EP4673226A1 - Rna-based inhibitors of trna modifying enzymes - Google Patents

Rna-based inhibitors of trna modifying enzymes

Info

Publication number
EP4673226A1
EP4673226A1 EP24764660.7A EP24764660A EP4673226A1 EP 4673226 A1 EP4673226 A1 EP 4673226A1 EP 24764660 A EP24764660 A EP 24764660A EP 4673226 A1 EP4673226 A1 EP 4673226A1
Authority
EP
European Patent Office
Prior art keywords
trna
cancer
rna molecule
rna
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24764660.7A
Other languages
German (de)
French (fr)
Inventor
Austin DRAYCOTT
Cole LEWIS
Wendy GILBERT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yale University
Original Assignee
Yale University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yale University filed Critical Yale University
Publication of EP4673226A1 publication Critical patent/EP4673226A1/en
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
    • C12Y103/01091—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1) tRNA-dihydrouridine20 synthase (NAD(P)+)(1.3.1.91)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/0107—Pseudouridylate synthase (4.2.1.70)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/18—Type of nucleic acid acting by a non-sequence specific mechanism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/335—Modified T or U

Definitions

  • 5- fluorouracil is a known anticancer drug that can be misincorporated into DNA of drug- treated cells, and it is believed that accumulation of 5-FU in the genome is correlated with 5-FU cytotoxicity in mammalian cells. As such, these compounds have significant off target effects.
  • the present invention is directed to an RNA molecule including at least one nucleotide capable of forming a covalent bond with a tRNA modifying enzyme, wherein formation of the covalent bond inhibits the tRNA modifying enzyme.
  • the present invention is directed to a composition including the RNA molecule.
  • the composition is a pharmaceutical composition.
  • the present invention is directed to a method for killing a cell.
  • the method includes: contacting the RNA molecule with the tRNA modifying enzyme in the cell.
  • the formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cell.
  • the present invention is directed to a method for treating a cancer in a subject in need thereof.
  • the method includes: administering to the subject an effective amount of the pharmaceutical composition.
  • the RNA molecule contacts the tRNA modifying enzyme in a cancer cell of the cancer, and formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cancer cell.
  • RNA molecule molecule
  • the present invention is directed to an RNA molecule.
  • the RNA molecule comprises at least one nucleotide capable of forming a covalent bond with a tRNA modifying enzyme.
  • the formation of the covalent bond inhibits the tRNA modifying enzyme.
  • the RNA molecule is a tRNA molecule.
  • the at least one nucleotide comprises a non-natural base.
  • the at least one nucleotide is 5-halouridine (5-haloU), 5- halocytidine (5-haloC), 5-aza-cytidine (5-azaC), 8-haloadenosine (8-haloA), 8-azanebularine, 8- aza-adenosine (8-azaA), or 8-haloguanosine (8-haloG).
  • the tRNA modifying enzyme is a dihydrouridine synthase (DUS) or a pseudouridine synthase (PUS).
  • the tRNA modifying enzyme is dihydrouridine synthase 1 (DUS1), dihydrouridine synthase 2 (DUS2), dihydrouridine synthase 3 (DUS3), dihydrouridine synthase 4 (DUS4), dihydrouridine synthase 1-like (DUS IL), dihydrouridine synthase 3 like (DUS3L), dihydrouridine synthase 4 like (DUS4L), pseudouridine synthase 1 (PUS1), pseudouridine synthase like l(PUSLl), pseudouridine synthase 3 (PUS3), TruB pseudouridine synthase family member 1 (TRUB1), TruB pseudouridine synthase family member 2 (TRUB2), dyskerin pseudouridine synthase 1 (DKC1), pseudouridine synthase 1 (DKC1),
  • the non-natural base is 5-haloU
  • the tRNA modifying enzyme is DUS2, DUS1L, DUS3L, DUS4L, ISCU, PUS1, PUS3, PUS7, PUS10, PUSL1, PUS7L, RPUSD1, RPUSD2, RPUSD4, TRMT2A, TRMT2B, TRUB1, TRUB2, or combinations thereof.
  • the nucleotide comprising the non-natural base is 5-azaC
  • the tRNA modifying enzyme is DNMT2, NSUN2, NSUN3, NSUN6, or combinations thereof.
  • the nucleotide comprising the non-natural base is 8-halo-G
  • the tRNA modifying enzyme is METTL1, WDR4, or combinations thereof.
  • the nucleotide comprising the non-natural base is 8-azaA
  • the tRNA modifying enzyme is ADAT1, ADAT2, ADAT3, or combinations thereof.
  • the at least one nucleotide is at a position corresponding to a natural position of a natural nucleotide of a natural tRNA that is modified by the tRNA modifying enzyme.
  • the RNA molecule is a tRNA molecule, and the non-natural modified base is a 5-halouracil in the D-loop, the t-psi-c loop, the anticodon loop, the RNA stems, and other portions of the tRNA molecule.
  • the RNA molecule comprises the sequence of any one of SEQ ID NOs:l-53; or comprises at least about 80% sequence identity to the sequence of any one of SEQ ID NOs:l-53.
  • the RNA molecule is an isolated tRNA molecule.
  • the two or more non-natural bases inhibit two or more different tRNA modifying enzymes.
  • the composition comprises the RNA molecule herein.
  • the formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cell.
  • the cell is a cancer cell in a culture.
  • the method comprises: administering to the subject an effective amount of the pharmaceutical composition herein.
  • the RNA molecule contacts the tRNA modifying enzyme in a cancer cell of the cancer.
  • the formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cancer cell.
  • the cancer is a bladder cancer, a breast cancer, a cervix cancer, a bile duct cancer, a colon cancer, an esophageal cancer, a head/neck cancer, a kidney clear cancer, a kidney papillary cancer, a liver cancer, a lung non small cell cancer, a lung small cell cancer, a prostate cancer, a rectum cancer, a sarcoma cancer, a stomach cancer, a uterine cancer, a liquid tumor (e.g., acute myeloid leukemia/ AML and the like), and the like.
  • a liquid tumor e.g., acute myeloid leukemia/ AML and the like
  • the method further comprises administering a chemotherapy to the subject.
  • Figs. 1A-1B depict non-limiting examples of tRNA modifying enzymes that are upregulated in cancers.
  • Fig. 1A dihydrouridine synthases (DUS) upregulated in cancers.
  • Fig. IB pseudouridine synthases (PUS) upregulated in cancers.
  • Fig. 2 depicts 5-halouracil forming covalent bound with a DUS, in accordance with some embodiments.
  • Fig. 3 depicts a non-limiting example of a tRNA molecule of the invention covalently bound to a tRNA modifying enzyme, in accordance with some embodiments.
  • Fig. 4 depicts a western blot confirming that a non-limiting example of the RNA molecule described herein was able to form covalent bond with a non-limiting tRNA modifying enzyme, DUS2, in accordance with some embodiments.
  • Fig. 5 is a graph showing the RNA molecule of Fig. 4 was able to kill cells of the lung adenocarcinoma cell line PC9, in accordance with some embodiments.
  • Figs. 6A-6I depict that DUS2 is overexpressed in lung cancer and loss of DUS2 sensitizes cells to ferroptosis, in accordance with some embodiments.
  • Fig. 6A Schematic of key regulators and chemical effectors of ferroptosis.
  • Fig. 6B High DUS2 mRNA expression correlates with resistance to chemical ferroptosis inducers in a panel of 860 cancer cell lines.
  • Fig. 6C DUS enzymes convert uridine to dihydrouridine.
  • Fig. 6D DUS2 RNA levels are significantly higher in lung adenocarcinoma (LU AD) tumor samples compared to normal tissue (two tailed Mann-Whitney U test, CysGCA p ⁇ 0001).
  • Fig. 6A-6I depict that DUS2 is overexpressed in lung cancer and loss of DUS2 sensitizes cells to ferroptosis, in accordance with some embodiments.
  • Fig. 6A Schematic of key regulators and chemical effect
  • FIG. 6E High DUS2 expression predicts worse outcomes for NSCLC patients.
  • Fig. 6F DUS2 protein expression is absent in clonal A549 DUS2 KO cells.
  • Fig. 6G Increased cell death (Annexin V+/PI+ cells) in DUS2 KOs compared to WT A549 following treatment with 2pM RSL-3.
  • Fig. 6H Elevated lipid ROS (Cl 1-BODIPY staining) in DUS2 KOs compared to WT A549 following treatment with 2pM RSL3.
  • Fig. 61 Quantification of Fig. 6H (two tailed t-test, * p ⁇ 0001).
  • Figs. 7A-7F depict that DUS2 is required to sustain levels of a specific tRNA, tRNACysGCA, in accordance with some embodiments.
  • Fig. 7A DUS2 targets position U20 in tRNA.
  • Fig. 7B Quantification of tRNA charging levels in DUS2 WT and KO cells. tRNA charging is unaffected by loss of DUS2, whereas Gin starvation reduces tRNAGln charging levels >50%.
  • Fig. 7C Changes in tRNA levels in DUS2 KO cells, black dots (FDR ⁇ 0.05), grey (FDR >0.05), red (tRNACysGCA with FDR ⁇ .05).
  • Fig. 7A-7F depict that DUS2 is required to sustain levels of a specific tRNA, tRNACysGCA, in accordance with some embodiments.
  • Fig. 7A DUS2 targets position U20 in tRNA.
  • Fig. 7B Quantification of tRNA charging
  • Example isodecoder tRNACysGCA10-l shows -50% lower expression in DUS2 KOs.
  • Fig. 7E Analysis of tRNA levels in TCGA LU AD data. tRNACysGCA but not tRNAGlnCTG levels are significantly higher in tumors compared to normal tissue (two tailed Mann-Whitney U test, CysGCA p ⁇ 0001).
  • Figs. 8A-8I depict that loss of DUS2 impairs translation of cysteine rich proteins, including metallothioneins, which leads to ferroptosis sensitivity, in accordance with some embodiments.
  • Fig. 8A Cysteine translation reporter.
  • Fig. 8B DUS2 KO impairs translation of TGT and TGC cysteine codons (two tailed t-test, *p ⁇ 03).
  • Fig. 8C Rescue of TGT translation by transfection of tRNACysGCA into DUS2 KO cells (two tailed t-test, * p ⁇ 0001).
  • Fig. 8D Proteins with high (>5%) Cys content are reduced in DUS2 KO cells.
  • Fig. 8E Metal lothi on ein translation reporter.
  • Fig. 8F Metal lothi on eins (MT1 A and MT1 G) are translated less well in DUS2 KOs.
  • Fig. 8G Increased cell death (Annexin V+/PI+ cells) in DUS2 KOs compared to WT A549 following addition of 62.5pM ZnCh.
  • Fig. 8H DUS2 KO cells have lower levels of reduced GSH (two tailed t-test, * p ⁇ 004).
  • Fig. 81 Model of anti-ferroptotic function of DUS2.
  • Figs. 9A-9G depict that combined loss of DUS2 and ferroptosis induction extends lifespan in a mouse xenograft NSCLC model, in accordance with some embodiments.
  • Fig. 9A A549 tumors grow faster than DUS2 KO tumors.
  • Fig. 9B Example tumor from A549 and DUS2 KO-2.
  • Fig. 9C DUS2 KO tumors have higher expression of a marker of ferroptosis, PTGS2, than A549 cells by qRT-PCR (ANOVA, * p ⁇ 002).
  • Fig. 9D Dosing scheme for xenograft experiments.
  • Fig. 9A-9G depict that combined loss of DUS2 and ferroptosis induction extends lifespan in a mouse xenograft NSCLC model, in accordance with some embodiments.
  • Fig. 9A A549 tumors grow faster than DUS2 KO tumors.
  • Fig. 9B Example tumor from A5
  • FIG. 9E Oral JKE-1674 treatment induces PTGS2 mRNA in mouse lungs (ANOVA, * p ⁇ 03).
  • Fig. 9F JKE-1674 treatment increases PTGS2 mRNA expression in DUS2 KO tumors (ANOVA, * p ⁇ 02).
  • Fig. 9G Mice implanted with DUS2 KO xenograft tumors survive longer than mice implanted with A549 tumors when treated with JKE-1674 (Mantel-Cox test * p ⁇ .03).
  • Figs. 10A-10E depict that fraction of dead (Annexin V+/PI+) DUS2 KO cells is reduced with pre-treated with DUS2 expression plasmid, Ferrostatin-1, Trolox but not ZVAD-FMK when treated with 2pM RSL-3.
  • Fig. 10D DUS2 KOs have higher levels of lipid ROS measured by Cl 1-BODIPY staining when treated with 200nM ML162.
  • Fig. 10E DUS2 KOs have higher levels of cellular ROS measured by H2DCFDA staining when treated with 2pM RSL-3.
  • Fig. 11 depicts that, in aggregate, the total pool of tRNACysGCA is reduced -40% in DUS2 KO clones.
  • Fig. 12B mRNAs encoding cysteine rich proteins are reduced in DUS2 KO cells consistent with RQC. Cumulative distribution of changes in mRNA abundance (log2 fold change, K-S test, p ⁇ 0001).).
  • Fig. 12C Depletion of RQC factor GIGYF2 (siGIGYF2) rescues MT1A mRNA levels in DUS2 KO cells compared to non-targeting control (siNT) (two tailed t-test, * p ⁇ 04).
  • Figs. 13A-13B depict that mouse weights generally increased over time with vehicle and JKE-1674.
  • Fig. 13B Mice receiving JKE-1674 had shorter median survival than mice receiving vehicle.
  • Figs. 14A-14B demonstrate that a non-limiting example of the RNA-based DUS inhibitors herein, CLB-001, was able to kill cancer cells with nanomolar level IC50, in accordance with some embodiments.
  • the hepatocellular carcinoma cell line, HepG2 (Fig. 14A), and the non-small cell lung cancer cell line, A549 (Fig. 14B) were subjected to various concentrations of CLB-001. The viabilities of the cells are plotted against the CLB-001 concentrations.
  • Fig. 15 demonstrates that the non-limiting example of the RNA-based DUS inhibitors herein, CLB-001, does not kill non-cancerous cells, in accordance with some embodiments.
  • the non-transformed hepatocyte cell line, AML 12 was subjected to various concentrations of CLB- 001.
  • the viabilities of the cells are plotted against the CLB-001 concentrations.
  • Fig. 16 demonstrates that the non-limiting example of the RNA-based DUS inhibitors herein, CLB-001, is vastly more potent than 5-FU, in accordance with some embodiments.
  • HepG2 was subjected to various concentrations of CLB-001.
  • the viabilities of the cells are plotted against the CLB-001 concentrations.
  • Figs. 17A-17C demonstrate that tRNA modifying enzymes are upregulated in hepatocellular carcinoma, in accordance with some embodiments.
  • the mRNA levels of various tRNA modifying enzymes from hepatocellular carcinoma (HCC) tumor tissues were compared with those from normal liver tissues.
  • the tRNA modifying enzymes were found to be consistently overexpressed in the HCC tumors.
  • Figs. 18A-18G demonstrate that high expression of tRNA modifying enzymes predicts worse outcomes in hepatocellular carcinoma (HCC), in accordance with some embodiments.
  • HCC patients are grouped according to the expression levels of the tRNA modifying enzymes in the tumor tissues, and the patient survival percentages with time are plotted according to the grouping.
  • Fig 19 demonstrates that a non-limiting example of the RNA-based DUS and PUS inhibitors herein, CLB-001, was able to kill cancer cells with nanomolar level IC50, in accordance with some embodiments.
  • the hepatocellular carcinoma cell lines, SNU-387, HepG2, and PLC/PRF/5 and the non-small cell lung cancer cell line, A549 were subjected to various concentrations of CLB-001 .
  • the viabilities of the cells are plotted against the CLB-001 concentrations.
