044] According to one aspect, the present disclosure contemplates treating or preventing a respiratory virus infection in a subject, such as a human subject, in need thereof. Respiratory viruses contemplated by the present disclosure include coronavirus, human rhinovirus, influenza virus, human parainfluenza virus, respiratory syncytial virus, metapneumo virus and the like.
[00045] According to one aspect, the respiratory virus infection is a coronavirus infection. Coronaviruses include, but are not limited to, alphacoronaviruses including human coronavirus 229E (HCoV-229E), and human coronavirus NL63 (HCoV-NL63), and betacoronaviruses including human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKUl), Middle East respiratory syndrome-related coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including variants thereof of the aforementioned alphacoronaviruses and betacoronaviruses. It is to be understood that the disclosure is not limited to particular genera, species, or variants of coronaviruses but includes any and all genera, species and variants of coronaviruses.
[00046] According to one aspect, the respiratory virus infection is a human rhinovirus infection. Rhinoviruses include, but are not limited to, human rhinovirus (HRV) species A, HRV species B and HRV species C, including any types, strains and variants thereof of the aforementioned species of HRV. It is to be understood that the disclosure is not limited to particular species, types, strains or variants of human rhinoviruses but includes any and all species, types, strains and variants of human rhinoviruses. [00047] According to one aspect, the respiratory virus infection is an influenza virus infection. Influenza viruses include, but arc not limited to, Influenza A virus (IAV) and Influenza B Virus (IBV), including any strains and variants thereof of the aforementioned types of influenza virus. It is to be understood that the disclosure is not limited to particular species, types, strains, or variants of influenza viruses but includes any and all species, types, strains and variants of influenza viruses.
[00048] According to one aspect, the respiratory virus infection is a parainfluenza virus infection. Parainfluenza viruses include, but are not limited to, human parainfluenza virus type 1 (PIV1), human parainfluenza type 2 (PIV2), human parainfluenza type 3 (PIV3), and human parainfluenza type 4 (PIV4), including any strains and variants thereof of the aforementioned types of parainfluenza viruses. It is to be understood that the disclosure is not limited to particular species, types, strains or variants of parainfluenza viruses but includes any and all species, types, strains and variants of parainfluenza viruses.
[00049] According to one aspect, the respiratory virus infection is a respiratory syncytial virus (RSV) infection. Respiratory syncytial viruses include, but are not limited to, RSV sub-types or groups A and B, including any strains and variants thereof of the aforementioned subtypes or groups of respiratory syncytial vims. It is to be understood that the disclosure is not limited to particular sub-types or groups, strains, or variants of respiratory syncytial viruses but includes any and all sub-types or groups, strains and variants of respiratory syncytial viruses.
[00050] According to one aspect, the respiratory virus infection is a metapneumovirus infection. Metapneumoviruses include, but are not limited to, human metapneumovirus genotype or lineage A and human metapneumovirus genotype or lineage B, and sub-groups or sub-lineages Al and A2, and Bl and B2 thereof, including any strains and variants of the aforementioned genotypes, lineages, sub-groups and sub-lineages of human metapneumovirus. It is to be understood that the disclosure is not limited to particular genotypes, lineages, sub-groups, sub-lineages, strains or variants of human metapneumoviruses but includes any and all genotypes, lineages, sub-groups, sub-lineages, strains and variants of human metapneumoviruses.
[00051] This list is exemplary only and is not exhaustive. Other human respiratory viruses may be identified based on the present disclosure and consulting relevant literature sources. [00052] Tn accordance with certain aspects, methods for treating symptoms associated with respiratory virus infections arc disclosed. Treatment of one or more symptoms associated with respiratory virus infections is intended to include, but is not limited to, the use of a compound, composition, or system described herein to reduce or alleviate one or more symptoms of a respiratory virus infection. As used herein, the term “symptom associated with a respiratory virus infection” refers to the host’s response to infection by one or more respiratory viruses. Symptoms associated with respiratory virus infections are well known in the art. Such responses include, but are not limited to, coughing, sneezing, runny nose, sore throat, fever, rapid breathing, trouble breathing, wheezing, congestion of nasal sinuses and/or lungs, body aches, fatigue, decrease in appetite, and exacerbation of other underlying respiratory conditions (e.g., asthma, COPD). This list is exemplary only and is not exhaustive, as additional symptoms associated with respiratory virus infections based on the disclosure herein can be identified.
[00053] According to one aspect, efficacy of the treatment and/or prevention methods described herein can be determined. Based on the present disclosure, known assays or modified known assays can be used for this particular purpose. For example, common endpoints in the viral respiratory field can be assayed and used to determine whether a treatment is effective, including reduction in viral load (e.g., quantifying viral load in nasal swabs, throat swabs, bronchioalveolar lavage fluid, etc.), reduction in duration of illness, reduction in symptoms (either severity of symptoms or number of symptoms as determined from either a diagnostic point of view of a physician or patient experience/perception), decreased percentage of individuals requiring hospitalization, decreased percentage of individuals requiring supplemental oxygen, decreased number of deaths, decreased intensive care unit (ICU) admissions, reduced number of days in the hospital and/or ICU, etc., and the like.
DEOXYHYPUSINE SYNTHASE PROTEIN
[00054] In accordance with the present disclosure, the term “deoxyhypusine synthase” or “DHPS” refers to an enzyme in the hypusination pathway of eIF5A that transfers a 4-amino butyl moiety from spermidine to the -NH2 of lysine of eIF5A.
[00055] According to one aspect, deoxyhypusine synthase includes at least the amino acid sequence: EGSLEREAPAGALAAVLKHSSTLPPESTQVRGYDFNRGVNYRALLEAFGTTGFQATNFG RAVQQVNAMIEKKLEPLSQDEDQHADLTQSRRPLTSCTIFLGYTSNLISSGIRETIRYLVQ HNMVDVLVTTAGGVEEDLIKCLAPTYLGEFSLRGKELRENGINRIGNLLVPNENYCKFE DWLMPILDQMVMEQNTEGVKWTPSKMIARLGKEINNPESVYYWAQKNHIPVFSPALTD GSLGDMIFFHSYKNPGLVLDIVEAQEFDGSDSGARPDEAVSWGKIRVDAQPVKVYADA
SLVFPLLVAETFAQKMDAFMHEKNED. (SEQ ID NO:1). According to one aspect, SEQ ID NO:1 may include an NT-terminal amino acid M at the beginning of SEQ ID NO:1.
[00056] According to one aspect, deoxyhypusine synthase includes at least the amino acid sequence: EGSLEREAPAGALAAVLKHSSTLPPESTQVRGYDFNRGVNYRALLEAFGTTGFQATNFG RAVQQVNAMIEKKLEPLSQDEDQHADLTQSRRPLTSCTIFLGYTSNLISSGIRETIRYLVQ HNMVDVLVTTAGGVEEDLIKCLAPTYLGEFSLRGKELRENGINRIGNLLVPNENYCKFE DWLMPILDQMVMEQNTEGVKWTPSKMIARLGKEINNPESVYYWAQKNHIPVFSPALTD GSLGDMIFFHSYKNPGLVLDIVEAQEFDGSDSGARPDEAVSWGKIRVDAQPVKVYADA SLVFPLLVAETFAQKMDAFMHEKNED. (SEQ ID NO:2). According to one aspect, SEQ ID NO:2 may include an N-terminal amino acid M at the beginning of SEQ ID NO:2.
[00057] According to one aspect, deoxyhypusine synthase includes at least the amino acid sequence: PIIPAFWEAEAGGSREEEFETSLANMIEKKLEPLSQDEDQHADLTQSRRPLTSCTIFLGYT SNLISSGIRETIRYLVQHNMVDVLVTTAGGVEEDLIKCLAPTYLGEFSLRGKELRENGINR IGNLLVPNENYCKFEDWLMPILDQMVMEQNTEGVKWTPSKMIARLGKEINNPESVYYW AQKNHIPVFSPALTDGSLGDMIFFHSYKNPGLVLDIVEDLRLINTQAIFAKCTGMIILGGG VVKHHIANANLMRNGADYAVYINTAQEFDGSDSGARPDEAVSWGKIRVDAQPVKVYA DASLVFPLLVAETFAQKMDAFMHEKNED. (SEQ ID NO: 3). According to one aspect, SEQ ID NO:3 may include an N-terminal amino acid M at the beginning of SEQ ID NO:3.