  • the invention is based in part on the discovery that substrate mimetic transfer RNAs (tRNAs) that incorporate non-natural nucleotides at specific positions can be used to covalently trap tRNA modifying enzymes e.g., tRNA modifying enzymes that drive cancer progression and/or metastasis), thereby inhibiting them.
  • tRNAs substrate mimetic transfer RNAs
  • an element means one element or more than one element.
  • isolated refers to molecules, which are isolated from other cellular components and is meant to encompass both purified and recombinant molecules.
  • isolated RNA molecule(s) thus refers to, for example, a RNA molecule(s) that is/are substantially free of cellular material, viral material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • Identity refers to the subunit sequence identity between two polymeric molecules, for example between two nucleic acid molecules, such as, between two RNA molecules.
  • two polynucleotide sequences have the same nucleotides at the same positions, e.g., if a position in each of two nucleic acid molecules is occupied by a uracil, then they are identical at that position.
  • the identity or extent to which two sequences have the same, for example nucleotide, at the same positions in an alignment is often expressed as a percentage.
  • two polynucleotides may each comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and may further comprise a sequence that is divergent between the two polynucleotides
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity.
  • Homology or identity can be determined by sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL known in the art.
  • the term “inhibit(s)” or “inhibiting” an tRNA modifying enzyme refers to any statistically significant decrease in biological activity of the tRNA modifying enzyme, including full blocking of the activity.
  • non-natural and “non-naturally occurring” refer to that which is not present in nature.
  • a “non-natural” or “non-naturally occurring nucleic acid or nucleotide refers to a nucleic acid or nucleotide that is not present in nature.
  • non-naturally occurring nucleic acids can include one or more non-natural base, sugar, and/or inter-subunit linkage, e.g., a sugar, base, and/or linkage that has been modified or substituted with respect to that found in a naturally occurring nucleic acid molecule.
  • non-naturally occurring nucleic acids include more than one type of modification, e.g., but not limited to, sugar and base modifications, sugar and linkage modifications, base and linkage modifications, or base, sugar, and linkage modifications.
  • a “subject” or “patient,” as used therein, may be a human or non -human mammal.
  • Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals.
  • wildtype is meant a non-mutated version of a gene, allele, genotype, nucleic acid, polypeptide, or phenotype, or a fragment of any of these. It may occur in nature or produced recombinantly.
  • the term “consisting essentially of’ when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”
  • the term “consisting essentially of’ refers to a composition, whose only active ingredient is the indicated active ingredient(s) (e.g., the indicated RNA molecule(s)), however, other components may be included which are for stabilizing, preserving, etc. the formulation, but are not involved directly in the therapeutic effect of the indicated active ingredient.
  • the terms “treat,” treating,” “ameliorating,” “treatment,” and the like refer to reducing or improving a disease or condition and/or one or more symptoms associated therewith. It will be appreciated that, although not precluded, treating a disease or condition and/or one or more symptoms associated therewith does not require that the disease, condition, or symptoms associated therewith be completely ameliorated or eliminated. It means that the clinical signs and/or the symptoms associated with a disease or condition are lessened as a result of the actions performed. The signs or symptoms to be monitored will be well known to the skilled clinician.
  • ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • the present invention provides an RNA molecule comprising at least one nucleotide capable of forming a covalent bond to a tRNA modifying enzyme, wherein formation of the covalent bond inhibits the tRNA modifying enzyme.
  • RNA molecules can be prepared using methods known in the art including, but not limited to, in vitro transcriptions and chemical synthesis. Nelissen, E. et aL, Nucleic Acids Research, 40(13):el02 (2012), Milligan, J. F. et al., Nucleic Acids Research, 15:8783-8798 (1987), Marshall, W. S. et al., Curr. Opin. Chem. Biol., 8:222-229 (2004), Ponchon, L. et aL, Nat. Protoc., 4:947-959 (2009), Ponchon, L. et al., Nat. Methods, 4:571-576 (2007), Ponchon, L. et l., Methods, 54:267-273 (2011), and U.S. Patent Application No, 2009/0298920, each of which is herein incorporated by reference in its entirety, describe methods for production of RNA.
  • the RNA molecule comprises any ribonucleic chain. In another embodiment, the RNA molecule comprises about 1,000 ribonucleotides in length, illustratively, about 5 to about 1,000, about 10 to about 900, about 20 to about 500, about 30 to about 300, about 40 to about 200, and about 70 to about 100 ribonucleotides.
  • the RNA molecule comprises about 76 to about 96 ribonucleotides.
  • the RNA molecule comprises about 60 to about 86 ribonucleotides.
  • formation of a bond between the at least nucleotide and the tRNA modifying enzyme can be achieved through any of a variety of direct or indirect covalent associations or attachments.
  • the at least one nucleotide is capable of forming a covalent bond to a tRNA modifying enzyme through direct or indirect formation of a covalent bond between a base of the at least one nucleotide and an amino acid residue of the tRNA modifying enzyme.
  • the covalent attachment of the at least one nucleotide to the tRNA modifying enzyme is irreversible.
  • the covalent attachment of the at least one nucleotide to the tRNA modifying enzyme inhibits the tRNA modifying enzyme.
  • inhibition comprises a decrease of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% in tRNA modifying enzyme activity.
  • inhibition comprises a decrease of 100% in tRNA modifying enzyme activity.
  • formation of the bond covalently traps the tRNA modifying enzyme, irreversibly inhibiting.
  • the at least one nucleotide is a non-natural nucleotide. In one embodiment, the at least one nucleotide comprises a non-natural base.
  • the RNA molecule comprises no more than 1, 2, 3, 4, ,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 non-natural nucleotides, independently each nucleotide comprising a non-natural base.
  • RNA molecule can include one or several heterocyclic bases other than the principal five base components of naturally-occurring nucleic acids.
  • the heterocyclic base includes, but is not limited to, uracil-5-yl cytosine-5-yl, adenine-7-yl, adenine-8-yl, guanine-7-yl, guanine-8-yl, 4-aminopyrrolo[2,3- d]pyrimidin-5-yl, 2-amino-4-oxopyrolo[2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3- d]pyrimidin-3-yl groups, where the purines are attached to the sugar moiety of the ISS via the 9- position, the pyrimidines via the 1 -position, the pyrrolopyrimidines via the 7-position and the pyrazolopyrimidines via the 1 -position.
  • the at least one nucleotide comprises at least one modified base.
  • base modifications include, but are not limited to, uracils modified at C-5 and/or C- 6, preferably with a halogen, including, but are not limited to, fluorouracil such as 5 -fluorouracil (5-FU), bromouracil such as 5-bromouracil, chlorouracil such as 5 -chlorouracil, and iodouracil such as 5-iodouracil and hydroxyuracil.
  • fluorouracil such as 5 -fluorouracil (5-FU)
  • bromouracil such as 5-bromouracil
  • chlorouracil such as 5 -chlorouracil
  • iodouracil such as 5-iodouracil and hydroxyuracil.
  • base modifications include 8- azaadenosine (8-aza-Ad), 7-deazaadenosine, N6-methyl-7-deazaadenosine, N6methyl-8- azaadenosine, 7-deaza-8 -azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N625 amino-7- deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-azaadenosine, N6- hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7- deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6thio-7- deazaguanosine, 6-thio-8 -azaguanosine, 7-
  • azacytosine 5 -bromocytosine, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, 5,6-dihydrocytosine, 5- iodocytosine, 5 -nitrocytosine, 5-hydroxy-cytosine, 6-thio-guanine, and 4-thiouracil.
  • the amino acid residue of the tRNA modifying enzyme is a conserved catalytic amino acid residue.
  • the amino acid residue of the tRNA modifying enzyme is a reactive amino acid residue. In one embodiment, the amino acid residue of the tRNA modifying enzyme is a reactive nucleophilic amino acid residue.
  • the reactive nucleophilic amino acid residue is a reactive cysteine residue, wherein formation of the covalent bond occurs via nucleophilic attack of the at least one nucleotide by the reactive cysteine residue.
  • the at least one nucleotide of the RNA molecule is a 5- halopyrmidine, wherein the at least one nucleotide is capable of forming a covalent bond with a conserved catalytic cysteine residue of the tRNA modifying enzyme, wherein the covalent bond crosslinks the RNA molecule to the tRNA modifying enzyme.
  • the crosslink begins with reduction (e.g., enzymatic reduction) of 5-halouridine to 5-halodihydrouridine followed by nucleophilic attack of a conserved catalytic cysteine of the tRNA modifying enzyme on the C5 position with halide serving as leaving group.
  • reduction e.g., enzymatic reduction
  • nucleophilic attack of a conserved catalytic cysteine of the tRNA modifying enzyme on the C5 position with halide serving as leaving group.
  • the RNA molecule is a tRNA molecule.
  • the tRNA molecule of the present invention is a single ribonucleotide chain which is capable of folding to adopt a characteristic, so-called cloverleaf secondary structure.
  • the secondary structure comprises (i) an acceptor stem composed of a first 7 ribonucleotides of the 5’ end of the ribonucleotide chain and 7 ribonucleotides that precede the last 4 ribonucleotides of the 3’ end of the ribonucleotide chain, thus forming a double-stranded structure comprising about 6 or 7 pairs of ribonucleotides, it being possible for the ribonucleotides comprising the first ribonucleotide of the 5’ end of the ribonucleotide chain and the ribonucleotide that precedes the last 4 ribonucleotides of the 3’ end of the ribonucleotide chain not to be paired; (ii) a D arm comprising 4 pairs of ribonucleotides and a D loop comprising about 8 to 10 ribonucleotides, formed by the folding of a part of the ribon
  • the tRNA modifying enzyme is a dihydrouridine synthase (DUS) or a pseudouridine synthase (PUS). Both DUS and PUS are known to contain highly conserved cysteine or aspartic acid residues in their respective active sites.
  • DUS dihydrouridine synthase
  • PUS pseudouridine synthase
  • the tRNA modifying enzyme is dihydrouridine synthase 1 (DUS1), dihydrouridine synthase 2 (DUS2), dihydrouridine synthase 3 (DUS3), dihydrouridine synthase 4 (DUS4), dihydrouridine synthase 1 -like (DUS1L), dihydrouridine synthase 3 like (DUS3L), dihydrouridine synthase 4 like (DUS4L), pseudouridine synthase 1 (PUS1), pseudouridine synthase like l(PUSLl), pseudouridine synthase 3 (PUS3), TruB pseudouridine synthase family member 1 (TRUB1), TruB pseudouridine synthase family member 2 (TRUB2), dyskerin pseudouridine synthase 1 (DKC1), pseudouridine synthase 7 (PUS7), pseudouridine synthase 7 like (PUS7L), RNA pseudouridylate synthase domain containing 1 (RP1), dihydr
  • the non-natural base in the RNA molecule is selected based on the target tRNA modifying enzyme that the RNA molecule is to inhibit.
  • a non-limiting list of nucleotides comprising non-natural bases together with tRNA modifying enzymes is shown below in Table 1.
  • the non-natural base is 5-haloU
  • the tRNA modifying enzyme is DUS2, DUS1L, DUS3L, DUS4L, ISCU, PUS1, PUS3, PUS7, PUS10, PUSL1, PUS7L, RPUSD1, RPUSD2, RPUSD4, TRMT2A, TRMT2B, TRUB1, TRUB2, or combinations thereof.
  • the tRNA molecule of the present invention comprises the sequence of a naturally occurring tRNA molecule, wherein the at least one nucleotide is a non-natural nucleotide located at a nucleotide position of the tRNA molecule that corresponds to the naturally occurring position, or is at a position that is no more than five, four, three, two, or one nucleotide position away from the naturally occurring position, in the naturally occurring tRNA molecule.
  • the location of the non-natural base is not limited, as tRNA modifying enzymes, such as those described herein, are known to modify bases located in virtually all the locations of tRNA molecules.
  • the location of the non-natural base is determined based on the enzyme to be inhibited, the sequence of the parent natural tRNA, as well as the non-natural base.
  • DUS enzymes can modify uridines in the D-loop (as well as uridines outside the D- loop such as the tRNA stems) of tRNAs and can be inhibited by 5-halo uridine.
  • inhibitors specific for DUS enzymes can be designed by modifying natural tRNA by incorporating a 5-halo uridine in the D-loop.
  • PUS can modify uridines in the D-loop, the t-psi-c loop, or the anticodon loop (as well as uridines outside the D-loop such as the tRNA stems).
  • inhibitors specific for PUS enzymes can be designed by modifying natural tRNA by incorporating a 5-halo uridine in the D-loop, the t-psi-c loop, and/or the anticodon loop.
  • the tRNA molecule comprises an anticodon-arm and an acceptor arm, wherein the anticodon-arm comprises a trinucleotide anticodon, wherein the anticodon recognizes a stop codon.
  • the anticodon recognizes a codon for alanine, arginine, aspartic acid, asparagine, cysteine, glycine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.
  • the anticodon recognizes a codon for cysteine.
  • the acceptor arm comprises a 3'-terminal sequence of 5’-cytidine-cytidine- adenosine (CCA)-3’ that overhangs the end.
  • the RNA molecule comprises the sequence: GGGGGXAXAGCXCAGXGGXAGAGCAXXXGACXGCAGAXCAAGAGGXCCCCGGXX CAAAXCCGGGXGCCCCC, wherein one or more of the Xs any nucleotide comprising a nonnatural base such as, for example, 5-halouracil (SEQ ID NO: 1).
  • the RNA molecule comprises the sequence of SEQ ID NO: 1, with the proviso that X is a U, C, or G, and one or more U, C, or G of SEQ ID NO: 1 comprises a non-natural base.
  • the RNA molecule comprises a sequence of a natural tRNA molecule including, but not limited to, any one of the tRNA molecules listed in Table 2, with the proviso that the RNA molecule comprises at least one nucleotide capable of forming a covalent bond with a tRNA modifying enzyme at any one of the nucleotide positions.
  • at least one U, A, G or C residue in the natural tRNA molecule is substituted with a 5-haloU, 8-haloG, 5-halo-C, 5-aza-C, or 8-azaA.
  • the RNA molecule comprises about 80% identity or more with the parent tRNA, such as about 85% identity or more, about 90% identity or more, about 92% identity or more, about 95% identity or more, about 97% identity or more, about 98% identity or more, about 99% identity or more, or identical.
  • a RNA molecule of the present invention comprises a percent degree of sequence identity to the sequence of any one of SEQ ID NOs: 1-53, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of any one of SEQ ID NOs: 1-53.
  • the specified degree of sequence identity retains one or more characteristics, e.g., substrate for a tRNA modifying enzyme and/or structure (e.g., cloverleaf secondary structure), as the RNA molecule of SEQ ID NOs: 1-53.
  • substrate for a tRNA modifying enzyme and/or structure e.g., cloverleaf secondary structure
  • the RNA molecule is an isolated or purified tRNA molecule.
  • the RNA molecule is purified.
  • the contaminants can be cellular proteins remaining after expression of the RNA molecule of interest in cell systems, or chemicals remaining after chemical synthesis. Suitable methods to purify the RNA molecule from a mixture of contaminants are known in the art.
  • the purity of the RNA molecule(s) of the invention is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% purity.
  • the RNA molecule (such as the tRNA molecule) comprises two or more non-natural bases, wherein the two or more non-natural bases inhibit two or more different tRNA modifying enzymes.
  • the non-natural modified base is a 5-halouracil in the D-loop, the t-psi-c loop, and/or the anticodon loop of the tRNA molecule.
  • the regimen for administration may affect what constitutes an effective amount.
  • the therapeutic formulations contemplated within the disclosure may be administered to the subject either prior to or after the onset of a disease and/or disorder contemplated herein. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations contemplated within the disclosure may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • a non-limiting example of an effective dose range for a therapeutic compound contemplated within the disclosure is from about 1 and 5,000 mg/kg of body weight/per day.
  • One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • RNA molecules of the invention for administration may be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 3050 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.
  • the administered dose of the RNA molecules is about 1 mg to about 2,500 mg. In some embodiments, the dose used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg.