[00058] According to one aspect, deoxyhypusine synthase includes at least the amino acid sequence: EGSLEREAPAGALAAVLKHSSTLPPESTQVRGYDFNRGVNYRALLEAFGTTGFQATNFG RAVQQVNAMIEKKLEPLSQDEDQHADLTQSRRPLTSCTIFLGYTSNLISSGIRETIRYLVQ HNMVDVLVTTAGGVEEDLIKCLAPTYLGEFSLRGKELRENGINRIGNLLVPNENYCKFE DWLMPILDQMVMEQNTEGVKWTPSKMIARLGKEINNPESVYYWAQKNHIPVFSPALTD GSLGDMTFFHSYKNPGLVLDIVEDLRLTNTQAIFAKCTGMITLGGGVVKHHIANANLMVP DQTRLSPGARSGWMHSPSRSMLTPPWSSPCLWLKPLPRRWMPSCMRRTRTERLRSQEG LTPSSIY. (SEQ ID N0:4). According to one aspect, SEQ ID NO:4 may include an N-terminal amino acid M at the beginning of SEQ ID NO: 4.
[00059] According to one aspect, deoxyhypusine synthase includes at least the amino acid sequence:
EGSLEREAPAGALAAVLKHSSTLPPESTQVRGYDFNRGVNYRALLEAFGTTGFQATNFG RAVQQVNAMIEKKLEPLSQDEDQHADLTQSRRPLTSCTIFLGYTSNLISSGIRETIRYLVQ HNMVDVLVTTAGGVEEDLIKCLAPTYLGEFSLRGKELRENGINRIGNLLVPNENYCKFE DWLMPILDQMVMEQNTEGVKWTPSKMIARLGKEINNPESVYYWAQKNHIPVFSPALTD GSLGDMIFFHSYKNPGLVLDIVEEDGCLHA. (SEQ ID. NO:5). According to one aspect, SEQ ID NO:5 may include an N-terminal amino acid M at the beginning of SEQ ID NO:5.
[00060] According to one aspect, deoxyhypusine synthase includes at least the amino acid sequence: PILDQMVMEQNTEGVKWTPSKMIARLGKEINNPESVYYWAQKNHIPVFSPALTDGSLG DMIFFHSYKNPGLVLDIVEDLRLINTQAIFAKCTGMIILGGGVVKHHIANANLMRNGAD YAVYINTAQEFDGSDSGARPDEAVSWGKIRVDAQPVKVYADASLVFPLLVAETFAQKM DAFMHEKNED. (SEQ ID NO:6). According to one aspect, SEQ ID NO:6 may include an N- terminal amino acid M at the beginning of SEQ ID NO: 6.
SMALL MOLECULE COMPETITIVE INHIBITORS
[00061] According to aspects of the disclosure, small molecules may be used as competitive inhibitors. An exemplary competitive inhibitor is a poly amine, such as a poly amine having structural similarity to spermidine. Representative competitive inhibitors include N1- guanyl-l,7-diaminoheptane, N ’-guanyl- 1.8-diaminooctanc. N’-guanylcaldinc. N1- guanylspermidine, or N8-guanylspermidine and the like.
[00062] According to one aspect, an exemplary competitive inhibitor is N,N’- bis [3 ,5 -bis [ 1 (aminoiminomethyl)hydrazonoethyl]phenyl] decanediamidetetrahydrochloride also known in the art as CNL1493, AXD455 or semapimod. SMALL MOLECULE ALLOSTERIC INHIBITORS
[00063] According to aspects of the disclosure, small molecules may be used as allosteric inhibitors.
[00064] According to one aspect, an exemplary allosteric inhibitor is
Figure imgf000021_0001
ANTIBODIES
[00066] According to certain aspects, a deoxyhypusine synthase inhibitor is an antibody. According to aspects of the present disclosure, antibodies or antibody formats may be used as competitive inhibitors or allosteric inhibitors. The term “antibody” is used herein in the broadest sense to refer to molecules with an immunoglobulin-like domain (for example IgG, IgM, IgA, IgD or IgE) and includes monoclonal, recombinant, synthetic, polyclonal, chimeric, human, humanized, multispccific antibodies, including bispccific antibodies, and hctcroconjugatc antibodies; a single variable domain, antigen binding antibody fragments (e.g., Fab, F(ab')2, Fv, disulphide linked Fv, single chain Fv, disulphide-linked scFv, diabodies, TANDAB, etc.) and modified versions of any of the foregoing.
[00067] The term “domain” refers to a folded protein structure which retains its tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain. The term “single variable domain” refers to a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains such as VH, VHH and VL and modified antibody variable domains, for example, in which one or more loops have been replaced by sequences which arc not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C- terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain. A single variable domain that is capable of binding an antigen or epitope independently of a different variable region or domain may be referred to as a “domain antibody” or “dAB.” A single variable domain may be a human single variable domain, but also includes single variable domains from other species such as rodent, nurse shark and Camelid VHH dABs. Camelid VHH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Such VHH domains may be humanized according to standard techniques available in the art, and such domains are considered to be “single variable domains.” As used herein VH includes camelid VHH domains.
[00068] “Alternative antibody formats” are those where the CDRs are arranged onto a suitable non-immunoglobulin protein scaffold or skeleton. The non-immunoglobulin scaffold may be a derived from the group consisting of CTLA-4, lipocalin, Protein A derived molecules such as Z-domain of Protein A (Affibody, SpA), A-domain (Avimer/Maxibody); heat shock proteins such as GroEl and GroES; transferrin (trans-body); ankyrin repeat protein (DARPin); peptide aptamer; C-type lectin domain (Tetranectin); human y-cry stallin and human ubiquitin (affilins); PDZ domains; LDL receptor class A domains; EGF domains; scorpion toxin kunitz type domains of human protease inhibitors; and fibronectin/adnectin.
APTAMERS
[00069] According to certain aspects, a deoxyhypusine synthase inhibitor is an aptamer. Aptamers can block activity of a protein by mechanisms such as hydrogen bonding, electrostatic complementarity, hydrophobic contacts, and steric exclusion. An aptamer may be a peptide aptamer or a nucleic acid aptamer. PEPTIDE APTAMERS
[00070] According to certain aspects, a dcoxyhypusinc synthase inhibitor is a peptide aptamer. A “peptide aptamer” as used herein is a small combinatorial polypeptide, about 5-20 amino acid residues long, containing one or more short variable peptide domains. Peptide aptamers typically are embedded as a loop within a protein scaffold. Methods of generating, selecting, synthesizing, and administering peptide aptamers are known.
NUCLEIC ACID APTAMERS
[00071] According to certain aspects, a deoxyhypusine synthase inhibitor is a nucleic acid aptamer. A “nucleic acid aptamer” as used herein is a single- stranded nucleic acid molecule, typically about 20-100 bases in length, that folds into a three-dimensional structure that selectively binds to a molecular target (e.g., a small molecule, a protein, a nucleic acid, a cell). Nucleic acid aptamers can be RNA aptamers, DNA aptamers, or XNA aptamers (synthetic xeno nucleic acids, having a different sugar backbone than naturally occurring DNA and RNA). Methods of generating, selecting, synthesizing, and administering nucleic acid aptamers are known.
CRISPR-CAS SYSTEMS
[00073] In some embodiments, a deoxyhypusine synthase inhibitor is a CRISPR- Cas system. In some embodiments, a CRISPR-Cas system may be used as a nucleic acid modifying agent. A “CRISPR-Cas system” as used herein uses a guide RNA (gRNA) complexed with a Cas protein to cleave a nucleic acid molecule encoding deoxyhypusine synthase at specific sites. In some embodiments, the Cas protein creates a double-strand break. In some embodiments, the Cas protein creates a single-strand break.