  • a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
  • the present disclosure is directed to a packaged pharmaceutical composition
  • a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a RNA molecule of the invention, alone or in combination with a second pharmaceutical agent; and instructions for treating or preventing, or reducing one or more symptoms of cancer in a subject.
  • Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for intracranially, oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art.
  • the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.
  • routes of administration of any of the compositions described herein include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical.
  • the compounds for use in the disclosure may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
  • compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein. Oral Administration
  • compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets.
  • excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.
  • the tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.
  • the compounds described herein may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e. , polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fdlers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate).
  • the tablets may be coated using suitable methods and coating materials such as OPADRYTM film coating systems available from Colorcon, West Point, Pa.
  • Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions.
  • the liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g, methyl or propyl p-hydroxy benzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats
  • emulsifying agent e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters or ethyl alcohol
  • preservatives e.g, methyl or propyl p-hydroxy benzoates or sorbic acid
  • the present disclosure also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds described herein, and a further layer providing for the immediate release of another medication.
  • a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.
  • RNA molecules described herein may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion.
  • Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used.
  • Additional dosage forms of the present invention include dosage forms as described in U.S. Patents Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos.
  • the formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
  • sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period.
  • the period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
  • the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds.
  • the compounds for use the method described herein may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
  • the RNA molecules described herein are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
  • delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.
  • pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
  • immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
  • short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
  • rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
  • RNA molecule of the present invention depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of the cancer in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.
  • a suitable dose of a RNA molecule described herein may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day.
  • the dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.
  • RNA molecule dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days.
  • a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
  • the administration of the modulator of the disclosure is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday").
  • the length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days.
  • the dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the patient's condition, to a level at which the improved disease is retained.
  • patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection.
  • RNA molecules for use in the methods described herein may be formulated in unit dosage form.
  • unit dosage form refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier.
  • the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
  • Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50.
  • Capsid assembly modulators exhibiting high therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human.
  • the dosage of such capsid assembly modulators lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity.
  • the dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.
  • RNA-based inhibitor In the tRNACys, position 20 (the 19 th nucleoside), a uridine, is naturally modified by tRNA modifying enzymes such as DUS2 into a dihydrouridine.
  • tRNA modifying enzymes such as DUS2 into a dihydrouridine.
  • the above-mentioned uridine was replaced with the non-natural modified nucleoside 5-fluorouridine (5-FU).
  • 5-FU tRNA or tRNACys-5FU This non-limiting example of RNA molecule inhibitor has the sequence set forth in SEQ ID NO: 1.
  • Dihydrouridine is a universally conserved tRNA modification installed by enzymes that are important for human health for reasons that are yet unclear.
  • High expression of dihydrouridine synthase 2 (DUS2) predicts poor patient outcomes in lung adenocarcinomal.
  • DUS2 dihydrouridine synthase 2
  • the present study shows in human cells and mouse xenografts that DUS2 suppresses ferroptosis, a metal -dependent non-apoptotic form of cell death to which many lung cancers are unusually sensitive, which is emerging as a therapeutic target in lung cancer.
  • high expression of DUS2 correlates with increased resistance to ferroptosis inducers.
  • DUS2 Loss of DUS2 causes increased sensitivity with concomitant accumulation of toxic lipid peroxides, a hallmark of ferroptotic cell death.
  • DUS2 is required to maintain tRNA CysGCA levels and support translation of cysteine-rich proteins including metallothioneins that serve as key regulators of both metal and redox homeostasis.
  • Metallothionein deficiency in DUS2 knockout cells leads to increased susceptibility to zinc intoxication and lower levels of reduced glutathione, which partially explains their sensitivity to ferroptosis. The results here reveal a tRNA-specific vulnerability and demonstrate the therapeutic potential of targeting DUS2.
  • Non-small cell lung cancers use a number mechanisms to avoid apoptosis including loss of expression of the pro-apoptotic gene Bcl-2-like protein (BIM) and amplification of an anti- apoptotic gene, induced myeloid leukemia cell differentiation protein (MCLl).
  • BIM pro-apoptotic gene Bcl-2-like protein
  • MCLl induced myeloid leukemia cell differentiation protein
  • ferroptosis is a form of non-apoptotic cell death that is emerging as a therapeutic target in lung cancer.
  • Example 2-2 DUS2 is overexpressed in lung cancer and loss of DUS2 sensitizes cells to ferroptosis
  • Hallmarks of ferroptotic cell death include dependence on redox active iron and accumulation of toxic lipid peroxides.
  • Several compounds have been described to induce ferroptosis, including class I ferroptosis inducers that inhibit import of cystine (erastin) and class II ferroptosis inducers which inhibit activity of the phospholipid hydroperoxidase GPX4 ((1S,3R)-RSL3, M162, and ML210) (Fig. 6A).
  • GPX4 phospholipid hydroperoxidase GPX4
  • NSCLC cell lines are sensitive to chemical ferroptosis inducers, both in vitro and in vivo, and development of ferroptosis modulating drugs is an active area of research.
  • Dihydrouridine synthases install a modified form of uridine in RNA (Fig. 6C).
  • Dihydrouridine (D) is the most common modified nucleotide in tRNA, and is found in tRNA from organisms from all branches of the tree of life. In tRNAs, D is thought to stabilize to the correct folding of the D-loop. Eukaryotes including humans express four D synthases, and each DUS has unique target nucleotides in multiple individual tRNAs. Disturbance of D levels and/or DUS expression are implicated in lung, brain and kidney cancer. DUS2 is known to modify tRNAs at position in the tRNA D-loop in yeast.
  • DUS2 is frequently overexpressed in non-small cell lung cancer (NSCLC) tumors (Fig. 6D), and NSCLC patients whose tumors express high levels of DUS2 have shorter survival time when compared to patients whose tumors do not express high levels of DUS2 (Fig. 6E).
  • NSCLC non-small cell lung cancer
  • a key feature of ferroptotic cell death is a buildup of toxic lipid peroxides.
  • the present study measured the levels of lipid peroxidation after GPX4 inhibitor treatment using an oxidation-sensitive fluorescent lipid peroxidation probe, Cl 1-BODIPY26.
  • the DUS2 KO cells had 6-8 fold higher levels of lipid peroxides when treated with RSL3 (Figs. 6H-6I), or a second GPX4 inhibitor, ML162 (Fig. 10D).
  • Example 2-3 DUS2 is required to sustain levels of a specific tRNA, tRNACysGCA
  • DUS2 is known to modify tRNAs at position 20 in the tRNA D-loop in yeast (Fig. 7 A), and D is known to stabilize tRNA folding
  • the present study next investigated if DUS2 is required to sustain tRNA expression or function in NSCLC cells.
  • the present study performed tRNA sequencing using a combination of the ARM-seq and DM-TIGRT-seq protocols.
  • the DUS2 KO cells showed no significant change in charging fraction of any tRNA.
  • As a positive control a -70% reduction of tRNAGln charging was detected after starving cells of glutamine (Fig. 7B).
  • both DUS2 KO cell lines showed a reproducible decrease in expression of nearly every tRNACysGCA isodecoder expressed in A549 cells (Figs. 7C-7D).
  • tRNACysGCA levels are summed across all isodecoders, the DUS2 KO cells have a -40% decrease in the total pool of tRNACysGCA (Fig. 11).
  • tRNACysGCA small RNA seq data from TCGA lung adenocarcinoma (LU AD) samples for tRNA expression.
  • tRNACysGCA the present study found statistically significant increased levels in patient tumor samples as compared to nontumor samples (Fig. 7E).
  • tRNAGlnCTG another U20 containing tRNA
  • Fig. 7F levels of another U20 containing tRNA
  • Example 2-4 Loss of DUS2 impairs translation of cysteine rich proteins, including known anti-ferroptotic oncoproteins
  • cysteine codon translation using luciferase reporters.
  • An array of 15 cysteine codons was prepended to a P2A sequence followed by firefly luciferase as a proxy for production of the cysteine repeat peptide, with an IRES driven renilla luciferase as a normalization control (Fig. 8A).
  • cysteine is encoded by two independent codons (UGU and UGC) that are decoded by the same pool of GCA anticodon tRNA, the present study generated versions of the cysteine repeat reporter with either UGU or UGC codons.
  • the present study then used SILAC proteomics to measure changes in the levels of endogenous proteins in DUS2 KO cells.
  • proteins with greater than 5% Cys content showed a significant decrease in abundance (Fig. 8D) consistent with deficient translation of cysteine codons in cells lacking DUS2.
  • the SILAC experiment detected only relatively abundant proteins with moderate cysteine content.
  • the present study did not observe any peptides corresponding to many cysteine rich proteins, including any of the metallothioneins.
  • Metallothioneins are a class of very cysteine rich proteins (-35% cysteine content) that have been linked to ferroptosis and cancer progression.
  • the present study therefore measured metallothionein translation in DUS2 KO cells using a dual luciferase reporter similar to the cysteine codon repeat reporter by replacing the arrays of cysteine codons with the coding sequences of MT 1A or MT 1G (Fig. 8E).
  • Production of metallothionein proteins was impaired in the DUS 2 KO cells (Figs. 8G-8H), demonstrating that loss of DUS2 activity leads to defects in production of an endogenously expressed cysteine rich oncoprotein known to inhibit ferroptosis and lipid peroxidation.
  • metallothionein mRNAs were decreased in abundance in the DUS2 KO cells and partially restored by knocking down the RQC factor GIGYF2 (Fig. 12C).
  • Metal lothi on eins utilize their high thiol content to play two interrelated roles in cells: first, through direct coordination of Zn 2+ and Cu 2+ ions, they are key regulators of cellular zinc and copper levels.
  • ferroptosis was initially characterized as an iron-dependent form of cell death, more recent studies show that defects in zinc homeostasis which elevate cytosolic zinc concentrations sensitize cells to ferroptosis.
  • Example 2-5 Combined loss of DUS2 and ferroptosis induction extends lifespan in a mouse xenograft NSCLC model
  • ferroptosis induction might be a more effective strategy to treat NSCLC in combination with inhibition of DUS2.
  • ferroptosis was induced in mice with established tumors by administration of a GPX4 inhibitor (Fig. 9D).
  • Most GPX4 inhibitors (RSL3, ML162, ML210) suffer from poor pharmacological properties and have limited utility in vivo.
  • a new class of GPX4 inhibitors with improved physiochemical and pharmacokinetic properties was recently developed.
  • DUS2 is frequently over expressed in NSCLC, and patients whose tumors express high levels of DUS2 have worse outcomes.
  • NSCLC cells depleted for DUS2 it was demonstrated that DUS2 is required to support the levels of a specific family of tRNAs, tRNA CysGCA. This result highlights the outsized roles that specific tRNA substrates can play in the biological functions of tRNA modifying enzymes.
  • Loss of CysGCA expression in DUS2 KO cells leads to defects in translation of cysteine codons, which reduces steady state levels of many cysteine rich proteins, including metallothioneins that play key roles in regulating cellular zinc levels and responding to oxidative stress. Loss of metallothionein expression in DUS2 KO cells sensitizes the cells to ferroptosis both in vitro and in vivo.
  • the data here supports that, to fend off ferroptosis, lung cancer cells require both cysteine incorporation into GSH and into cysteine rich metallothionein proteins. Inhibiting either DUS2 or the MT family could increase ferroptosis sensitivity in patients and hold therapeutic value.
  • A549 cells were maintained in a 50:50 mixture of DMEM:F12 medium (Gibco), supplemented with lx penicillin/ streptomycin (Gibco) and 10% FBS (Sigma). Cells were grown at 37°C with 5% CO2 and maintained at subconfluency. CRISPR knockout generation
  • DUS2 CRISPR knockout A549 cells were generated using a single-guide LentiCRISPRv2 strategy to cause deletions in the third and fourth exons of DUS2.
  • Oligos for each guide RNA were phosphorylated and annealed and then cloned into pLentiGuide-Puro (Addgene) digested with BsmBI.
  • Cas9/guideRNA lentiviruses were generated by transfection of pLentiGuide-Puro, psPAX2 (Addgene), and pdr8.2 (Addgene) into 293T cells. Viral supernatant was harvested, filtered and flash frozen 48 and 96 hours post transfection.
  • ImL of 48hr viral supernatant was placed in a 6-well dish with A549 cells at 50% confluency. At 90% confluency, the A549 cells were split in to a 10cm dish and selected for stable integrations using lug/mL puromycin (Sigma). After a stable puro resistant population was generated, single clones were isolated using serial dilution and colony picking. Single cell clones were expanded, screened for lack of expression of DUS2 protein, and frozen.
  • Whole cell lysates were made by pelleting A549 cells and re-suspending fresh or frozen (-80°C) pellets in RIPA buffer (50mM Tris pH 8, 150 mM NaCl, sodium deoxycholate 0.5%, sodium dodecyl sulfate 0.1%, NP-40 1%), lysed on ice for 10 min with vortexing. Lysates were clarified by centrifugation at 4°C and maximum speed (22,500 x g) for 15 min. Approximately 20ug of whole cell lysate, as determined by BCA assay, was run on a 7% Tris-Acetate Gel and transferred to nitrocellulose membranes using wet transfer.
  • RIPA buffer 50mM Tris pH 8, 150 mM NaCl, sodium deoxycholate 0.5%, sodium dodecyl sulfate 0.1%, NP-40 1%
  • Membranes were blocked in 5% milk for 1 hour and incubated with primary antibodies overnight at 4C in 5% milk low-salt TBST (50 mM Tris pH 7.5 150 mM NaCl 0.1% Tween-20).
  • Antibodies used for Western blot were as follows: anti-DUS2 at 1: 10,000, anti-GAPDH at 1 :10,000 (Sigma-Aldrich G9545). Secondary antibody incubation was for 1 hour at room temperature using HRP conjugated goat anti-rabbit IgG at 1 :3000 (Promega W4011). Washes were with high-salt TBST (50 mM Tris pH 7.5 400 mM NaCl 0.1% Tween-20).
  • Each IL pellet was resuspended in 20mL fresh AlkB lysis buffer (50 mM HEPES pH8.0, lOmM Fe(II) sulfate, 300mM NaCl and 5mM imidazole). Cells were lysed by sonication and addition of lysozyme (Sigma). Lysates were clarified with a 12,000 x g spin for 30min at 4°C. Lysates were filtered through a 0.2uM filter and loaded onto a HisTrap 5mL nickel column (Cytiva).
  • Unbound protein and RNA were removed with extensive washing with lysis buffer, and crude alkB protein was eluted from the Ni column using AlkB Lysis buffer with 250mM imidazole.
  • AlkB protein containing fractions were pooled and desalted using a Zeba spin desalting column (Thermo). Desalted protein was purified away from bound RNA using a MonoS column (Cytiva) with a lOOmM-lM NaCl gradient.
  • AlkB protein containing fractions were pooled and concentrated using Amicon Ultra- 15 10KMWCO filters (Milipore). Concentrated AlkB protein was fractionated over a HiLoad 16/60 Superdex S200 column (Cytiva). S200 fractions containing AlkB were again concentrated using Amicon Ultra- 15 10KMWCO filters (Milipore), diluted to 50% glycerol, and flash frozen.
  • A549 cells were harvested by pelleting and resuspending fresh or frozen (-80°C) pellets in ImL of QIAzol (Qiagen). Total RNA was harvested according to the manufacturer’s protocol. tRNA sequencing
  • RNA from A549 cells was resuspended in lOOmM NaOAc/HOAc pH 4.8. 3pL IM NaICU (50mM FC) was added and the mixture was incubated at 22°C. After 30 minutes, 6.65pL IM glucose was added. Total RNA was then recovered by EtOH precipitation. Briefly, lOpL 3M NaOAc, ImL EtOH were added, incubated at -20°C for 15min, and then spun at 4°C and maximum speed (22,500 x g) for 30 min. The RNA pellet was washed with 70% EtOH and spund again for 5min. The pellet was resuspended in 50pL of Na Borate pH 9.5 and incubated at 45°C for 90 minutes.
  • RNAs were depleted from the total RNA with Qiagen miRNeasy spin colums using manufactures recommendations.
  • Small RNAs were demethylated with AlkB and AlkB D135S in AlkB buffer (50 mM HEPES KOH, pH 8, 75 pM ferrous ammonium sulfate pH 5, 1 mM a-ketoglutarate, 2 mM sodium ascorbate, 50 pg/ml BSA) with with 4* molar ratio of wtAlkB and 4* molar ratio of D135S at 37°C for 100 minutes. RNA was recovered with denaturing SILANE bead cleanup.
  • a 3’ adapter was ligated onto the small RNA using T4Rnl2.
  • T7 template DNA was constructed using PCR to fuse the T7 promoter sequence to the tRNA CysGCA sequence with CCA tail added.
  • tRNA CysGCA RNA was prepared by run off transcription with T7 RNAP at 37°C for 8 hours followed by template removal with DNAsel (Ambion) at 37°C for 30 minutes.
  • Full length tRNA CysGCA was purified on an 8% denaturing urea-PAGE gel, eluted overnight, precipitated with ethanol, and resuspended water.
  • 2pg tRNACysGCA and 2pg translational reporter plasmid were co-transfected into cells using TransIT-X2 (Mirus).
  • DUS2 Full length DUS2 was cloned into pcDNA3.1 (CMV promoter, C-terminal FLAG tag) and 2pg of DUS2 plasmid was transfected into cells using TransIT-X2 (Mirus). Forty-eight (48) hours after transfection, cells were split into 6 well plates and allowed to recover for 24 hours. At 40-50% confluency, cells were treated with indicated concentrations of ferroptosis inducing compounds for 12 hours. Cells then stained with Annexin/PI as below. tRNAseq data analysis
  • Demultiplexed reads were adapter trimmed using BBTools bbduk.sh. Adapter trimmed reads were then PCR-duplicate collapsed based on unique molecular identifier (UMI) using dedupe, sh. The UMI was then force trimmed with a second round of trimming. Adapter trimmed and duplicate collapsed reads were then aligned to a single copy of each isodecoder pseudogenome using bbmap.sh. tRNA expression was quantified by counting the number of uniquely mapping reads that mapped to a tRNA, and differential expression analysis was performed using limma-voom. tRNAs with less than 100 uniquely mapping reads were not considered during expression analysis. tRNA charging ratio was determined using custom python scripts ratioing the number of reads terminating with CC-3’ or CCA-3’.
  • Equal amounts of DUS2 KO and wt cells were seeded into 6 well plates. Cells were allowed to grow to -80% confluency, and media was switched to DMEM -Met for 20m. lOpL lOOuCi/mL 35S Met was added to each well, and incubated at 37°C for 30m. To harvest, cells were washed in IX PBS 2x, and harvested in 200pL RIPA with lx PMSF and lx cOmplete. Lysates were freeze thawed 2x, and spun at 4°C at 22,500 x g for 15min to pellet cellular debris. Equal amounts of whole cell lysate, as determined by BCA assay were loaded on a 4-20% SDS- PAGE gel, dried for 2hrs and exposed overnight on a storage phosphor screen.
  • Dual luciferase assay pCMV:codonarray:P2A:Fluc:IRES:Rluc or pCMV:metallothionein:P2A:Fluc:IRES:Rluc constructs were constructed by gibson assembly into pTwist CMV Hygro, and successful assembly was confirmed by sanger sequencing. 2pg of each plasmid was transfected into cells using TransIT-X2 (Minis). Forty-eight (48) hours after transfection, cells were harvested in IX passive lysis buffer (Promega), and flash frozen.
  • Lysates were freeze thawed 2x, and then 75 pL of lysate was used to measure firefly and renilla luciferase activity with the dual -luciferase reporter assay system (Promega) according to manufacturer’s instructions.
  • the SILAC experiment was configured as a two channel experiment: Cells were grown in either 1 :1 DMEM:F12 with dialyzed FBS (Gibco) supplemented with either un-labeled Arg and Lys (Invitrogen) or 13C6 ,15N4 Arg and 13C6 ,15N2 Lys (Invitrogen). Cells were maintained in isotopically labeled medium for 10 doublings, and then were harvested in RIPA supplemented with ImM PMSF and IX HALT phosphatase/protease cocktail (Pierce). Lysates were clarified at 4°C and 22,500 x g for 10 minutes.
  • Total protein was quantified using a BCA assay, and 120pg total protein was submitted to the Yale MS & Proteomics Resource where they were processed and analyzed.
  • Total protein samples were filtered through a 3-kDa Amicon Ultra filter, and the retentate was SpeedVac dried and used for downstream proteomics preparation.
  • Dried protein pellets were reduced with DTT, alkylated with iodoacetamide, enzymatically digested with trypsin, and desalted using C18 RP microspin column.
  • High-resolution liquid chromatography mass spectrometry MS/MS data were collected on an Orbitrap Fusion mass spectrometer coupled to aNanoACQUITY UPLC.
  • RNAseq Total RNA was isolated for three replicates of A549 and both DUS2 KO cell lines as described above. Stranded poly(A)+ selected mRNA-seq libraries were prepared by Genewiz and sequenced on a HiSeq X 10 with paired end 150-bp reads. qRT-PCR
  • siRNA knockdown experiments cells were seeded into 6-well plates, and transfected with siGIGFY2 or siNT siRNAs using TransIT- X2 (Mirus) for 48 hours.
  • Total RNA was DNAse treated using TURBO DNAse (Thermo) according to manufacture instructions.
  • One-step qRT-PCR was performed with gene specific forward and reverse primers using Luna Universal One-Step RT-qPCR (NEB) reagents on a CFX96 Real-Time PCR instrument (Bio-Rad). Fold change was calculated using the Pfaffl method, with GAPDH as the housekeeping gene.
  • Cells were counted and seeded into 6-well plates (Corning). At 40-50% confluency, cells were treated with ferroptosis inducing or inhibiting compounds (RSL3, Cayman Chemical, ML 162 Cayman Chemical, Trolox, Sigma, ZVAD-FMK, Promega, ZnC12, Sigma)for 12 hours. Cells were harvested by trypsinization and centrifugation, washed once with IX Hanks Buffered Salt Solution (HBSS), and resuspended in IX annexin-binding buffer (Thermo) and stained with Annexin V/Propidium Iodide according to manufacturer’s instructions. Cells were fdtered through 70 micron filters and analyzed on a BD LSR II FACS analyzer using FITC and Propidium Iodide filter sets.
  • ferroptosis inducing or inhibiting compounds RSL3, Cayman Chemical, ML 162 Cayman Chemical, Trolox, Sigma, ZVAD-FMK,
  • mice began dosing once either tumor was at least 5 mm long in at least one dimension. Mice within each group were randomized before dosing. Mice were given 10 mg/mL JKE-1674 (MedChemExpress) (10% 100 mg/mL JKE-1674 dissolved in DMSO, 90% 2O%- - cyclodextran in IX PBS) to a concentration of 50 mg JKE-1674 per kg body weight, or vehicle solution (10% DMSO, 90% 20%-P-cyclodextran in IX PBS) by oral gavage. Three of the six mice injected with only A549 or A549 DUS2 KO cells were dosed with JKE-1674, and the remaining mice were dosed with vehicle solution. Mice were dosed twice weekly. Survival endpoints were defined by death (either naturally or as required by veterinary technicians based on the health of each mouse), a 15% decrease in body weight, or a tumor reaching 2 cm in length in any dimension.
  • JKE-1674 MedChemExpress
  • Example 3 In the study described in Example 3, the effects of a non-limiting example of the RNA based DUS/PUS inhibitors herein on various types of cells were studied.
  • Example 3 HepG2 cells were maintained in DMEM plus 10% FBS. A549 cells were maintained in 50:50 DMEM:F12 plus 10% FBS. CLB-001 tRNA was transfected using Lipofectamine RNAiMAX. Cell viability was measured using Cell TiterGlo 2.0.
  • Example 3-1 RNA-based inhibitor for tRNA modifying enzymes kills cancer cells but not non-transformed cells
  • the cultured hepatocellular carcinoma cell line, HepG2 was treated with various concentrations of CLB-001. At higher concentrations, CLB-001 was found to be able to almost complete kill the hepatocellular carcinoma cells.
  • the IC50 was calculated to be around 5 nM.
  • the cultured non-small cell lung cancer cell line, A549 was treated with various concentrations of CLB-001. At higher concentrations, CLB-001 was found to be able to almost complete kill the non-small cell lung cancer cells.
  • the IC50 was calculated to be around 10 nM.
  • the IC50 was calculated to be between 3 nM and 30 nM.
  • CLB-001 did not show significant toxicity toward non-cancer cells.
  • AML12 non-transformed hepatocyte cell line
  • CLB-001 no statistically significant cell death caused by the DUS inhibitor was observed. This is true even at the relatively high 1000 nM CLB-001 concentration.
  • Example 3-2 CLB-001 was vastly more potent than 5-fluorouracil in killing cancer cells The present study discovered that CLB-001 is vastly more potent than 5FU in killing HepG2 cancer cells.
  • HepG2 cells were separated into two groups. The first group was subjected to various concentrations of 5-fluorouracil (5FU), and the second group was subjected to various concentrations of CLB-001, in which 5FU was incorporated into the tRNACys molecule. The experiment demonstrates that CLB-001 is more than 7000 times more potent than 5FU in killing the HepG2 cancer cells.
  • 5FU 5-fluorouracil
  • Example 3-3 High tRNA modifying enzymes levels are associated with hepatocellular carcinoma and worse outcomes in hepatocellular carcinoma
  • various modifying enzymes (PUS1, PUSL1, PUS7, RPUSD1, RPUSD2, TRMT2A, TRMT2B, DUS IL, DUS2, DUS3L, and DUS4L) are upregulated in hepatocellular carcinoma tumors, as assayed by the mRNA levels of these enzymes.
  • Figs. 18A-18G the upregulations of various modifying enzymes generally predict worse outcomes in hepatocellular carcinoma.
  • the present invention is directed to the following non-limiting embodiments:
  • Embodiment 1 An RNA molecule comprising at least one nucleotide capable of forming a covalent bond with a tRNA modifying enzyme, wherein formation of the covalent bond inhibits the tRNA modifying enzyme.
  • Embodiment 2 The RNA molecule of Embodiment 1, wherein the RNA molecule is a tRNA molecule.
  • Embodiment 3 The RNA molecule of Embodiment 1 or 2, wherein the at least one nucleotide comprises a non-natural base.
  • Embodiment 4 The RNA molecule of Embodiment 3, wherein the at least one nucleotide is 5-halouridine (5-haloU), 5-halocytidine (5-haloC), 5-aza-cytidine (5-azaC), 8-haloadenosine (8-haloA), 8-azanebularine, 8-aza-adenosine (8-azaA), or 8-haloguanosine (8-haloG).
  • Embodiment 5 The RNA molecule of any one of Embodiment 1-4, wherein the tRNA modifying enzyme is a dihydrouridine synthase (DUS) or a pseudouridine synthase (PUS).
  • DUS dihydrouridine synthase
  • PUS pseudouridine synthase
  • Embodiment 6 The RNA molecule of any one of Embodiment 1-5, wherein the tRNA modifying enzyme is dihydrouridine synthase 1 (DUS1), dihydrouridine synthase 2 (DUS2), dihydrouridine synthase 3 (DUS3), dihydrouridine synthase 4 (DUS4), dihydrouridine synthase 1 -like (DUS1L), dihydrouridine synthase 3 like (DUS3L), dihydrouridine synthase 4 like (DUS4L), pseudouridine synthase 1 (PUS1), pseudouridine synthase like l(PUSLl), pseudouridine synthase 3 (PUS3), TruB pseudouridine synthase family member 1 (TRUB1), TruB pseudouridine synthase family member 2 (TRUB2), dyskerin pseudouridine synthase 1 (DKC1), pseudouridine synthase 7 (PUS7), pseudouridine synthase 7 like (PUS7
  • Embodiment 7 The RNA molecule of any one of Embodiment 1-6, wherein the non-natural base is 5-haloU, and the tRNA modifying enzyme is DUS2, DUS IL, DUS3L, DUS4L, ISCU, PUS1, PUS3, PUS7, PUS10, PUSL1, PUS7L, RPUSD1, RPUSD2, RPUSD4, TRMT2A, TRMT2B, TRUB1, or TRUB2; the nucleotide comprising the non-natural base is 5-azaC, and the tRNA modifying enzyme is DNMT2, NSUN2, NSUN3, or NSUN6; the nucleotide comprising the non-natural base is 8-halo-G, and the tRNA modifying enzyme is METTL1 or WDR4; or the nucleotide comprising the non-natural base is 8-azaA, and the tRNA modifying enzyme is ADAT1, ADAT2, or ADAT3.
  • the tRNA modifying enzyme is DUS2, D
  • Embodiment 8 The RNA molecule of any one of Embodiments 3-7, wherein the at least one nucleotide is at a position corresponding to a natural position of a natural nucleotide of a natural tRNA that is modified by the tRNA modifying enzyme.
  • Embodiment 9 The RNA molecule of any one of Embodiments 3-8, wherein the RNA molecule is a tRNA molecule, and wherein the non-natural modified base is a 5-halouracil in the D-loop, the t-psi-c loop, the anticodon loop, or a stem of the tRNA molecule.
  • Embodiment 10 The RNA molecule of any one of Embodiments 1-9, wherein the RNA molecule comprises the sequence of any one of SEQ ID NOs: 1-53; or comprises at least about 80% sequence identity to the sequence of any one of SEQ ID NOs: 1-53.
  • Embodiment 11 The RNA molecule of any one of Embodiments 1-10, wherein the RNA molecule is an isolated tRNA molecule.
  • Embodiment 12 The RNA molecule of any one of Embodiments 1-11, wherein the RNA molecule comprises two or more non-natural bases, and wherein the two or more non-natural bases inhibit two or more different tRNA modifying enzymes.
  • Embodiment 13 A composition comprising the RNA molecule of any one of Embodiments 1-12.
  • Embodiment 14 The composition of Embodiment 13, further comprising a pharmaceutically acceptable carrier, wherein the composition is a pharmaceutical composition.
  • Embodiment 15 A method for killing a cell, the method comprising: contacting the RNA molecule of any one of Embodiments 1-12 with the tRNA modifying enzyme in the cell, wherein formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cell.
  • Embodiment 16 The method of Embodiment 15, wherein the cell is a brain cancer cell, a digestive tract cancer cell, a kidney cancer cell, a liver cancer cell, or a lung cancer cell.
  • Embodiment 17 The method of any one of Embodiments 15-16, wherein the cell is a cancer cell in a culture.
  • Embodiment 18 A method for treating a cancer in a subject in need thereof, the method comprising: administering to the subject an effective amount of the pharmaceutical composition of Embodiment 14, wherein the RNA molecule contacts the tRNA modifying enzyme in a cancer cell of the cancer, wherein formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cancer cell.
  • Embodiment 19 The method of Embodiment 18, wherein the cancer is a lung cancer, a brain cancer, a digestive tract cancer, a kidney cancer, or a liver cancer.
  • Embodiment 20 The method of Embodiment 18, wherein the cancer is a bladder cancer, a breast cancer, a cervix cancer, a bile duct cancer, a colon cancer, an esophageal cancer, a head/neck cancer, a kidney clear cancer, a kidney papillary cancer, a liver cancer, a lung non small cell cancer, a lung small cell cancer, a prostate cancer, a rectum cancer, a sarcoma cancer, a stomach cancer, a uterine cancer, or a liquid tumor.