[00074] Cas proteins typically interact with gRNAs via at least one RNA binding or recognition domain. In some embodiments, a Cas protein also comprises one or more of a nuclease domain (e.g., RNase or DNase), DNA binding domain, helicase domain, protein-protein interaction domain, or dimerization domain.
[00075] Examples of Cas proteins include a wild-type Cas9 protein, a wild -type Cpfl protein (e.g., FnCpfl), Casl, CaslB, Cast, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, CasF, CasG, CasH, Csy 1 , Csy2, Csy3, Csel (CasA), Cse2 (Cas6), Cse3 (CasE), Cse4 (CasC), Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions thereof. In some embodiments, a Cas protein is linked to a heterologous polypeptide, such as a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain.
[00076] In some embodiments, a Cas protein and at least one gRNA form a colocalization complex with a gRNA recognition sequence (also known as a protospacer sequence) in a nucleic acid molecule encoding deoxyhypusine synthase. gRNAs can be designed and synthesized using methods known in the art. According to one aspect, a gRNA spacer sequence complementary to a target protospacer sequence includes from about 17 to about 23 nucleotides, from about 18 to about 22 nucleotides, or from about 19 to about 21 nucleotides. In some embodiments, a gRNA spacer sequence includes 20 nucleotides.
[00077] In some embodiments, a gRNA recognizes a site in a deoxyhypusine synthase genomic nucleic acid molecule that encodes a deoxyhypusine synthase protein having the amino acid sequence SEQ ID NO: 1. In some embodiments, a gRNA recognizes a site in a deoxyhypusine synthase genomic nucleic acid molecule that encodes a deoxyhypusine synthase protein having the amino acid sequence SEQ ID NO:2. In some embodiments, a gRNA recognizes a site in a deoxyhypusine synthase genomic nucleic acid molecule that encodes a deoxyhypusine synthase protein having the amino acid sequence SEQ ID NO:3. In some embodiments, a gRNA recognizes a site in a deoxyhypusine synthase genomic nucleic acid molecule that encodes a deoxyhypusine synthase protein having the amino acid sequence SEQ ID NO:4. In some embodiments, a gRNA recognizes a site in a deoxyhypusine synthase genomic nucleic acid molecule that encodes a deoxyhypusine synthase protein having the amino acid sequence SEQ ID NO:5. In some embodiments, a gRNA recognizes a site in a deoxyhypusine synthase genomic nucleic acid molecule that encodes a deoxyhypusine synthase protein having the amino acid sequence SEQ ID NO:6. In some embodiments, cleavage occurs on one or both strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the nucleic acid sequence present in the deoxyhypusine synthase genomic nucleic acid molecule to which a DNA-targeting segment of a gRNA binds. l ' l ZINC FINGER NUCLEASES
[00078] In some embodiments, a dcoxyhypusinc synthase inhibitor is a zinc finger nuclease. In some embodiments, a zinc finger nuclease or system may be used as a nucleic acid modifying agent in which arrays of zinc finger (ZF) modules, each targeting three DNA bases, are assembled into a zinc finger protein (ZFP). Examples of using ZFPs to target genes are known.
TALE SYSTEMS
[00079] In some embodiments, a deoxyhypusine synthase inhibitor is a TALE system. TALEs (Transcription Activator- Like Effector proteins) are nucleic acid binding proteins secreted by proteobacteria. They contain a nucleic acid binding domain composed of tandem repeats of highly conserved monomer polypeptides, typically 33, 34, or 35 amino acids long, that differ from each other at amino acids 12 and 13 (“repeat variable di-residues” or RVD). The nucleotide binding affinity of a TALE monomer is determined by the amino acids in its RVD. TALE proteins can be fused to a nuclease domain (e.g., FokI) to generate a TALE system, or TALEN system, for gene editing applications. Examples of using TALE systems to effect gene modifications are known.
MEGANUCLEASES
[00080] In some embodiments, a deoxyhypusine synthase inhibitor is a meganuclease, i.e., an endodeoxyribonuclease characterized by a large recognition site (e.g., double-stranded DNA sequences of 12-40 base pairs). Examples of using meganucleases are known.
ANTISENSE MOLECULES
[00081] In some embodiments, a deoxyhypusine synthase inhibitor is an antisense molecule. “Antisense molecules” are oligonucleotide molecules containing sequence complementarity to target RNA molecules that bind to the target RNA and inhibit its function. Antisense molecules can be used to reduce expression of deoxyhypusine synthase mRNA, thereby reducing levels of deoxyhypusine synthase protein. Any region of deoxyhypusine synthase mRNA can be used as the target region. [00082] Antisense molecules include, but are not limited to, single-stranded DNA molecules (antisense oligodcoxyribonuclcotidcs), small interfering RNA (siRNA), ribozymes, and DNAzymes. siRNA
[00083] In some embodiments, a deoxyhypusine synthase inhibitor is an siRNA. An siRNA (“short interfering RNA”) is a double-stranded RNA that can induce RNAi, thereby inhibiting the activity of a gene, in this case deoxyhypusine synthase. An siRNA can be obtained by chemical synthesis, biochemical synthesis, or biosynthesis. Methods of making and using siRNAs are known.
ANTISENSE OLIGODEOXYRIBONUCLEOTIDES
[00084] In some embodiments, a deoxyhypusine synthase inhibitor is an antisense oligodeoxyribonucleotide. An “antisense oligodeoxyribonucleotide” or “antisense oligonucleotide” is an oligonucleotide in which at least a portion of the nucleotide sequence is complementary to a target nucleic acid and hybridizes to the target nucleic acid. Methods of making and using antisense oligonucleotides are known.
DNAZYMES
[00084] In some embodiments, a deoxyhypusine synthase inhibitor is a DNAzyme. A “DNAzyme,” also called a DNA enzyme, deoxyribozyme, or catalytic DNA, are non-naturally occurring DNA oligonucleotides that can perform a specific chemical reaction and are often, but not always, catalytic. Methods of making and using DNAzymes are known.
RIBOZYMES
[00085] In some embodiments, a deoxyhypusine synthase inhibitor is a ribozyme. As used herein the term “ribozyme,” also known as an RNA enzyme or catalytic RNA, is an RNA molecule that participates in RNA processing reactions, such as RNA splicing and transfer RNA biosynthesis. Examples of ribozymes include hammerhead ribozymes, hairpin ribozymes, hepatitis delta virus ribozymes, Group I intron ribozymes, RNAse P ribozymes, VS ribozymes, and Leadzymes. Methods of using ribozymes are known. THERAPEUTIC USES
[00086] In some embodiments, the present disclosure provides a use of, or a method of administering, an inhibitor of human deoxyhypusine synthase to treat or prevent respiratory virus infections, such as coronavirus, human rhinovirus, influenza virus, human parainfluenza virus, respiratory syncytial virus, and metapneumo virus infections. The present disclosure provides for both prophylactic and therapeutic methods of treating a subject for a respiratory virus infection or symptoms associated therewith.
[00087] In one aspect, the invention provides a method for preventing in a subject, a respiratory virus infection or symptom associated with a respiratory virus infection by administering to the subject one or more inhibitory compounds, compositions, or systems as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms, such that a respiratory virus infection or symptom associated therewith is prevented or, alternatively, delayed in its progression.
[00088] The term “prevent” or “preventing” as used herein includes preventing the severity or frequency of a symptom of a viral respiratory infection, such as coughing, sneezing, runny nose, sore throat, fever, trouble breathing, congestion of nasal sinuses and/or lungs, body aches, and fatigue or preventing the onset of the respiratory virus infection itself. According to one aspect, a subject who is at risk of being infected with a respiratory virus infection, such as an individual in an at-risk population, such as immunocompromised individuals or individuals having underlying conditions, is provided with a deoxyhypusine synthase inhibitor or composition described herein. According to another aspect, a subject who has been exposed to a respiratory virus infection is provided with a deoxyhypusine synthase inhibitor or composition described herein for post-exposure prophylaxis to prevent the severity or frequency of a symptom of a viral respiratory infection, or to prevent the onset of the respiratory virus infection itself.
[00089] In one aspect, the invention provides a method for treating in a subject, a respiratory virus infection or symptom associated with a respiratory virus infection by administering to the subject one or more inhibitory compounds such as a deoxyhypusine synthase inhibitor, compositions, or systems as described herein. Administration of a treatment agent can occur during or after the manifestation of symptoms, such that a respiratory virus infection or symptom associated therewith is reduced or, alternatively, delayed in its progression. [00090] The term “treatment” or “treating” refers to ameliorating or stabilizing the specified condition, reducing or eliminating the symptoms of the condition, slowing or eliminating the progression of the condition, and preventing or delaying reoccurrence of the condition in a previously afflicted patient or subject. “Treatment” or “treating” as used herein includes reducing the severity or frequency of a symptom of a viral respiratory infection, such as coughing, sneezing, runny nose, sore throat, fever, trouble breathing, congestion of nasal sinuses and/or lungs, body aches, and fatigue and/or reducing the respiratory virus infection itself. In some embodiments, “treatment” or “treating” as used herein refers to one or more of reducing viral load (e.g., quantifying viral load in nasal swabs, throat swabs, bronchioalveolar lavage fluid, etc.), reducing duration of illness, reducing symptoms (either severity of symptoms or number of symptoms as determined from either a diagnostic point of view of a physician or patient experience/perception), decreasing percentage of individuals requiring hospitalization, decreasing percentage of individuals requiring supplemental oxygen, decreasing number of deaths, decreasing ICU admissions, reducing number of days in the hospital and/or ICU, etc., and the like.
COMBINATION THERAPIES
[00091] In some embodiments, the inhibitor of human deoxyhypusine synthase is administered in combination with at least one other (i.e., at least a second) therapeutic agent used to treat symptoms of a respiratory virus infection. Such agents include, but are not limited to, acetaminophen, nonsteroidal anti-inflammatory drugs, antihistamines, nasal ipratropium, antitussives, steroids, decongestants, and combinations thereof.
[00092] In some embodiments, the inhibitor of human deoxyhypusine synthase is administered in combination with at least one other (i.e., at least a second) therapeutic agent used to treat a respiratory virus infection, as opposed to symptoms of the respiratory virus infection, such as antivirals (such as nucleos(t)ide inhibitors, protease inhibitors, etc.)
[00093] In some embodiments, the inhibitor of human deoxyhypusine synthase and the other therapeutic agent(s) may be administered together or separately and, when administered separately, administration may occur simultaneously or sequentially, in any order. Simultaneous administration may be achieved by administration of (1) a unitary pharmaceutical composition including the therapeutic agents; or (2) simultaneous administration of separate pharmaceutical compositions each including one of the therapeutic agents. Alternatively, the combination may be administered separately in a sequential manner wherein one treatment agent is administered first and the other second or vice versa. Such sequential administration may be close in time or remote in time.
PHARMACEUTICAL COMPOSITIONS
[00094] An inhibitor of deoxyhypusine synthase may be administered by any convenient route. In some embodiments, the inhibitor of deoxyhypusine synthase may be administered orally, parenterally, intranasally, or by inhalation. In some embodiments, the inhibitor of deoxyhypusine synthase is administered in a pharmaceutical composition. In some embodiments, the inhibitor of deoxyhypusine synthase is formulated in a pharmaceutical composition adapted for oral or parenteral administration, or for administration intranasally or by inhalation.
[00095] According to one aspect, the invention provides a pharmaceutical composition comprising an inhibitor of deoxyhypusine synthase and a pharmaceutically acceptable excipient. According to another aspect, the invention provides a process for the preparation of a pharmaceutical composition comprising admixing an inhibitor of deoxyhypusine synthase with a pharmaceutically acceptable excipient.
[00096] Pharmaceutical formulations adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.
[00097] Pharmaceutical formulations adapted for nasal administration can comprise a coarse powder having a particle size for example in the range 20 to 500 microns that is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the inhibitor of deoxyhypusine synthase.
[00098] Pharmaceutical formulations adapted for administration by inhalation include fine particle dusts or mists, which may be generated by means of various types of metered, dose pressurized aerosols, nebulizers or insufflators, all of which are well known in the art.
[00099] Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain anti-oxidants, buffers, l ' l bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets using known techniques.
[000100] It should be understood that in addition to the ingredients particularly mentioned above, the formulations described herein may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
[000101] Also provided are unitary pharmaceutical compositions in which a deoxyhypusine synthase inhibitor and one or more other therapeutic agent(s) may be administered together. When a deoxyhypusine synthase inhibitor is used in combination with a second therapeutic agent, the dose of each therapeutic agent may differ from the dose of that therapeutic agent when used alone.
[000102] A deoxyhypusine synthase inhibitor may be administered by an effective route, according to the type of inhibitor. In some embodiments, the deoxyhypusine synthase inhibitor may be administered by an injection for example subcutaneously or intravenously.
[000103] According to one aspect, provided herein is a pharmaceutical composition comprising a deoxyhypusine synthase inhibitor and a pharmaceutically acceptable excipient. According to another aspect, the present disclosure provides a process for the preparation of a pharmaceutical composition comprising admixing a deoxyhypusine synthase inhibitor with a pharmaceutically acceptable excipient.
[000104] Pharmaceutical formulations may be presented as units such as solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions. In some embodiments, the inhibitor is present in an aqueous solution.
[000105] Pharmaceutical formulations adapted for injection include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example scaled ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
[000106] The present disclosure also provides unitary pharmaceutical compositions in which a deoxyhypusine synthase inhibitor and one or more other therapeutic agent(s) disclosed herein may be administered in combination, concurrently or sequentially.
DOSAGES
[000107] According to one aspect, a method of treating or preventing a respiratory virus infection or symptom associated therewith includes the step of administering a therapeutically and/or prophylactically effective amount of a compound or system to a subject, such as a human subject, in need of such treatment or prevention. According to this aspect, a subject is administered a therapeutically and/or prophylactically effective amount that is effective to treat or prevent a respiratory virus infection or symptom associated therewith. The term “therapeutically effective amount” refers to the quantity of a compound or a pharmaceutically acceptable salt thereof, which will elicit the desired biological response in an animal or human body. It may vary depending on the compound, the disease and its severity and the age and weight of the subject to be treated. Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the respiratory virus infection or symptom associated therewith, previous treatments, the general health and/or age of the subject, and other diseases present. Treatment of a subject with a therapeutically and/or prophylactically effective amount of a compound or system can include a single treatment or can include a series of treatments. It will also be appreciated that the effective dosage used for treatment or prevention may increase or decrease over the course of a particular treatment.
[000108] In the Examples, the following deoxyhypusine synthase inhibitor compounds were selected and assayed for their cytoprotective potential using cytopathic effect (CPE) as the endpoint. Nl -guanyl-1 ,7-diamine-heptane Compound 1
Figure imgf000032_0001
p
EXAMPLE 1
Inhibition of Deoxyhypusine Synthase Reduces the Cytopathic Effect of Coronavirus Infection
[000109] MRC-5 human epithelial cells were seeded at a density of 5 x 103 per well in 96-well tissue culture plates and allowed to adhere overnight. The following day, six different dilutions of Compound 1, Compound 2, and Compound 3 ranging from about 10 nM to 500 pM, i.e., 50 pM, 15.8 pM, 5 pM, 1.58 pM, 0.5 pM and 0.16 pM were added for a pre-treatment period of three hours at 37 °C and 5 % CO2. Human coronavirus strain 229E (HCoV-229E), diluted to a pre-determined titre to yield 85 % to 95 % killing at six days post-infection, was added to the plates. Cell viability was measured by XTT tetrazolium dye staining following six days of incubation at 37 °C and 5 % CO2, with optical density measured spectrophotometrically at 450 nm and 650 nm using SOFTMAX Pro 4.6 software. Cellular toxicity due to compound treatment alone was measured in parallel with uninfected cells. The concentrations of compound that reduced the CPE by 50% (EC 50) and uninfected cell viability by 50% (CC50) were calculated using parameter curve fit analysis, and are provided in Table 1 below for HCoV-229E.