  • the cancer is a bladder cancer, a breast cancer, a cervix cancer, a bile duct cancer, a colon cancer, an esophageal cancer, a head/neck cancer, a kidney clear cancer, a kidney papillary cancer, a liver cancer, a lung non small cell cancer, a lung small cell cancer, a prostate cancer, a rectum cancer, a sarcoma cancer, a stomach cancer, a
  • Embodiment 21 The method of any one of Embodiments 18-20, further comprising administering a chemotherapy to the subject.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The disclosure provides RNA molecules comprising at least one nucleotide capable of forming a covalent bond with a tRNA modifying enzyme, wherein formation of the covalent bond inhibits the tRNA modifying enzyme, and compositions comprising the RNA molecules. The disclosure further provides methods for killing cells, in particular cancer cells, and for treating or preventing cancer in a subject.

Description

RNA-BASED INHIBITORS OF tRNA MODIFYING ENZYMES
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/487,980, filed March 02, 2023, which is incorporated herein by reference in its entirety.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
This invention was made with government support under F31CA254339 and 5R21 CA2461 18-02 awarded by National Institutes of Health. The government has certain rights in the invention.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
The XML file named " 047162-7443 WO 1 Seq Listing.xml" created on March 1, 2024, comprising 56.7 Kbytes, is hereby incorporated by reference in its entirety.
BACKGROUND
Many known anti cancer chemotherapies have significant side effects that result from off target inhibition of cellular pathways such as, for example, DNA replication. For example, 5- fluorouracil (5-FU) is a known anticancer drug that can be misincorporated into DNA of drug- treated cells, and it is believed that accumulation of 5-FU in the genome is correlated with 5-FU cytotoxicity in mammalian cells. As such, these compounds have significant off target effects.
A need exists in the art for compositions and methods effective against cancer and/or have reduced off-target effects. The present invention addresses this need. SUMMARY
In some aspects, the present invention is directed to an RNA molecule including at least one nucleotide capable of forming a covalent bond with a tRNA modifying enzyme, wherein formation of the covalent bond inhibits the tRNA modifying enzyme.
In some aspects, the present invention is directed to a composition including the RNA molecule. In some embodiments, the composition is a pharmaceutical composition.
In some aspects, the present invention is directed to a method for killing a cell. In some embodiments, the method includes: contacting the RNA molecule with the tRNA modifying enzyme in the cell. In some embodiments, the formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cell.
In some aspects, the present invention is directed to a method for treating a cancer in a subject in need thereof. In some embodiments, the method includes: administering to the subject an effective amount of the pharmaceutical composition. In some embodiments, the RNA molecule contacts the tRNA modifying enzyme in a cancer cell of the cancer, and formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cancer cell.
In some aspects, the present invention is directed to the following non-limiting embodiments: RNA molecule
In some embodiments, the present invention is directed to an RNA molecule.
In some embodiments, the RNA molecule comprises at least one nucleotide capable of forming a covalent bond with a tRNA modifying enzyme.
In some embodiments, the formation of the covalent bond inhibits the tRNA modifying enzyme.
In some embodiments, the RNA molecule is a tRNA molecule.
In some embodiments, the at least one nucleotide comprises a non-natural base.
In some embodiments, the at least one nucleotide is 5-halouridine (5-haloU), 5- halocytidine (5-haloC), 5-aza-cytidine (5-azaC), 8-haloadenosine (8-haloA), 8-azanebularine, 8- aza-adenosine (8-azaA), or 8-haloguanosine (8-haloG).
In some embodiments, the tRNA modifying enzyme is a dihydrouridine synthase (DUS) or a pseudouridine synthase (PUS). In some embodiments, the tRNA modifying enzyme is dihydrouridine synthase 1 (DUS1), dihydrouridine synthase 2 (DUS2), dihydrouridine synthase 3 (DUS3), dihydrouridine synthase 4 (DUS4), dihydrouridine synthase 1-like (DUS IL), dihydrouridine synthase 3 like (DUS3L), dihydrouridine synthase 4 like (DUS4L), pseudouridine synthase 1 (PUS1), pseudouridine synthase like l(PUSLl), pseudouridine synthase 3 (PUS3), TruB pseudouridine synthase family member 1 (TRUB1), TruB pseudouridine synthase family member 2 (TRUB2), dyskerin pseudouridine synthase 1 (DKC1), pseudouridine synthase 7 (PUS7), pseudouridine synthase 7 like (PUS7L), RNA pseudouridylate synthase domain containing 1 (RPUSD1), RNA pseudouridylate synthase domain containing 2 (RPUSD2), RNA pseudouridylate synthase domain containing 4 (RPUSD4), pseudouridine synthase 10 (PUS 10), tRNA methyltransferase 2 homolog A (TRMT2A), tRNA methyltransferase 2 homolog B (TRMT2B), NOP2/Sun RNA methyltransferase 2 (NSUN2), NOP2/Sun RNA methyltransferase 3 (NSUN3), NOP2/Sun RNA methyltransferase 6 (NSUN6), DNA methyltransferase 2 (DNMT2), Methyltransferase-Like Protein 1 (METTL1), WD repeat domain 4 (WDR4), adenosine deaminase TRNA specific 1 (ADAT1), adenosine deaminase TRNA specific 2 (ADAT2), adenosine deaminase TRNA specific 2 (ADAT3), ISCU, or combinations thereof.
In some embodiments, the non-natural base is 5-haloU, and the tRNA modifying enzyme is DUS2, DUS1L, DUS3L, DUS4L, ISCU, PUS1, PUS3, PUS7, PUS10, PUSL1, PUS7L, RPUSD1, RPUSD2, RPUSD4, TRMT2A, TRMT2B, TRUB1, TRUB2, or combinations thereof.
In some embodiments, the nucleotide comprising the non-natural base is 5-azaC, and the tRNA modifying enzyme is DNMT2, NSUN2, NSUN3, NSUN6, or combinations thereof.
In some embodiments, the nucleotide comprising the non-natural base is 8-halo-G, and the tRNA modifying enzyme is METTL1, WDR4, or combinations thereof.
In some embodiments, the nucleotide comprising the non-natural base is 8-azaA, and the tRNA modifying enzyme is ADAT1, ADAT2, ADAT3, or combinations thereof.
In some embodiments, the at least one nucleotide is at a position corresponding to a natural position of a natural nucleotide of a natural tRNA that is modified by the tRNA modifying enzyme.
In some embodiments, the RNA molecule is a tRNA molecule, and the non-natural modified base is a 5-halouracil in the D-loop, the t-psi-c loop, the anticodon loop, the RNA stems, and other portions of the tRNA molecule. In some embodiments, the RNA molecule comprises the sequence of any one of SEQ ID NOs:l-53; or comprises at least about 80% sequence identity to the sequence of any one of SEQ ID NOs:l-53.
In some embodiments, the RNA molecule is an isolated tRNA molecule.
In some embodiments, the RNA molecule comprises two or more non-natural bases.
In some embodiments, the two or more non-natural bases inhibit two or more different tRNA modifying enzymes.
Composition
In some embodiments, the present invention is directed to a composition.
In some embodiments, the composition comprises the RNA molecule herein.
In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.
In some embodiments, the composition is a pharmaceutical composition.
Method for killing a cell
In some embodiments, the present invention is directed to a method for killing a cell.
In some embodiments, the method comprises contacting the RNA molecule herein with the tRNA modifying enzyme in the cell .
In some embodiments, the formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cell.
In some embodiments, the cell is a brain cancer cell, a digestive tract cancer cell, a kidney cancer cell, a liver cancer cell, or a lung cancer cell.
In some embodiments, the cell is a bladder cancer cell, a breast cancer cell, a cervix cancer cell, a bile duct cancer cell, a colon cancer cell, an esophageal cancer cell, a head/neck cancer cell, a kidney clear cell cancer cell, a kidney papillary cancer cell, a liver cancer cell, a lung non small cell cancer cell, a lung small cell cancer cell, a prostate cancer cell, a rectum cancer cell, a sarcoma cancer cell, a stomach cancer cell, a uterine cancer cell, a liquid tumor (e.g., acute myeloid leukemia/ AML and the like) cell, and the like.
In some embodiments, the cell is a cancer cell in a culture. Method for treating cancer
In some embodiments, the present invention is directed to a method for treating a cancer in a subject in need thereof.
In some embodiments, the method comprises: administering to the subject an effective amount of the pharmaceutical composition herein.
In some embodiments, the RNA molecule contacts the tRNA modifying enzyme in a cancer cell of the cancer.
In some embodiments, the formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cancer cell.
In some embodiments, the cancer is a lung cancer, a brain cancer, a digestive tract cancer, a kidney cancer, a liver cancer, or combinations thereof.
In some embodiments, the cancer is a bladder cancer, a breast cancer, a cervix cancer, a bile duct cancer, a colon cancer, an esophageal cancer, a head/neck cancer, a kidney clear cancer, a kidney papillary cancer, a liver cancer, a lung non small cell cancer, a lung small cell cancer, a prostate cancer, a rectum cancer, a sarcoma cancer, a stomach cancer, a uterine cancer, a liquid tumor (e.g., acute myeloid leukemia/ AML and the like), and the like.
In some embodiments, the method further comprises administering a chemotherapy to the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
Figs. 1A-1B depict non-limiting examples of tRNA modifying enzymes that are upregulated in cancers. Fig. 1A: dihydrouridine synthases (DUS) upregulated in cancers. Fig. IB: pseudouridine synthases (PUS) upregulated in cancers.
Fig. 2 depicts 5-halouracil forming covalent bound with a DUS, in accordance with some embodiments.
Fig. 3 depicts a non-limiting example of a tRNA molecule of the invention covalently bound to a tRNA modifying enzyme, in accordance with some embodiments.
Fig. 4 depicts a western blot confirming that a non-limiting example of the RNA molecule described herein was able to form covalent bond with a non-limiting tRNA modifying enzyme, DUS2, in accordance with some embodiments. Fig. 5 is a graph showing the RNA molecule of Fig. 4 was able to kill cells of the lung adenocarcinoma cell line PC9, in accordance with some embodiments.
Figs. 6A-6I depict that DUS2 is overexpressed in lung cancer and loss of DUS2 sensitizes cells to ferroptosis, in accordance with some embodiments. Fig. 6A: Schematic of key regulators and chemical effectors of ferroptosis. Fig. 6B: High DUS2 mRNA expression correlates with resistance to chemical ferroptosis inducers in a panel of 860 cancer cell lines. Fig. 6C: DUS enzymes convert uridine to dihydrouridine. Fig. 6D: DUS2 RNA levels are significantly higher in lung adenocarcinoma (LU AD) tumor samples compared to normal tissue (two tailed Mann-Whitney U test, CysGCA p< 0001). Fig. 6E: High DUS2 expression predicts worse outcomes for NSCLC patients. Fig. 6F: DUS2 protein expression is absent in clonal A549 DUS2 KO cells. Fig. 6G: Increased cell death (Annexin V+/PI+ cells) in DUS2 KOs compared to WT A549 following treatment with 2pM RSL-3. Fig. 6H: Elevated lipid ROS (Cl 1-BODIPY staining) in DUS2 KOs compared to WT A549 following treatment with 2pM RSL3. Fig. 61: Quantification of Fig. 6H (two tailed t-test, * p< 0001).
Figs. 7A-7F depict that DUS2 is required to sustain levels of a specific tRNA, tRNACysGCA, in accordance with some embodiments. Fig. 7A: DUS2 targets position U20 in tRNA. Fig. 7B: Quantification of tRNA charging levels in DUS2 WT and KO cells. tRNA charging is unaffected by loss of DUS2, whereas Gin starvation reduces tRNAGln charging levels >50%. Fig. 7C: Changes in tRNA levels in DUS2 KO cells, black dots (FDR< 0.05), grey (FDR >0.05), red (tRNACysGCA with FDR< .05). Fig. 7D: Example isodecoder tRNACysGCA10-l shows -50% lower expression in DUS2 KOs. Fig. 7E: Analysis of tRNA levels in TCGA LU AD data. tRNACysGCA but not tRNAGlnCTG levels are significantly higher in tumors compared to normal tissue (two tailed Mann-Whitney U test, CysGCA p< 0001).
Figs. 8A-8I depict that loss of DUS2 impairs translation of cysteine rich proteins, including metallothioneins, which leads to ferroptosis sensitivity, in accordance with some embodiments. Fig. 8A: Cysteine translation reporter. Fig. 8B: DUS2 KO impairs translation of TGT and TGC cysteine codons (two tailed t-test, *p< 03). Fig. 8C: Rescue of TGT translation by transfection of tRNACysGCA into DUS2 KO cells (two tailed t-test, * p< 0001). Fig. 8D: Proteins with high (>5%) Cys content are reduced in DUS2 KO cells. Cumulative distribution of changes in protein abundance (log2 fold change, Kolmogorov-Smirnov test, p< 05). Fig. 8E: Metal lothi on ein translation reporter. Fig. 8F: Metal lothi on eins (MT1 A and MT1 G) are translated less well in DUS2 KOs. Fig. 8G: Increased cell death (Annexin V+/PI+ cells) in DUS2 KOs compared to WT A549 following addition of 62.5pM ZnCh. Fig. 8H: DUS2 KO cells have lower levels of reduced GSH (two tailed t-test, * p< 004). Fig. 81: Model of anti-ferroptotic function of DUS2.
Figs. 9A-9G depict that combined loss of DUS2 and ferroptosis induction extends lifespan in a mouse xenograft NSCLC model, in accordance with some embodiments. Fig. 9A: A549 tumors grow faster than DUS2 KO tumors. Fig. 9B: Example tumor from A549 and DUS2 KO-2. Fig. 9C: DUS2 KO tumors have higher expression of a marker of ferroptosis, PTGS2, than A549 cells by qRT-PCR (ANOVA, * p< 002). Fig. 9D: Dosing scheme for xenograft experiments. Fig. 9E: Oral JKE-1674 treatment induces PTGS2 mRNA in mouse lungs (ANOVA, * p< 03). Fig. 9F: JKE-1674 treatment increases PTGS2 mRNA expression in DUS2 KO tumors (ANOVA, * p< 02). Fig. 9G: Mice implanted with DUS2 KO xenograft tumors survive longer than mice implanted with A549 tumors when treated with JKE-1674 (Mantel-Cox test * p<.03).
Figs. 10A-10E depict that fraction of dead (Annexin V+/PI+) DUS2 KO cells is reduced with pre-treated with DUS2 expression plasmid, Ferrostatin-1, Trolox but not ZVAD-FMK when treated with 2pM RSL-3. Fig. 10D: DUS2 KOs have higher levels of lipid ROS measured by Cl 1-BODIPY staining when treated with 200nM ML162. Fig. 10E: DUS2 KOs have higher levels of cellular ROS measured by H2DCFDA staining when treated with 2pM RSL-3.
Fig. 11 depicts that, in aggregate, the total pool of tRNACysGCA is reduced -40% in DUS2 KO clones.
Fig. 12A depicts that the total protein synthesis is unimpaired in DUS2 KO cells as measured by 35S-Met incorporation (two tailed t-test, * p=.67). Fig. 12B: mRNAs encoding cysteine rich proteins are reduced in DUS2 KO cells consistent with RQC. Cumulative distribution of changes in mRNA abundance (log2 fold change, K-S test, p< 0001).). Fig. 12C: Depletion of RQC factor GIGYF2 (siGIGYF2) rescues MT1A mRNA levels in DUS2 KO cells compared to non-targeting control (siNT) (two tailed t-test, * p< 04).
Figs. 13A-13B depict that mouse weights generally increased over time with vehicle and JKE-1674. Fig. 13B: Mice receiving JKE-1674 had shorter median survival than mice receiving vehicle. Figs. 14A-14B demonstrate that a non-limiting example of the RNA-based DUS inhibitors herein, CLB-001, was able to kill cancer cells with nanomolar level IC50, in accordance with some embodiments. The hepatocellular carcinoma cell line, HepG2 (Fig. 14A), and the non-small cell lung cancer cell line, A549 (Fig. 14B), were subjected to various concentrations of CLB-001. The viabilities of the cells are plotted against the CLB-001 concentrations.