Table 1
Figure imgf000032_0002
Figure imgf000033_0001
[000110] These results demonstrate that deoxyhypusine synthase inhibitors can reduce the cytopathic effect of coronavirus infection.
EXAMPLE 2
Inhibition of Deoxyhypusine Synthase Reduces the Cytopathic Effect of Human Rhinovirus Infection
[000111] Hl-HeLa human epithelial cells were seeded at a density of 5 x 10 per well in 96-well tissue culture plates and allowed to adhere overnight. The following day, six different dilutions of Compound 1, Compound 2, and Compound 3 ranging from about 10 nM to 500 pM, i.e., 50 pM, 15.8 pM, 5 pM, 1.58 pM, 0.5 pM and 0.16 pM were added for a pre-treatment period of three hours at 37 °C and 5 % CO2. Human rhinovirus type 16 strain 11757 (HRV 16), diluted to a pre-determined titre to yield 85 % to 95 % killing at six days post-infection, was added to the plates. Cell viability was measured by XTT tetrazolium dye staining following six days of incubation at 37 °C and 5 % CO2, with optical density measured spectrophotometrically at 450 nm and 650 nm using SOFTMAX Pro 4.6 software. Cellular toxicity due to compound treatment alone was measured in parallel with uninfected cells. The concentrations of compound that reduced the CPE by 50% (EC 50) and uninfected cell viability by 50% (CC50) were calculated using parameter curve fit analysis, and the EC50 data are provided in Table 2 below for HRV 16. indicates that a reduction in CPE by 50% was not achieved over the compound concentration range tested. Table 2
Figure imgf000033_0002
[000112] These results demonstrate that deoxyhypusine synthase inhibitors can reduce the cytopathic effect of human rhinovirus infection. EXAMPLE 3
Inhibition of Deoxyhypusine Synthase Reduces the Cytopathic Effect of Influenza Virus Infection
[000113] RPMI2650 cells were seeded at a density of 5 x 103 per well in 96-well tissue culture plates and allowed to adhere overnight. The following day, the cells were washed and six different dilutions of Compound 1, Compound 2, and Compound 3 ranging from about 10 nM to 500 pM, i.e., 50 pM, 15.8 pM, 5 pM, 1.58 pM, 0.5 pM and 0.16 pM were added for a pretreatment period of three hours at 37 °C and 5 % CO2. Human Influenza virus strain A/PR/34 (IAV PR/34), diluted to a pre-determined titre to yield 85 % to 95 % killing at six days post-infection, was added to the plates. Cell viability was determined following four days of incubation at 37°C and 5% CO2 using CELLTITER-GLO (CTG) reagent, which is based on the chemiluminescent detection of ATP as an indicator of metabolically active cells. Cellular toxicity due to compound treatment alone was measured in parallel with uninfected cells. The concentrations of compound that reduced the CPE by 50% (EC 50) and uninfected cell viability by 50% (CC50) were calculated using parameter curve fit analysis. A reduction in CPE by 50% (EC50) for each of Compound 1, Compound 2, and Compound 3 was not observed over the concentration range tested under these experimental conditions, thus the highest percentage reduction in IAV CPE observed along with the CC50 data are provided in Table 3 below. Moreover, the utility of these compounds to reduce the cytopathic effect of influenza virus can be determined using alternate concentration ranges and/or different experimental conditions.
Table 3
Figure imgf000034_0001
[000114] These results demonstrate that deoxyhypusine synthase inhibitors can reduce the cytopathic effect of influenza virus infection.
EXAMPLE 4 Inhibition of Deoxyhypusine Synthase Reduces the Cytopathic Effect of Human Parainfluenza Virus Infection
[000115] Human lung epithelial A549 cells were harvested, counted, and resuspended in phenol red-free DMEM medium containing 10% FBS, HEPES, glutamine and pyruvate. Six different dilutions of compounds ranging from about 10 nM to 500 pM, i.e., 50 pM, 15.8 pM, 5 pM, 1.58 pM, 0.5 pM and 0.16 pM were added were added for a pre-treatment period of 24 hours at 37 °C and 5 % CO2. PIV3-GFP virus was applied to the cells at a target multiplicity- of-infection (MOI) to yield 40-60% infection of cells by GFP fluorescence two days after infection. Following 44 hours of incubation at 37 °C and 5 % CO2, the cells were fixed in 2% paraformaldehyde containing 10 pM Hoechst dye. After one-hour incubation, plates were washed three times with PBS and imaged at 4x using blue and green fluorescence channels on a PerkinElmer ENSIGHT imager. Hoechst-stained nuclei were identified using a segmentation algorithm. GFP-positive nuclei were quantified using a minimum fluorescence threshold determined from positive controls. The maximum and minimum GFP positive nuclei were calculated from positive and negative controls and used to normalize data for putative inhibitors against PIV3. EC50 values were calculated by fitting a sigmoidal variable slope nonlinear regression model to the data. For compound cytotoxicity measurements, nuclei counts were normalized to positive controls for EC50 calculation. The EC50 data and CC50 data are provided in Table 4 below for PIV3-GFP. indicates that a reduction in CPE by 50% was not achieved over the compound concentration range tested.
Table 4
Figure imgf000035_0001
[000116] These results demonstrate that deoxyhypusine synthase inhibitors can reduce the cytopathic effect of human parainfluenza virus infection.
EXAMPLE 5 Inhibition of Deoxyhypusine Synthase Reduces the Cytopathic Effect of Respiratory Syncytial Virus Infection
[000117] HEp-2 human epithelial cells were seeded at a density of 5 x 103 per well in 96-well tissue culture plates and allowed to adhere overnight. The following day, six different dilutions of Compound 1, Compound 2, and Compound 3 ranging from about 10 nM to 500 pM, i.e., 50 pM, 15.8 pM, 5 pM, 1.58 pM, 0.5 pM and 0.16 pM were added for a pre-treatment period of three hours at 37 °C and 5 % CCh. Human RSV strain A2 (RSV A2), diluted to a pre -determined titre to yield 85 % to 95 % killing at six days post-infection, was added to the plates. Cell viability was measured by XTT tetrazolium dye staining following six days of incubation at 37 °C and 5 % CO2, with optical density measured spectrophotometrically at 450 nm and 650 nm using SOFTMAX Pro 4.6 software. Cellular toxicity due to compound treatment alone was measured in parallel with uninfected cells. The concentrations of compound that reduced the CPE by 50% (EC 50) and uninfected cell viability by 50% (CC50) were calculated using parameter curve fit analysis, and the EC50 data are provided in Table 5 below for RSV A2. indicates that a reduction in CPE by 50% was not achieved over the compound concentration range tested.
Table 5
Figure imgf000036_0001
[000118] These results demonstrate that inhibitors can reduce the cytopathic effect of respiratory syncytial virus infection.
EXAMPLE 6
Inhibition of Deoxyhypusine Synthase Reduces the Cytopathic Effect of Human Metapneumovirus Infection
[000119] LLC-MK2 (American Type Culture Collection CCL-7) cells are seeded at an appropriate density per well in 96-well tissue culture plates and allowed to adhere overnight. The following day, dilutions of Compound 1, Compound 2, and Compound 3 ranging from about 10 nM to 500 p M are added for a pre-treatment period of three hours at 37 °C and 5 % CO2. Human mctapncumovirus, diluted to an appropriate prc-dctcrmincd titre, is added to the plates. Cell viability is measured by XTT tetrazolium dye staining following four to seven days of incubation at 37 °C and 5 % CO2, with optical density measured spectrophotometrically at 450 nm and 650 nm using SOFTMAX Pro 4.6 software. Cellular toxicity due to compound treatment alone is measured in parallel with uninfected cells. The concentrations of compound that reduced the CPE by 50% (EC50) and uninfected cell viability by 50% (CC50) are calculated using parameter curve fit analysis, and the EC50 data and CC50 data are collected.
EXAMPLE 7
Deoxyhypusine Synthase is Abundantly Expressed in Human Lung Epithelial Cells
[000120] The ability of human lung epithelial cells to express deoxyhypusine synthase was determined.
[000121] Cells of the human lung epithelial line A549 (American Type Culture Collection CCL-185) were seeded in 24-well plates in DMEM + 10% FBS + 1% penicillin / streptomycin and incubated for 24 hours at 37 °C and 5% CO2. Culture medium was removed from each well, cells were washed with phosphate buffered saline, and fresh culture medium (DMEM + 10% FBS) was added to each well. Cells were incubated at 37 °C and 5 % CO2. Total cellular RNA was extracted and purified using the Qiagen RNEASY Plus Mini Kit. Quantitative PCR (qPCR) was used to quantify transcript levels of deoxyhypusine synthase and of the housekeeping gene beta-actin (ACTB) using TAQMAN Gene Expression assays (Applied Biosystems). Threshold cycle (Ct) values were calculated for each gene to provide an indication of relative transcript abundance and reported in Table 6 below. Ct values < 29 are generally considered to be indicative of abundant target sequence in the sample.
Table 6
Figure imgf000037_0001
EXAMPLE 8 CRISPR Knockout of Deoxyhypusine Synthase Increases Cell Survival or Decreases Virus Infection
[000123] A CRISPR-Cas system was used to genetically knock out deoxyhypusine synthase in cells and the effect of deoxyhypusine synthase genetic knock out on viral infection/replication of respiratory viruses was determined.
[000124] Genome-wide CRISPR knockout (KO) screens were performed in cellular infection models for human rhinovirus (HRV), parainfluenza virus (PIV), respiratory syncytial virus (RSV) and Influenza A vims (IAV). CRISPR library generation was performed as previously described. Briefly, CRISPR KO sgRNA libraries were introduced into cell lines via lentivirus transduction. Cas9/sgRNA expressing cells were selected via antibiotic resistance and expanded. For each screening experiment, cells were infected with virus and enriched for a specific phenotype of interest (susceptibility/resi stance to virus infection-dependent gene expression and/or virus- induced cytopathic effect.) Details of each cellular infection model are described in Table 7 below.
Table 7
Figure imgf000038_0001
[000125] Next-generation-sequencing (NGS) was employed to determine sgRNA sequence abundance in each cell population of interest using standard techniques. Sequencing data deconvolution and analysis was performed using MAGeCK computational tool to determine average changes in relative abundance across each sgRNA for each gene in the enriched population of interest compared to controls. In addition to the genome-wide libraries, a respiratory virus- focused subpool CRISPR KO library was generated using the top hits from the individual genomewide screens. The focused subpool library was re-screened against each cellular infection model. NGS and data analysis was performed as described for the genome-wide screens. Deoxyhypusine synthase was a statistically significant gene-level hit (MAGeCK score p-value < 0.001) in the genome-wide and/or focused custom CRISPR KO screens for each virus tested. The CRISPR screen results are summarized in Table 8 and Table 9 below. “X” denotes that the gene was a statistically significant screen hit (p-value <0.001).
Table 8
Figure imgf000039_0001
Table 9
Figure imgf000039_0002
[000126] Confirmation of deoxyhypusine synthase involvement in virus replication and/or virus induced cytopathic effects was conducted by assessing resistance to viral infection in individual genetically perturbed cell lines by knockout using CRISPR.
[000127] Details of individual CRISPR KO cell lines are provided below. CRISPR editing of deoxyhypusine synthase was performed by transducing cells with Cas9/sgRNA expressing lentivirus. Non-targeting (NT) sgRNA sequences were used where indicated as negative controls. A list of sgRNA sequences is provided in Table 10 below.
Table 10
Figure imgf000040_0001
[000128] Following lentivirus transduction, the CRISPR knockout cells were selected via antibiotic resistance, expanded, then tested individually in the cellular infection models. Gene editing efficiency was determined for each KO cell line as using standard methods
[000129] For HRV, Hl-HeLa cells were plated in 96- well plates and infected with HRV at various multiplicities of infection (MOI). Viral infection was measured by determining the level of virus-induced cytopathic effect using the CELLTITER-GLO (CTG) viability assay (Promega) and measuring luminescence signal in infected wells relative to uninfected controls. The impact of deoxyhypusine synthase gene perturbation on viral infection was measured by determining relative survival in deoxyhypusine synthase KO cells compared to NT controls.
[000130] For influenza virus, A549 cells were plated in 96-well plates and infected with IAV. The impact of deoxyhypusine synthase gene perturbation on IAV infection was measured by determining the percentage of cells that were positive for the influenza viral protein nucleoprotein (NP) in deoxyhypusine synthase KO cells compared to NT controls. In the CRISPR knockout experiments, cells were infected in suspension whereas in the knockdown experiments, cells were infected 48 hours after siRNA transfection.
[000131] For parainfluenza virus, A549 cells were plated and infected with PIV-GFP at various MOI. The impact of deoxyhypusine synthase gene perturbation on viral infection was measured by determining the percentage of cells that were positive for GFP expression in deoxyhypusine synthase KO cells compared to NT controls.
[000132] For respiratory syncytial virus, HEp-2 cells were plated in 96-well plates and infected with RSV-GFP. The impact of deoxyhypusine synthase gene editing on RSV infection was measured either by calculating the percentage of cells that were positive for GFP or relative survival (using the CTG viability assay) in deoxyhypusine synthase KO cells compared to NT controls. [000133] Results of the individual genetic perturbation experiments are summarized in Tabic 11 below.
Table 11
Figure imgf000041_0001
[000134] Deoxyhypusine synthase CRISPR knockout cell lines displayed resistance to virus infection validating deoxyhypusine synthase as a target for the treatment or prevention of a respiratory virus infection. Results in CRISPR KO cells for rhinovirus and parainfluenza virus are shown in Fig. 2 and Fig. 3.
[000135] Fig. 2 is directed to evaluating HRV infection (human rhinovirus 16) in CRISPR-mediated deoxyhypusine synthase knockout cell lines. Deoxyhypusine synthase knockout cells compared to NT (non-target) controls show increased resistance to human rhinovirus-induced cytopathic effect. sgRNAs targeting ICAM1 were used as a positive control against HRV (ICAM1) to evaluate the effect of known pro-pathogen genes in this infection model. The results are presented as means of four independent experiments. Error bars represent standard error of the mean (SEM). * = p-value <0.05 compared to NT1 and NT2 as determined by one-way ANOVA with Tukey comparison, ns = not significant.