Fig. 15 demonstrates that the non-limiting example of the RNA-based DUS inhibitors herein, CLB-001, does not kill non-cancerous cells, in accordance with some embodiments. The non-transformed hepatocyte cell line, AML 12, was subjected to various concentrations of CLB- 001. The viabilities of the cells are plotted against the CLB-001 concentrations.
Fig. 16 demonstrates that the non-limiting example of the RNA-based DUS inhibitors herein, CLB-001, is vastly more potent than 5-FU, in accordance with some embodiments. HepG2 was subjected to various concentrations of CLB-001. The viabilities of the cells are plotted against the CLB-001 concentrations. Even though the active moiety of CLB-001 is 5-FU, incorporating 5-FU into tRNACys molecule dramatically increased the potency of the drug, such that CLB-001 is more than 7000X more potent than 5FU in killing HepG2 cancer cells.
Figs. 17A-17C demonstrate that tRNA modifying enzymes are upregulated in hepatocellular carcinoma, in accordance with some embodiments. The mRNA levels of various tRNA modifying enzymes from hepatocellular carcinoma (HCC) tumor tissues were compared with those from normal liver tissues. The tRNA modifying enzymes were found to be consistently overexpressed in the HCC tumors.
Figs. 18A-18G demonstrate that high expression of tRNA modifying enzymes predicts worse outcomes in hepatocellular carcinoma (HCC), in accordance with some embodiments. In each panel of the figures, HCC patients are grouped according to the expression levels of the tRNA modifying enzymes in the tumor tissues, and the patient survival percentages with time are plotted according to the grouping.
Fig 19 demonstrates that a non-limiting example of the RNA-based DUS and PUS inhibitors herein, CLB-001, was able to kill cancer cells with nanomolar level IC50, in accordance with some embodiments. The hepatocellular carcinoma cell lines, SNU-387, HepG2, and PLC/PRF/5 and the non-small cell lung cancer cell line, A549 were subjected to various concentrations of CLB-001 . The viabilities of the cells are plotted against the CLB-001 concentrations.
DETAILED DESCRIPTION
Without being limited by theory, the invention is based in part on the discovery that substrate mimetic transfer RNAs (tRNAs) that incorporate non-natural nucleotides at specific positions can be used to covalently trap tRNA modifying enzymes e.g., tRNA modifying enzymes that drive cancer progression and/or metastasis), thereby inhibiting them.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice of an/or for the testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used according to how it is defined, where a definition is provided.
It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
It is also to be understood that the methods described in this disclosure are not limited to particular methods and experimental conditions disclosed herein as such methods and conditions may vary.
Furthermore, the experiments described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J. Sambrook, and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).
Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and/or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.
Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
So that the disclosure may be more readily understood, select terms are defined below.
As used herein, the articles “a” and “an” are used to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±20% or within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value, as such variations are appropriate to perform the disclosed methods. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
The term “isolated” as used herein in relation to any molecule, as in, for example, “isolated RNA molecule,” refers to molecules, which are isolated from other cellular components and is meant to encompass both purified and recombinant molecules. The term “isolated RNA molecule(s)” thus refers to, for example, a RNA molecule(s) that is/are substantially free of cellular material, viral material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
“Identity” as used herein refers to the subunit sequence identity between two polymeric molecules, for example between two nucleic acid molecules, such as, between two RNA molecules. When two polynucleotide sequences have the same nucleotides at the same positions, e.g., if a position in each of two nucleic acid molecules is occupied by a uracil, then they are identical at that position. The identity or extent to which two sequences have the same, for example nucleotide, at the same positions in an alignment is often expressed as a percentage. Since two polynucleotides may each comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. Homology or identity can be determined by sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL known in the art.
As used herein, the term “inhibit(s)” or “inhibiting” an tRNA modifying enzyme refers to any statistically significant decrease in biological activity of the tRNA modifying enzyme, including full blocking of the activity.
The terms “non-natural” and “non-naturally occurring” refer to that which is not present in nature. For example, a “non-natural” or “non-naturally occurring nucleic acid or nucleotide refers to a nucleic acid or nucleotide that is not present in nature. For example, non-naturally occurring nucleic acids can include one or more non-natural base, sugar, and/or inter-subunit linkage, e.g., a sugar, base, and/or linkage that has been modified or substituted with respect to that found in a naturally occurring nucleic acid molecule. In some embodiments, non-naturally occurring nucleic acids include more than one type of modification, e.g., but not limited to, sugar and base modifications, sugar and linkage modifications, base and linkage modifications, or base, sugar, and linkage modifications.
A “subject” or “patient,” as used therein, may be a human or non -human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals.
By “wildtype” is meant a non-mutated version of a gene, allele, genotype, nucleic acid, polypeptide, or phenotype, or a fragment of any of these. It may occur in nature or produced recombinantly.
In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U. S. Patent law and can mean "includes," "including," and the like; "consisting essentially of" or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. Use of the transitional phrase “consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of’ when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.” In some embodiments, the term “consisting essentially of’ refers to a composition, whose only active ingredient is the indicated active ingredient(s) (e.g., the indicated RNA molecule(s)), however, other components may be included which are for stabilizing, preserving, etc. the formulation, but are not involved directly in the therapeutic effect of the indicated active ingredient.
As used herein, the terms “treat,” treating,” “ameliorating,” “treatment,” and the like refer to reducing or improving a disease or condition and/or one or more symptoms associated therewith. It will be appreciated that, although not precluded, treating a disease or condition and/or one or more symptoms associated therewith does not require that the disease, condition, or symptoms associated therewith be completely ameliorated or eliminated. It means that the clinical signs and/or the symptoms associated with a disease or condition are lessened as a result of the actions performed. The signs or symptoms to be monitored will be well known to the skilled clinician.
Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
Molecules and Compositions
In some aspects, the present invention provides an RNA molecule comprising at least one nucleotide capable of forming a covalent bond to a tRNA modifying enzyme, wherein formation of the covalent bond inhibits the tRNA modifying enzyme.
RNA molecules can be prepared using methods known in the art including, but not limited to, in vitro transcriptions and chemical synthesis. Nelissen, E. et aL, Nucleic Acids Research, 40(13):el02 (2012), Milligan, J. F. et al., Nucleic Acids Research, 15:8783-8798 (1987), Marshall, W. S. et al., Curr. Opin. Chem. Biol., 8:222-229 (2004), Ponchon, L. et aL, Nat. Protoc., 4:947-959 (2009), Ponchon, L. et al., Nat. Methods, 4:571-576 (2007), Ponchon, L. et l., Methods, 54:267-273 (2011), and U.S. Patent Application No, 2009/0298920, each of which is herein incorporated by reference in its entirety, describe methods for production of RNA.
In one embodiment, the RNA molecule comprises any ribonucleic chain. In another embodiment, the RNA molecule comprises about 1,000 ribonucleotides in length, illustratively, about 5 to about 1,000, about 10 to about 900, about 20 to about 500, about 30 to about 300, about 40 to about 200, and about 70 to about 100 ribonucleotides.
In other embodiments, the RNA molecule comprises about 76 to about 96 ribonucleotides.
In other embodiments, the RNA molecule comprises about 60 to about 86 ribonucleotides.
In some embodiments, formation of a bond between the at least nucleotide and the tRNA modifying enzyme can be achieved through any of a variety of direct or indirect covalent associations or attachments.
In one embodiment, the at least one nucleotide is capable of forming a covalent bond to a tRNA modifying enzyme through direct or indirect formation of a covalent bond between a base of the at least one nucleotide and an amino acid residue of the tRNA modifying enzyme.
In other embodiments, the covalent attachment of the at least one nucleotide to the tRNA modifying enzyme is irreversible.
In another embodiment, the covalent attachment of the at least one nucleotide to the tRNA modifying enzyme inhibits the tRNA modifying enzyme. In one embodiment, inhibition comprises a decrease of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% in tRNA modifying enzyme activity. In another embodiment, inhibition comprises a decrease of 100% in tRNA modifying enzyme activity.
In some embodiments, formation of the bond covalently traps the tRNA modifying enzyme, irreversibly inhibiting.
In some embodiments, the at least one nucleotide is a non-natural nucleotide. In one embodiment, the at least one nucleotide comprises a non-natural base.
In one embodiment, the RNA molecule comprises no more than 1, 2, 3, 4, ,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 non-natural nucleotides, independently each nucleotide comprising a non-natural base.
One of ordinary skill in the art will recognize that a large number of “synthetic” non- natural nucleosides comprising various heterocyclic bases and/or various sugar moieties (and sugar analogs) are available in the art, and that as long as other criteria of the present invention are satisfied, the RNA molecule can include one or several heterocyclic bases other than the principal five base components of naturally-occurring nucleic acids.
In some embodiments, the heterocyclic base includes, but is not limited to, uracil-5-yl cytosine-5-yl, adenine-7-yl, adenine-8-yl, guanine-7-yl, guanine-8-yl, 4-aminopyrrolo[2,3- d]pyrimidin-5-yl, 2-amino-4-oxopyrolo[2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3- d]pyrimidin-3-yl groups, where the purines are attached to the sugar moiety of the ISS via the 9- position, the pyrimidines via the 1 -position, the pyrrolopyrimidines via the 7-position and the pyrazolopyrimidines via the 1 -position.
In other embodiments, the at least one nucleotide comprises at least one modified base. Examples of base modifications include, but are not limited to, uracils modified at C-5 and/or C- 6, preferably with a halogen, including, but are not limited to, fluorouracil such as 5 -fluorouracil (5-FU), bromouracil such as 5-bromouracil, chlorouracil such as 5 -chlorouracil, and iodouracil such as 5-iodouracil and hydroxyuracil. Other examples of base modifications include 8- azaadenosine (8-aza-Ad), 7-deazaadenosine, N6-methyl-7-deazaadenosine, N6methyl-8- azaadenosine, 7-deaza-8 -azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N625 amino-7- deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-azaadenosine, N6- hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7- deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6thio-7- deazaguanosine, 6-thio-8 -azaguanosine, 7-deaza-8 -azaguanosine, and 6-thio-7deaza-8- azaguanosine. azacytosine, 5 -bromocytosine, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, 5,6-dihydrocytosine, 5- iodocytosine, 5 -nitrocytosine, 5-hydroxy-cytosine, 6-thio-guanine, and 4-thiouracil.
In one embodiment, the at least one nucleotide comprising a non-natural base is 5-halo (halo=F/Cl/Br/I) uridine, 5-halo (halo=F/Cl/Br/I) cytidine, 5-aza-cytidine, 8-halo (halo=F/Cl/Br/I) adenosine, 8-azanebularine, 8-aza-adenosine, or 8-halo (halo=F/Cl/Br/I) guanosine.
In some embodiments, the amino acid residue of the tRNA modifying enzyme is a conserved catalytic amino acid residue.
In other embodiments, the amino acid residue of the tRNA modifying enzyme is a reactive amino acid residue. In one embodiment, the amino acid residue of the tRNA modifying enzyme is a reactive nucleophilic amino acid residue.
In some embodiments, the reactive nucleophilic amino acid residue is a reactive cysteine residue, wherein formation of the covalent bond occurs via nucleophilic attack of the at least one nucleotide by the reactive cysteine residue.
In another embodiment, the at least one nucleotide of the RNA molecule is a 5- halopyrmidine, wherein the at least one nucleotide is capable of forming a covalent bond with a conserved catalytic cysteine residue of the tRNA modifying enzyme, wherein the covalent bond crosslinks the RNA molecule to the tRNA modifying enzyme.
Without being limited by theory, in some embodiments, the crosslink begins with reduction (e.g., enzymatic reduction) of 5-halouridine to 5-halodihydrouridine followed by nucleophilic attack of a conserved catalytic cysteine of the tRNA modifying enzyme on the C5 position with halide serving as leaving group. Dai, W. et al., Nat Chem Biol., 17(11):1178-1187 (2021), which describe activity -based RNA modifying enzyme probing, is herein incorporated by reference in its entirety.
In one embodiment, the RNA molecule is a tRNA molecule.
The general characteristics of a naturally occurring tRNA are well-known to one of ordinary skill in the art. Sprinzl, M. et al., Nucleic Acids Research, 26(1): 148-153 (1998), which is herein incorporated by reference in its entirety, describe compilation of tRNA sequences and sequences of tRNA genes.
In some embodiments, the tRNA molecule of the present invention is a single ribonucleotide chain which is capable of folding to adopt a characteristic, so-called cloverleaf secondary structure.
In some embodiments, the secondary structure comprises (i) an acceptor stem composed of a first 7 ribonucleotides of the 5’ end of the ribonucleotide chain and 7 ribonucleotides that precede the last 4 ribonucleotides of the 3’ end of the ribonucleotide chain, thus forming a double-stranded structure comprising about 6 or 7 pairs of ribonucleotides, it being possible for the ribonucleotides comprising the first ribonucleotide of the 5’ end of the ribonucleotide chain and the ribonucleotide that precedes the last 4 ribonucleotides of the 3’ end of the ribonucleotide chain not to be paired; (ii) a D arm comprising 4 pairs of ribonucleotides and a D loop comprising about 8 to 10 ribonucleotides, formed by the folding of a part of the ribonucleotide chain that follows the first 7 ribonucleotides of the 5’ end of the ribonucleotide chain; (iii) a stem of the anticodon comprising 5 pairs of ribonucleotides, and a loop of the anticodon comprising about 7 ribonucleotides (stem-loop of the anticodon), formed by the folding of a part of the ribonucleotide chain that follows the D arm and the D loop; (iv) a variable loop comprising about 4 to about 21 ribonucleotides and formed by a part of the ribonucleotide chain that follows the stem of the anticodon and the loop of the anticodon; (v) a T arm comprising about 5 pairs of ribonucleotides, and a T loop comprising about 8 ribonucleotides, formed by the folding of a part of the ribonucleotide chain that follows the variable loop and precedes the ribonucleotides of the 3’ end of the ribonucleotide chain which comprising the acceptor stem.
In some embodiments, the tRNA modifying enzyme is a dihydrouridine synthase (DUS) or a pseudouridine synthase (PUS). Both DUS and PUS are known to contain highly conserved cysteine or aspartic acid residues in their respective active sites.
In some embodiments, the tRNA modifying enzyme is dihydrouridine synthase 1 (DUS1), dihydrouridine synthase 2 (DUS2), dihydrouridine synthase 3 (DUS3), dihydrouridine synthase 4 (DUS4), dihydrouridine synthase 1 -like (DUS1L), dihydrouridine synthase 3 like (DUS3L), dihydrouridine synthase 4 like (DUS4L), pseudouridine synthase 1 (PUS1), pseudouridine synthase like l(PUSLl), pseudouridine synthase 3 (PUS3), TruB pseudouridine synthase family member 1 (TRUB1), TruB pseudouridine synthase family member 2 (TRUB2), dyskerin pseudouridine synthase 1 (DKC1), pseudouridine synthase 7 (PUS7), pseudouridine synthase 7 like (PUS7L), RNA pseudouridylate synthase domain containing 1 (RPUSD1), RNA pseudouridylate synthase domain containing 2 (RPUSD2), RNA pseudouridylate synthase domain containing 4 (RPUSD4), pseudouridine synthase 10 (PUS 10), tRNA methyltransferase 2 homolog A (TRMT2A), tRNA methyltransferase 2 homolog B (TRMT2B), NOP2/Sun RNA methyltransferase 2 (NSUN2), NOP2/Sun RNA methyltransferase 3 (NSUN3), NOP2/Sun RNA methyltransferase 6 (NSUN6), DNA methyltransferase 2 (DNMT2), Methyltransferase-Like Protein 1 (METTL1), WD repeat domain 4 (WDR4), adenosine deaminase TRNA specific 1 (ADAT1), adenosine deaminase TRNA specific 2 (ADAT2), adenosine deaminase TRNA specific 2 (ADAT3), or ISCU.