[000136] Fig. 3 is directed to evaluating PIV infection (Parainfluenza virus 3 strain JS) in CRISPR-mediated deoxyhypusine synthase knockout cell lines. Deoxyhypusine synthase knockout cells show decreased percentage of Parainfluenza Virus Type 3 (PIV)-GFP positive cells compared to NT controls. sgRNAs targeting SLC35A1 were used as a positive control against PIV (SLC35A1) to evaluate the effect of known pro-pathogen genes in this infection model. The results are presented as means of four independent experiments. Error bars represent standard error of the mean (SEM). * = p-value <0.05 compared to NT1 and NT2 as determined by one-way ANOVA with Tukey comparison, ns = not significant. EXAMPLE 9 Embodiments
[000137] The present disclosure provides a method of treating or preventing a respiratory virus infection or one or more symptoms associated therewith or inhibiting or reducing viral protein synthesis, in a human subject in need thereof, which includes inhibiting activity of deoxyhypusine synthase within the human subject or decreasing an amount of deoxyhypusine synthase within the human subject in a manner to reduce the respiratory virus infection or one or more symptoms associated therewith. According to one aspect, inhibiting activity of deoxyhypusine synthase within the human subject is carried out by administering to the human subject an effective amount of a competitive inhibitor that binds to an active site of deoxyhypusine synthase and inhibits enzymatic activity of deoxyhypusine synthase. According to one aspect, the competitive inhibitor is a molecule that competes for binding with a molecule having structural similarity to spermidine and that binds to the active site of deoxyhypusine synthase and inhibits enzymatic activity of deoxyhypusine synthase. According to one aspect, inhibiting activity of deoxyhypusine synthase within the human subject is carried out by administering to the human subject an effective amount of an allosteric inhibitor that binds to deoxyhypusine synthase and inhibits enzymatic activity of deoxyhypusine synthase. According to one aspect, reducing the amount of deoxyhypusine synthase within the human subject is carried out by administering an effective amount of a degradation-promoting compound to the human subject that binds to deoxyhypusine synthase and promotes degradation of deoxyhypusine synthase. According to one aspect, the deoxyhypusine synthase includes an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO: 6. According to one aspect, the respiratory virus infection is caused by a coronavirus, a human rhinovirus, an influenza virus, a human parainfluenza virus, a respiratory syncytial virus or a human metapneumovirus. According to one aspect, reducing the respiratory virus infection is carried out by reducing viral load. According to one aspect, the competitive inhibitor is administered by inhalation, intranasally, transdermally, orally, rectally, transmucosally, intestinally, parenterally, intramuscularly, subcutaneously or intravenously. According to one aspect, the allosteric inhibitor is administered by inhalation, intranasally, transdermally, orally, rectally, transmucosally, intestinally, parenterally, intramuscularly, subcutaneously or intravenously. According to one aspect, the degradation-promoting compound is administered by inhalation, intranasally, transdermally, orally, rectally, transmucosally, intestinally, parenterally, intramuscularly, subcutaneously or intravenously.
[000138] The present disclosure provides a composition including at least a deoxyhypusine synthase inhibitor and a pharmaceutically acceptable excipient. According to one aspect, the deoxyhypusine synthase inhibitor is a competitive inhibitor, an allosteric inhibitor or a degradation-promoting compound. According to one aspect, the composition is provided as a single dose unit including an inhibitory effective amount of the deoxyhypusine synthase inhibitor.
[000139] The present disclosure provides use of a deoxyhypusine synthase inhibitor in the manufacture of a medicament for the treatment or prevention of a respiratory virus infection or one or more symptoms associated therewith or for reducing viral protein synthesis. According to one aspect, the deoxyhypusine synthase inhibitor is a competitive inhibitor, an allosteric inhibitor or a degradation-promoting compound. According to one aspect, the medicament is provided as a single dose unit including an inhibitory effective amount of the deoxyhypusine synthase inhibitor.
[000140] The present disclosure provides a deoxyhypusine synthase inhibitor for use in the treatment or prevention of a respiratory virus infection or one or more symptoms associated therewith or for use in inhibiting or reducing viral protein synthesis. According to one aspect, the treatment or prevention includes inhibiting activity of deoxyhypusine synthase or decreasing an amount of deoxyhypusine synthase in a manner to reduce the respiratory virus infection or one or more symptoms associated therewith. According to one aspect, the deoxyhypusine synthase inhibitor is a competitive inhibitor that competes for binding with a molecule having structural similarity to spermidine and binds to an active site of deoxyhypusine synthase and inhibits enzymatic activity of deoxyhypusine synthase. According to one aspect, the deoxyhypusine synthase inhibitor is an allosteric inhibitor that binds to deoxyhypusine synthase and inhibits enzymatic activity of deoxyhypusine synthase. According to one aspect, the deoxyhypusine synthase inhibitor is a degradation-promoting compound that binds to deoxyhypusine synthase and promotes degradation of deoxyhypusine synthase. According to one aspect, the deoxyhypusine synthase inhibitor is a competitive inhibitor, an allosteric inhibitor or a degradation-promoting compound. According to one aspect, the deoxyhypusine synthase includes an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. According to one aspect, the respiratory virus infection is caused by a coronavirus, a human rhinovirus, an influenza virus, a human parainfluenza virus, a respiratory syncytial virus or a human mctapncumovirus. According to one aspect, the treatment or prevention includes reducing the respiratory virus infection by reducing viral load. According to one aspect, the deoxyhypusine synthase inhibitor is administered by inhalation, intranasally, transdermally, orally, rectally, transmucosally, intestinally, parenterally, intramuscularly, subcutaneously or intravenously.
[000141] The invention now being fully described, it will be apparent that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.
SEQUENCE LISTING
SEQ ID NO: 1
EGSLEREAPAGALAAVLKHSSTLPPESTQVRGYDFNRGVNYRALLEAFGTTGFQATNFG RAVQQVNAMIEKKLEPLSQDEDQHADLTQSRRPLTSCTIFLGYTSNLISSGIRETIRYLVQ HNMVDVLVTTAGGVEEDLIKCLAPTYLGEFSLRGKELRENGINRIGNLLVPNENYCKFE DWLMPILDQMVMEQNTEGVKWTPSKMIARLGKEINNPESVYYWAQKNHIPVFSPALTD GSLGDMIFFHSYKNPGLVLDIVEAQEFDGSDSGARPDEAVSWGKIRVDAQPVKVYADA SLVFPLLVAETFAQKMDAFMHEKNED
SEQ ID NO: 2
EGSLEREAPAGALAAVLKHSSTLPPESTQVRGYDFNRGVNYRALLEAFGTTGFQATNFG RAVQQVNAMIEKKLEPLSQDEDQHADLTQSRRPLTSCTIFLGYTSNLISSGIRETIRYLVQ HNMVDVLVTTAGGVEEDLIKCLAPTYLGEFSLRGKELRENGINRIGNLLVPNENYCKFE DWLMPILDQMVMEQNTEGVKWTPSKMIARLGKEINNPESVYYWAQKNHIPVFSPALTD GSLGDMIFFHSYKNPGLVLDIVEAQEFDGSDSGARPDEAVSWGKIRVDAQPVKVYADA SLVFPLLVAETFAQKMDAFMHEKNED
SEQ ID NO: 3
PIIPAFWEAEAGGSREEEFETSLANMIEKKLEPLSQDEDQHADLTQSRRPLTSCTIFLGYT SNLISSGIRETIRYLVQHNMVDVLVTTAGGVEEDLIKCLAPTYLGEFSLRGKELRENGINR IGNLLVPNENYCKFEDWLMPILDQMVMEQNTEGVKWTPSKMIARLGKEINNPESVYYW AQKNHIPVFSPALTDGSLGDMIFFHSYKNPGLVLDIVEDLRLINTQAIFAKCTGMIILGGG VVKHHIANANLMRNGADYAVYINTAQEFDGSDSGARPDEAVSWGKIRVDAQPVKVYA DASLVFPLLVAETFAQKMDAFMHEKNED SEQ TD NO: 4
EGSLEREAPAGALAAVLKHSSTLPPESTQVRGYDFNRGVNYRALLEAFGTTGFQATNFG
RAVQQVNAMIEKKLEPLSQDEDQHADLTQSRRPLTSCTIFLGYTSNLISSGIRETIRYLVQ
HNMVDVLVTTAGGVEEDLIKCLAPTYLGEFSLRGKELRENGINRIGNLLVPNENYCKFE
DWLMPILDQMVMEQNTEGVKWTPSKM1ARLGKE1NNPESVYYWAQKNH1PVFSPALTD
GSLGDMIFFHSYKNPGLVLDIVEDLRLINTQAIFAKCTGMIILGGGVVKHHIANANLMVP
DQTRLSPGARSGWMHSPSRSMLTPPWSSPCLWLKPLPRRWMPSCMRRTRTERLRSQEG LTPSSIY
SEQ ID NO: 5
EGSEEREAPAGAEAAVEKHSSTEPPESTQVRGYDFNRGVNYRAEEEAFGTTGFQATNFG
RAVQQVNAMIEKKLEPLSQDEDQHADLTQSRRPLTSCTIFLGYTSNLISSGIRETIRYLVQ
HNMVDVLVTTAGGVEEDLIKCLAPTYLGEFSLRGKELRENGINRIGNLLVPNENYCKFE
DWLMPILDQMVMEQNTEGVKWTPSKMIARLGKEINNPESVYYWAQKNHIPVFSPALTD
GSLGDMIFFHSYKNPGLVLDIVEEDGCLHA
SEQ ID NO: 6
PILDQMVMEQNTEGVKWTPSKMIARLGKEINNPESVYYWAQKNHIPVFSPALTDGSLG
DMIFFHSYKNPGLVLDIVEDLRLINTQAIFAKCTGMIILGGGVVKHHIANANLMRNGAD
YAVYINTAQEFDGSDSGARPDEAVSWGKIRVDAQPVKVYADASLVFPLLVAETFAQKM
DAFMHEKNED
SEQ ID NO: 7
AGTTCTGTTCGATAGATGCC
SEQ ID NO: 8
TTGGGGATTAACCCAGAGCC
SEQ ID NO: 9
CGGGGCTACGACTTCAACCG
SEQ ID NO: 10
TGAACAGCATACACTAACGA
SEQ ID NO: 11
TGACGTGTGCAGTAATACTG

Claims

CLAIMS What is claimed is:
1. A method of treating or preventing a respiratory virus infection or one or more symptoms associated therewith or inhibiting or reducing viral protein synthesis, in a human subject in need thereof, comprising inhibiting activity of deoxyhypusine synthase within the human subject or decreasing an amount of deoxyhypusine synthase within the human subject in a manner to reduce the respiratory virus infection or one or more symptoms associated therewith.
2. The method of claim 1 wherein inhibiting activity of deoxyhypusine synthase within the human subject is carried out by administering to the human subject an effective amount of a competitive inhibitor that binds to an active site of deoxyhypusine synthase and inhibits enzymatic activity of deoxyhypusine synthase.
3. The method of claim 2 wherein the competitive inhibitor is a molecule that competes for binding with a molecule having structural similarity to spermidine and that binds to the active site of deoxyhypusine synthase and inhibits enzymatic activity of deoxyhypusine synthase.
4. The method of claim 1 wherein inhibiting activity of deoxyhypusine synthase within the human subject is carried out by administering to the human subject an effective amount of an allosteric inhibitor that binds to deoxyhypusine synthase and inhibits enzymatic activity of deoxyhypusine synthase.
5. The method of claim 1 wherein reducing the amount of deoxyhypusine synthase within the human subject is carried out by administering an effective amount of a degradationpromoting compound to the human subject that binds to deoxyhypusine synthase and promotes degradation of deoxyhypusine synthase.
6. The method of any one of claims 1-5 wherein the deoxyhypusine synthase comprises an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO: 6.
7. The method of any one of claims 1-6 wherein the respiratory virus infection is caused by a coronavirus, a human rhinovirus, an influenza virus, a human parainfluenza virus, a respiratory syncytial virus or a human metapneumovirus.
8. The method of any one of claim 1-7 comprising reducing the respiratory virus infection by reducing viral load.
9. The method of claim 2 or claim 3 wherein the competitive inhibitor is administered by inhalation, intranasally, transdermally, orally, rectally, transmucosally, intestinally, parenterally, intramuscularly, subcutaneously or intravenously.
10. The method of claim 4 wherein the allosteric inhibitor is administered by inhalation, intranasally, transdermally, orally, rectally, transmucosally, intestinally, parenterally, intramuscularly, subcutaneously or intravenously.
11. The method of claim 5 wherein the degradation-promoting compound is administered by inhalation, intranasally, transdermally, orally, rectally, transmucosally, intestinally, parenterally, intramuscularly, subcutaneously or intravenously.
12. A composition comprising a deoxyhypusine synthase inhibitor and a pharmaceutically acceptable excipient.
13. The composition of claim 12 wherein the deoxyhypusine synthase inhibitor is a competitive inhibitor, an allosteric inhibitor or a degradation-promoting compound.
14. The composition of claim 13 provided as a single dose unit including an inhibitory effective amount of the deoxyhypusine synthase inhibitor.
15. Use of a deoxyhypusine synthase inhibitor in the manufacture of a medicament for the treatment or prevention of a respiratory virus infection or one or more symptoms associated therewith or for reducing viral protein synthesis.
16. The use of claim 15 wherein the deoxyhypusine synthase inhibitor is a competitive inhibitor, an allosteric inhibitor or a degradation-promoting compound.
17. The use of claim 15 or claim 16 wherein the medicament is provided as a single dose unit including an inhibitory effective amount of the deoxyhypusine synthase inhibitor.
18. A deoxyhypusine synthase inhibitor for use in the treatment or prevention of a respiratory virus infection or one or more symptoms associated therewith or for use in inhibiting or reducing viral protein synthesis.
19. The deoxyhypusine synthase inhibitor for use of claim 18, wherein the treatment or prevention comprises inhibiting activity of deoxyhypusine synthase or decreasing an amount of deoxyhypusine synthase in a manner to reduce the respiratory virus infection or one or more symptoms associated therewith.
20. The deoxyhypusine synthase inhibitor for use of claim 18 or claim 19, wherein the deoxyhypusine synthase inhibitor is a competitive inhibitor that competes for binding with a molecule having structural similarity to spermidine and binds to an active site of deoxyhypusine synthase and inhibits enzymatic activity of deoxyhypusine synthase.
21. The deoxyhypusine synthase inhibitor for use of claim 18 or claim 19, wherein the deoxyhypusine synthase inhibitor is an allosteric inhibitor that binds to deoxyhypusine synthase and inhibits enzymatic activity of deoxyhypusine synthase.
22. The deoxyhypusine synthase inhibitor for use of claim 18 or claim 19, wherein the dcoxyhypusinc synthase inhibitor is a degradation-promoting compound that binds to deoxyhypusine synthase and promotes degradation of deoxyhypusine synthase.
23. The deoxyhypusine synthase inhibitor for use of claim 18, wherein the deoxyhypusine synthase inhibitor is a competitive inhibitor, an allosteric inhibitor or a degradation-promoting compound.
24. The deoxyhypusine synthase inhibitor for use of any one of claims 18-23, wherein the deoxyhypusine synthase comprises an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.
25. The deoxyhypusine synthase inhibitor for use of any one of claims 18-24, wherein the respiratory virus infection is caused by a coronavirus, a human rhinovirus, an influenza virus, a human parainfluenza virus, a respiratory syncytial virus or a human metapneumovirus.
26. The deoxyhypusine synthase inhibitor for use of any one of claims 18-25, wherein the treatment or prevention comprises reducing the respiratory virus infection by reducing viral load.
27. The deoxyhypusine synthase inhibitor for use of any one of claims 18-26, wherein the deoxyhypusine synthase inhibitor is administered by inhalation, intranasally, transdermally, orally, rectally, transmucosally, intestinally, parenterally, intramuscularly, subcutaneously or intravenously.
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Citations (1)

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WO2001056553A2 (en) * 2000-02-02 2001-08-09 Axxima Pharmaceuticals Ag Pharmaceutically active aromatic guanylhydrazones

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WO2001056553A2 (en) * 2000-02-02 2001-08-09 Axxima Pharmaceuticals Ag Pharmaceutically active aromatic guanylhydrazones

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FIRPO MASON R. ET AL: "Targeting Polyamines Inhibits Coronavirus Infection by Reducing Cellular Attachment and Entry", ACS INFECTIOUS DISEASES, vol. 7, no. 6, 23 September 2020 (2020-09-23), US, pages 1423 - 1432, XP093071492, ISSN: 2373-8227, DOI: 10.1021/acsinfecdis.0c00491 *
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