In some embodiments, the non-natural base in the RNA molecule is selected based on the target tRNA modifying enzyme that the RNA molecule is to inhibit. A non-limiting list of nucleotides comprising non-natural bases together with tRNA modifying enzymes is shown below in Table 1.
Table 1: Examples of enzymes and nucleotides comprising non-natural bases
In some embodiments, the non-natural base is 5-haloU, and the tRNA modifying enzyme is DUS2, DUS1L, DUS3L, DUS4L, ISCU, PUS1, PUS3, PUS7, PUS10, PUSL1, PUS7L, RPUSD1, RPUSD2, RPUSD4, TRMT2A, TRMT2B, TRUB1, TRUB2, or combinations thereof.
Base modifications of natural tRNA molecules by tRNA modifying enzymes in humans and other species have been extensively mapped, and there is a large amount of available information regarding natural tRNA molecules modified by these enzymes, as well as the specific naturally occurring nucleotides of the naturally occurring tRNA molecule that are modified by these enzymes. As such, in some embodiments, the tRNA molecule of the present invention comprises the sequence of a naturally occurring tRNA molecule, wherein the at least one nucleotide is a non-natural nucleotide located at a nucleotide position of the tRNA molecule that corresponds to the naturally occurring position, or is at a position that is no more than five, four, three, two, or one nucleotide position away from the naturally occurring position, in the naturally occurring tRNA molecule.
The location of the non-natural base is not limited, as tRNA modifying enzymes, such as those described herein, are known to modify bases located in virtually all the locations of tRNA molecules. In some embodiments, the location of the non-natural base is determined based on the enzyme to be inhibited, the sequence of the parent natural tRNA, as well as the non-natural base. For example, DUS enzymes can modify uridines in the D-loop (as well as uridines outside the D- loop such as the tRNA stems) of tRNAs and can be inhibited by 5-halo uridine. As such, in some embodiments, inhibitors specific for DUS enzymes can be designed by modifying natural tRNA by incorporating a 5-halo uridine in the D-loop. For another example, PUS can modify uridines in the D-loop, the t-psi-c loop, or the anticodon loop (as well as uridines outside the D-loop such as the tRNA stems). In other embodiments, inhibitors specific for PUS enzymes can be designed by modifying natural tRNA by incorporating a 5-halo uridine in the D-loop, the t-psi-c loop, and/or the anticodon loop.
In some embodiments, the tRNA molecule comprises an anticodon-arm and an acceptor arm, wherein the anticodon-arm comprises a trinucleotide anticodon, wherein the anticodon recognizes a stop codon. In other embodiments, the anticodon recognizes a codon for alanine, arginine, aspartic acid, asparagine, cysteine, glycine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. In one embodiment, the anticodon recognizes a codon for cysteine. In another embodiment, the acceptor arm comprises a 3'-terminal sequence of 5’-cytidine-cytidine- adenosine (CCA)-3’ that overhangs the end.
In some embodiments, the RNA molecule comprises the sequence: GGGGGXAXAGCXCAGXGGXAGAGCAXXXGACXGCAGAXCAAGAGGXCCCCGGXX CAAAXCCGGGXGCCCCC, wherein one or more of the Xs any nucleotide comprising a nonnatural base such as, for example, 5-halouracil (SEQ ID NO: 1). In other embodiments, the RNA molecule comprises the sequence of SEQ ID NO: 1, with the proviso that X is a U, C, or G, and one or more U, C, or G of SEQ ID NO: 1 comprises a non-natural base.
In some embodiments, the RNA molecule comprises a sequence of a natural tRNA molecule including, but not limited to, any one of the tRNA molecules listed in Table 2, with the proviso that the RNA molecule comprises at least one nucleotide capable of forming a covalent bond with a tRNA modifying enzyme at any one of the nucleotide positions. In some embodiments, at least one U, A, G or C residue in the natural tRNA molecule is substituted with a 5-haloU, 8-haloG, 5-halo-C, 5-aza-C, or 8-azaA. In some embodiments, the RNA molecule comprises about 80% identity or more with the parent tRNA, such as about 85% identity or more, about 90% identity or more, about 92% identity or more, about 95% identity or more, about 97% identity or more, about 98% identity or more, about 99% identity or more, or identical.
Table 2: Non-limiting examples of parent tRNA molecules for designing the RNA based inhibitor
In some embodiments, a RNA molecule of the present invention comprises a percent degree of sequence identity to the sequence of any one of SEQ ID NOs: 1-53, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of any one of SEQ ID NOs: 1-53. In some embodiments, the specified degree of sequence identity retains one or more characteristics, e.g., substrate for a tRNA modifying enzyme and/or structure (e.g., cloverleaf secondary structure), as the RNA molecule of SEQ ID NOs: 1-53.
In some embodiments, the RNA molecule is an isolated or purified tRNA molecule.
In some embodiments, the RNA molecule is purified. The contaminants can be cellular proteins remaining after expression of the RNA molecule of interest in cell systems, or chemicals remaining after chemical synthesis. Suitable methods to purify the RNA molecule from a mixture of contaminants are known in the art. In certain embodiments, the purity of the RNA molecule(s) of the invention is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% purity.
In one embodiment, the RNA molecule (such as the tRNA molecule) comprises two or more non-natural bases, wherein the two or more non-natural bases inhibit two or more different tRNA modifying enzymes.
In other aspects, the present invention provides a composition comprising the RNA molecules described herein. In some embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. Suitable carriers and/or formulations of pharmaceutical compositions are described elsewhere herein.
Methods
In one aspect, the present invention provides a method for inhibiting a tRNA modifying enzyme. The method comprises contacting the RNA molecule described herein with the tRNA modifying enzyme in the cell. The formation of the covalent bond inhibits the tRNA modifying enzyme.
In some embodiments, the method is an in vitro or ex vivo method.
In one embodiment, the at least one nucleotide comprises a non-natural base.
In another embodiment, the non-natural base comprises 5-halouracil or 8-azaadenosine.
In some embodiments, the non-natural modified base is a 5-halouracil in the D-loop, the t-psi-c loop, and/or the anticodon loop of the tRNA molecule.
In other embodiments, the tRNA modifying enzyme is a dihydrouridine synthase (DUS) or a pseudouridine synthase (PUS).
In other aspects, the present invention provides a method for killing a cell. The method comprises contacting the RNA molecule described herein with the tRNA modifying enzyme in the cell. The formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cell.
In some embodiments, the method is an in vitro or ex vivo method.
In one embodiment, the cell is a brain cancer cell, a digestive tract cancer cell, a kidney cancer cell, a liver cancer cell, or a lung cancer cell.
In another embodiment, the cell is a cancer cell in a culture.
In one embodiment, the at least one nucleotide comprises a non-natural base.
In another embodiment, the non-natural base comprises 5-halouracil or 8-azaadenosine.
In some embodiments, the non-natural modified base is a 5-halouracil in the D-loop, the t-psi-c loop, and/or the anticodon loop of the tRNA molecule.
In another aspect, the present invention provides a method for treating or preventing cancer in a subject in need thereof. The method comprises administering to the subject an effective amount of a pharmaceutical composition comprising the RNA molecule described herein. The RNA molecule contacts the tRNA modifying enzyme in a cancer cell of the cancer, wherein formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cancer cell.
In some embodiments, the cancer is a lung cancer, a brain cancer, a digestive tract cancer, a kidney cancer, or a liver cancer.
In other embodiments, the method further comprises administering a chemotherapy to the subject before, after, or concurrently with the administration of the pharmaceutical composition comprising the RNA molecule described herein.
In some embodiments, the subject is a mammal. In one embodiment, the subject is a human.
In other embodiments, the method comprises administering the pharmaceutical composition in combination with a therapeutic that induces ferroptosis in the cancer cell. Compounds that induce ferroptosis are described in, for example, Wang et al., Genes & Diseases, 9(2): 334-346 (March 2022), which is herein incorporated by reference in its entirety. Non-limiting examples of ferroptosis-inducing compounds include, but are not limited to, GPX4 inhibitors such as, for example, RSL3, ML 162, ML210, JKE-1674, and the like.
In some embodiments, the RNA molecule herein is further modified, conjugated or combined with a delivery vehicle for delivery into a cell or administration to a subject in need thereof. Non-limiting modifications include, but are not limited to, backbone modifications, such as introducing phosphorothioate (PS) or phosphodi ester (PO) linkages to the backbone of the RNA molecule; modifying nucleobase to improve delivery (in addition to the non-natural bases for inhibiting tRNA modifying enzymes), such as by introducing pyrimidine methylation; modifying termini of the RNA molecule to improve delivery, such as by introducing '-(E)- vinylphosphonate or abasic ribonucleotides to reduce exonuclease digestion; modifying ribose sugar groups such as by introducing 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E) and 2'- Fluoro (2'-F) to improve the resistance of the RNA molecule to nuclease digestion; conjugating the RNA molecule with lipids, peptides, aptamers, antibodies, or sugars to improve the cellular intake; or packing the RNA molecules with nanoparticles. In some embodiments, the modification, conjugation or delivery vehicle is the same as or similar to those already used for delivering oligonucleotide drugs, which is described in, for example, Roberts et al. (Nature Reviews Drug Discovery volume 19, pages673-694 (2020)) and Huang et al. (Biomaterials Research volume 26, Article number: 49 (2022).
Administration and Dosage
The regimen for administration may affect what constitutes an effective amount. The therapeutic formulations contemplated within the disclosure may be administered to the subject either prior to or after the onset of a disease and/or disorder contemplated herein. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations contemplated within the disclosure may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
Administration of the compositions contemplated within the disclosure to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease and/or disorder contemplated herein in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound contemplated within the disclosure to treat a disease and/or disorder contemplated herein in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound contemplated within the disclosure is from about 1 and 5,000 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
Actual dosage levels of the active ingredients in the pharmaceutical compositions contemplated within the disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.
A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds contemplated within the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms contemplated within the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disease and/or disorder contemplated herein.
In certain embodiments, the compositions described herein are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions described herein comprise a therapeutically effective amount of a compound described herein and a pharmaceutically acceptable carrier.
The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
In certain embodiments, the compositions described herein are administered to the patient in dosages that range from one to five times per day or more. In another embodiment, the compositions described herein are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions described herein varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.
The RNA molecules of the invention for administration may be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 3050 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.
In some embodiments, the administered dose of the RNA molecules is about 1 mg to about 2,500 mg. In some embodiments, the dose used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a RNA molecule of the invention, alone or in combination with a second pharmaceutical agent; and instructions for treating or preventing, or reducing one or more symptoms of cancer in a subject.
Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for intracranially, oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.
Routes of administration of any of the compositions described herein include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the disclosure may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein. Oral Administration
For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.
For oral administration, the compounds described herein may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e. , polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fdlers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g, methyl or propyl p-hydroxy benzoates or sorbic acid).
The present disclosure also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds described herein, and a further layer providing for the immediate release of another medication. Using a wax/pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release. Parenteral Administration
For parenteral administration, the RNA molecules described herein may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used.
Additional Administration Forms
Additional dosage forms of the present invention include dosage forms as described in U.S. Patents Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos. WO 03/35041; WO 03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO 02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO 98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.
Controlled Release Formulations and Drug Delivery Systems
In certain embodiments, the formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method described herein may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation. In certain embodiments of the disclosure, the RNA molecules described herein are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.
The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
Dosing
The therapeutically effective amount or dose of a RNA molecule of the present invention depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of the cancer in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.
A suitable dose of a RNA molecule described herein may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.
It is understood that the amount of RNA molecule dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the modulator of the disclosure is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the patient's condition, to a level at which the improved disease is retained. In certain embodiments, patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection.
The RNA molecules for use in the methods described herein may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Capsid assembly modulators exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such capsid assembly modulators lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.
Examples
The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the molecules and compositions of the present invention and practice the claimed methods. The following working examples therefore, specifically point out embodiments of the present invention, and are not to be construed as limiting in any way.
Example 1: Design and test of an RNA-based inhibitor for tRNA modifying enzymes
To test the hypothesis that non-specific chemotherapy compounds can be incorporated into tRNA molecules, and form covalent bonds with and inhibit tRNA modifying enzymes that modifies the tRNA molecules, the present study designed a non-limiting RNA molecule inhibitor based on a natural tRNA molecule.
Referring to Fig. 2, the non-limiting RNA molecule inhibitor was designed to utilize the non-natural modified base 5-halouracil, which is able to react with a cysteine residue in the active site of dihydrouridine synthases (DETS). Notably, the cysteine residue in the active site is conserved in all DUS (Rider et al., J Biol Chem. 2009 Apr 17;284(16): 10324-33). Referring to Fig. 3, the non-limiting RNA molecule inhibitor herein is modified from a natural tRNA decodes the cysteine codon (tRNACys). In the tRNACys, position 20 (the 19th nucleoside), a uridine, is naturally modified by tRNA modifying enzymes such as DUS2 into a dihydrouridine. In the RNA molecule inhibitor, the above-mentioned uridine was replaced with the non-natural modified nucleoside 5-fluorouridine (5-FU). The result RNA-based inhibitor is referred to as 5-FU tRNA or tRNACys-5FU. This non-limiting example of RNA molecule inhibitor has the sequence set forth in SEQ ID NO: 1.
Referring to Fig. 4, the present study confirmed that the RNA molecule inhibitor including the non-natural 5-FU nucleoside was able to form stable covalent bond with DUS2.
Referring to Figs. 5, 14A, the present study further confirmed that tRNACys-5FU was able to significantly reduce the viability of a lung cancer cell line, PC9.
Example 2-1: Dihydrouridine synthase 2 sustains levels of tRNACys and prevents ferroptosis in lung cancer
Dihydrouridine is a universally conserved tRNA modification installed by enzymes that are important for human health for reasons that are yet unclear. High expression of dihydrouridine synthase 2 (DUS2) predicts poor patient outcomes in lung adenocarcinomal. Here, the present study shows in human cells and mouse xenografts that DUS2 suppresses ferroptosis, a metal -dependent non-apoptotic form of cell death to which many lung cancers are unusually sensitive, which is emerging as a therapeutic target in lung cancer. Consistent with a positive role for DUS2 in lung adenocarcinoma growth and metastasis, high expression of DUS2 correlates with increased resistance to ferroptosis inducers. Loss of DUS2 causes increased sensitivity with concomitant accumulation of toxic lipid peroxides, a hallmark of ferroptotic cell death. Mechanistically, DUS2 is required to maintain tRNA CysGCA levels and support translation of cysteine-rich proteins including metallothioneins that serve as key regulators of both metal and redox homeostasis. Metallothionein deficiency in DUS2 knockout cells leads to increased susceptibility to zinc intoxication and lower levels of reduced glutathione, which partially explains their sensitivity to ferroptosis. The results here reveal a tRNA-specific vulnerability and demonstrate the therapeutic potential of targeting DUS2.
Many cancers display resistance to canonical apoptotic cell death pathways. Non-small cell lung cancers (NSCLC) use a number mechanisms to avoid apoptosis including loss of expression of the pro-apoptotic gene Bcl-2-like protein (BIM) and amplification of an anti- apoptotic gene, induced myeloid leukemia cell differentiation protein (MCLl).One of the cell death mechanism, ferroptosis, is a form of non-apoptotic cell death that is emerging as a therapeutic target in lung cancer.
Example 2-2: DUS2 is overexpressed in lung cancer and loss of DUS2 sensitizes cells to ferroptosis
Hallmarks of ferroptotic cell death include dependence on redox active iron and accumulation of toxic lipid peroxides. Several compounds have been described to induce ferroptosis, including class I ferroptosis inducers that inhibit import of cystine (erastin) and class II ferroptosis inducers which inhibit activity of the phospholipid hydroperoxidase GPX4 ((1S,3R)-RSL3, M162, and ML210) (Fig. 6A). Several studies have demonstrated that NSCLC cell lines are sensitive to chemical ferroptosis inducers, both in vitro and in vivo, and development of ferroptosis modulating drugs is an active area of research. Intriguingly, resistance to treatment with several class II ferroptosis inducers (RSL3, ML162 and ML210) correlates with expression of the tRNA modifying enzyme dihydrouridine synthase 2 (DUS2) in a panel of 860 cancer cell lines (Fig. 6B).
Dihydrouridine synthases (DUS) install a modified form of uridine in RNA (Fig. 6C). Dihydrouridine (D) is the most common modified nucleotide in tRNA, and is found in tRNA from organisms from all branches of the tree of life. In tRNAs, D is thought to stabilize to the correct folding of the D-loop. Eukaryotes including humans express four D synthases, and each DUS has unique target nucleotides in multiple individual tRNAs. Disturbance of D levels and/or DUS expression are implicated in lung, brain and kidney cancer. DUS2 is known to modify tRNAs at position in the tRNA D-loop in yeast. DUS2 is frequently overexpressed in non-small cell lung cancer (NSCLC) tumors (Fig. 6D), and NSCLC patients whose tumors express high levels of DUS2 have shorter survival time when compared to patients whose tumors do not express high levels of DUS2 (Fig. 6E).
Here, the present study investigated the role that DUS2 plays in NSCLC disease progression. Using CRISPR/Cas9, DUS2 was knock out in a NSCLC cell line (A549) that expresses high levels of DUS2 and show that loss of DUS2 leads to hypersensitivity to ferroptosis inducing compounds. Consistent with the in vitro sensitivity of the DUS2 KOs to ferroptosis, the present study shows that DUS2 KO cells form smaller tumors in a mouse xenograft model and are more sensitive to systemic administration of a ferroptosis inducer. The present study probed the role of DUS2 in gene expression and find that loss of DUS2 causes a -40% decrease in the level of a single tRNA, CysGCA. This loss of tRNACysGCA reduces levels of cysteine rich proteins proteome wide, including decreasing synthesis of a family of small, highly conserved cysteine rich proteins called metallothioneins (MTs). MTs are known to inhibit ferroptosis, and play two critical roles in cells: first, MTs directly inhibit the formation of lipid peroxides and defend the cell against oxidative stress. Second, MTs are major regulators of intracellular zinc levels. The results here establish the loss of MT expression as the likely basis for increased ferroptosis in the absence of DUS2 and suggest therapeutic potential for targeting DUS2 in lung cancer.
To investigate the link between high DUS2 expression and poor patient prognosis in NSCLC, the present study used CRISPR/Cas9 to generate knockout (KO) cell lines in a common NSCLC model cell line (A549) that expresses high levels of DUS2. Using two different lentiviral delivered guide RNAs targeting exons 3 and 4 of the DUS2 coding sequence, multiple independent clonal KO cell lines were recovered. As expected, the clonal DUS2 KO CRISPR lines did not have detectable DUS2 protein expression (Fig. 6F).
The correlation between DUS2 mRNA levels and resistance to known ferroptosis inducing compounds across a panel of 860 cell lines prompted the present study to test the sensitivity of DUS2 KO cells to ferroptosis. The DUS2 KO cells showed an approximately 2- fold increase in the fraction of dead cells after ferroptosis induction with the GPX4 inhibitor RSL3 (Fig. 6G). This sensitivity was reversed upon re-expression of DUS2 (Fig. 10A), or by treatment with known ferroptosis inhibitors, trolox and ferrostatin (Figs. 10A-10C), but not by treatment with Z-FAD-FMK, an apoptosis inhibitor (Figs. 10A-10C). A key feature of ferroptotic cell death is a buildup of toxic lipid peroxides. The present study measured the levels of lipid peroxidation after GPX4 inhibitor treatment using an oxidation-sensitive fluorescent lipid peroxidation probe, Cl 1-BODIPY26. When compared to WT A549 cells, the DUS2 KO cells had 6-8 fold higher levels of lipid peroxides when treated with RSL3 (Figs. 6H-6I), or a second GPX4 inhibitor, ML162 (Fig. 10D). Consistent with the higher levels of lipid peroxidation in the DUS2 KO cells, using a probe for cellular reactive oxygen species (ROS), 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), the present study observed higher cellular ROS levels in the DUS2 KOs when treated with RSL3 (Fig. 10E).
Example 2-3: DUS2 is required to sustain levels of a specific tRNA, tRNACysGCA
As DUS2 is known to modify tRNAs at position 20 in the tRNA D-loop in yeast (Fig. 7 A), and D is known to stabilize tRNA folding, the present study next investigated if DUS2 is required to sustain tRNA expression or function in NSCLC cells. The present study performed tRNA sequencing using a combination of the ARM-seq and DM-TIGRT-seq protocols. The DUS2 KO cells showed no significant change in charging fraction of any tRNA. As a positive control a -70% reduction of tRNAGln charging was detected after starving cells of glutamine (Fig. 7B). Notably, both DUS2 KO cell lines showed a reproducible decrease in expression of nearly every tRNACysGCA isodecoder expressed in A549 cells (Figs. 7C-7D). When tRNACysGCA levels are summed across all isodecoders, the DUS2 KO cells have a -40% decrease in the total pool of tRNACysGCA (Fig. 11).
The present study next re-analyzed small RNA seq data from TCGA lung adenocarcinoma (LU AD) samples for tRNA expression. For tRNACysGCA, the present study found statistically significant increased levels in patient tumor samples as compared to nontumor samples (Fig. 7E). Levels of another U20 containing tRNA (tRNAGlnCTG) were not significantly different in tumor samples (Fig. 7F). Together, these results show that DUS2 is required to sustain levels of a specific tRNA, tRNACysGCA, in NSCLC cells and suggest a role for tRNACysGCA levels in lung cancer disease.
Example 2-4: Loss of DUS2 impairs translation of cysteine rich proteins, including known anti-ferroptotic oncoproteins
To determine if the -40% decrease in tRNACysGCA expression in the DUS2 KO cells has a functional impact on translation, the present study measured cysteine codon translation using luciferase reporters. An array of 15 cysteine codons was prepended to a P2A sequence followed by firefly luciferase as a proxy for production of the cysteine repeat peptide, with an IRES driven renilla luciferase as a normalization control (Fig. 8A). Because cysteine is encoded by two independent codons (UGU and UGC) that are decoded by the same pool of GCA anticodon tRNA, the present study generated versions of the cysteine repeat reporter with either UGU or UGC codons. When transfected into the DUS2 KO cells, these reporters showed a -40% decrease in the ratio of firefly luciferase produced to renilla luciferase produced (Fig. 8B). This loss of efficient cysteine translation was partially rescued by transfection of in vitro transcribed tRNACysGCA into the DUS2 KO cells (Fig. 8C). The observed cysteine translation defects did not affect bulk protein synthesis as determined by 35S methionine incorporation (Fig. 12A).
The present study then used SILAC proteomics to measure changes in the levels of endogenous proteins in DUS2 KO cells. When analyzed by amino acid content, proteins with greater than 5% Cys content showed a significant decrease in abundance (Fig. 8D) consistent with deficient translation of cysteine codons in cells lacking DUS2. Due to the inherently limited coverage of shotgun proteomics and the fact that many cysteine rich proteins are secreted, the SILAC experiment detected only relatively abundant proteins with moderate cysteine content. The present study did not observe any peptides corresponding to many cysteine rich proteins, including any of the metallothioneins. Metallothioneins (MTs) are a class of very cysteine rich proteins (-35% cysteine content) that have been linked to ferroptosis and cancer progression. The present study therefore measured metallothionein translation in DUS2 KO cells using a dual luciferase reporter similar to the cysteine codon repeat reporter by replacing the arrays of cysteine codons with the coding sequences of MT 1A or MT 1G (Fig. 8E). Production of metallothionein proteins was impaired in the DUS 2 KO cells (Figs. 8G-8H), demonstrating that loss of DUS2 activity leads to defects in production of an endogenously expressed cysteine rich oncoprotein known to inhibit ferroptosis and lipid peroxidation.
In parallel the present study measured steady state mRNA levels in the DUS2 KO cells by RNA seq. Among the hundreds of mRNAs that were differentially expressed in the DUS2 KO cells (Fig. 12B), it was noticed that mRNAs encoding cysteine rich proteins were decreased in the DUS2 KO cells (Fig. 12B). Slow translation elongation has been shown to trigger mRNA degradation downstream of surveillance by the ribosome quality control (RQC) pathway. It was hypothesized that reduced tRNACysGCA levels, which impair cysteine translation in DUS2 KO cells, cause ribosomes to stall more frequently on cysteine codons, leading to RQC mediated degradation of cysteine rich mRNAs. Supporting this hypothesis, metallothionein mRNAs were decreased in abundance in the DUS2 KO cells and partially restored by knocking down the RQC factor GIGYF2 (Fig. 12C). Metal lothi on eins utilize their high thiol content to play two interrelated roles in cells: first, through direct coordination of Zn2+ and Cu2+ ions, they are key regulators of cellular zinc and copper levels. Although ferroptosis was initially characterized as an iron-dependent form of cell death, more recent studies show that defects in zinc homeostasis which elevate cytosolic zinc concentrations sensitize cells to ferroptosis. Given the established role of metallothioneins in regulating intracellular zinc levels, the role of zinc in the ferroptosis sensitivity of cells lacking DUS2 was explored. DUS2 KO cells are more sensitive to Zn2+ intoxication and Zn2+-induced cell death (Fig. 8G). The second function of the MTs is to defend the cell from oxidative stress and directly inhibit the accumulation of lipid peroxides, which is a hallmark of ferroptosis. It was hypothesized that because of the decreased MT levels in the DUS2 KOs there would be an increased demand for GPX4 mediated reduction of lipid peroxides, and lower cellular levels of reduced glutathione (GSH). Supporting this hypothesis, the present study found significantly lower GSH levels in the DUS2 KO cells (Fig. 8H). Together, these observations suggest that a major cause of ferroptosis sensitivity in the DUS2 KO cells is loss of MT synthesis, which in turn triggers defects in metal and redox homeostasis (Fig. 81).
Example 2-5: Combined loss of DUS2 and ferroptosis induction extends lifespan in a mouse xenograft NSCLC model
To characterize the impact of DUS2 on tumor growth and progression in vivo, the present study subcutaneously injected either A549 or DUS2 KO cells to develop xenograft tumors in nude mice. After injection, we monitored tumor size and mouse survival. The tumors derived from DUS2 KO cells took 33% longer to establish and were notably smaller than tumors from A549 cells (Fig. 9A-9B). Examining the DUS2 WT and KO tumors revealed modestly increased expression of the ferroptosis biomarker PTSG2 in the DUS2 KO tumors, suggesting endogenous induction of ferroptosis in the tumors (Fig. 9C) Ferroptosis inducers are a promising therapeutic approach for the treatment of some cancers. The in vitro experiments indicate that ferroptosis induction might be a more effective strategy to treat NSCLC in combination with inhibition of DUS2. To determine if the ferroptosis sensitivity of the DUS2 KO cells could be exploited for therapeutic benefit, ferroptosis was induced in mice with established tumors by administration of a GPX4 inhibitor (Fig. 9D). Most GPX4 inhibitors (RSL3, ML162, ML210) suffer from poor pharmacological properties and have limited utility in vivo. However, a new class of GPX4 inhibitors with improved physiochemical and pharmacokinetic properties was recently developed. The present study first tested if oral administration of one of these compounds (JKE- 1674) could induce ferroptosis in mouse lungs by measuring mRNA levels of a marker of ferroptosis, PTSG241 after oral JKE-1674 administration. JKE-1674 induced PTSG2 mRNA in lung tissue approximately 8.5-fold (Fig. 9E), similar to the level of induction by other GPX4 inhibitors. Treatment with JKE-1674 induced PTSG2 expression 3-fold in DUS2 KO tumors (Fig. 9F). Among the mice receiving JKE, mice with DUS2 KO tumors had significantly increased lifespan (Fig. 9G). Together, these data indicate that either inhibition of DUS2 or combinatorial inhibition of DUS2 and induction of ferroptosis could be a promising therapeutic strategy for treatment of NSCLC patients.
Example 2-6
The data here indicate that high expression of a ubiquitous tRNA modifying enzyme is a specific cancer vulnerability in NSCLC cells. DUS2 is frequently over expressed in NSCLC, and patients whose tumors express high levels of DUS2 have worse outcomes. Using NSCLC cells depleted for DUS2, it was demonstrated that DUS2 is required to support the levels of a specific family of tRNAs, tRNA CysGCA. This result highlights the outsized roles that specific tRNA substrates can play in the biological functions of tRNA modifying enzymes. Loss of CysGCA expression in DUS2 KO cells leads to defects in translation of cysteine codons, which reduces steady state levels of many cysteine rich proteins, including metallothioneins that play key roles in regulating cellular zinc levels and responding to oxidative stress. Loss of metallothionein expression in DUS2 KO cells sensitizes the cells to ferroptosis both in vitro and in vivo.
The data here supports that, to fend off ferroptosis, lung cancer cells require both cysteine incorporation into GSH and into cysteine rich metallothionein proteins. Inhibiting either DUS2 or the MT family could increase ferroptosis sensitivity in patients and hold therapeutic value.
Example 2-7: Methods
Cell culture
A549 cells were maintained in a 50:50 mixture of DMEM:F12 medium (Gibco), supplemented with lx penicillin/ streptomycin (Gibco) and 10% FBS (Sigma). Cells were grown at 37°C with 5% CO2 and maintained at subconfluency. CRISPR knockout generation
DUS2 CRISPR knockout A549 cells were generated using a single-guide LentiCRISPRv2 strategy to cause deletions in the third and fourth exons of DUS2. Oligos for each guide RNA were phosphorylated and annealed and then cloned into pLentiGuide-Puro (Addgene) digested with BsmBI. Cas9/guideRNA lentiviruses were generated by transfection of pLentiGuide-Puro, psPAX2 (Addgene), and pdr8.2 (Addgene) into 293T cells. Viral supernatant was harvested, filtered and flash frozen 48 and 96 hours post transfection. For infection, ImL of 48hr viral supernatant was placed in a 6-well dish with A549 cells at 50% confluency. At 90% confluency, the A549 cells were split in to a 10cm dish and selected for stable integrations using lug/mL puromycin (Sigma). After a stable puro resistant population was generated, single clones were isolated using serial dilution and colony picking. Single cell clones were expanded, screened for lack of expression of DUS2 protein, and frozen.
Western Blotting
Whole cell lysates were made by pelleting A549 cells and re-suspending fresh or frozen (-80°C) pellets in RIPA buffer (50mM Tris pH 8, 150 mM NaCl, sodium deoxycholate 0.5%, sodium dodecyl sulfate 0.1%, NP-40 1%), lysed on ice for 10 min with vortexing. Lysates were clarified by centrifugation at 4°C and maximum speed (22,500 x g) for 15 min. Approximately 20ug of whole cell lysate, as determined by BCA assay, was run on a 7% Tris-Acetate Gel and transferred to nitrocellulose membranes using wet transfer. Membranes were blocked in 5% milk for 1 hour and incubated with primary antibodies overnight at 4C in 5% milk low-salt TBST (50 mM Tris pH 7.5 150 mM NaCl 0.1% Tween-20). Antibodies used for Western blot were as follows: anti-DUS2 at 1: 10,000, anti-GAPDH at 1 :10,000 (Sigma-Aldrich G9545). Secondary antibody incubation was for 1 hour at room temperature using HRP conjugated goat anti-rabbit IgG at 1 :3000 (Promega W4011). Washes were with high-salt TBST (50 mM Tris pH 7.5 400 mM NaCl 0.1% Tween-20).
AlkB andAlkB D135S purification pET30a-AlkB and pET30a-AlkB(D135S) (Addgene) were transformed into BL21(DE3) (NEB). IL cultures were grown to OD .55, at 37°C with shaking. IPTG (Gold Bio) and FeSCL (Sigma) were added to ImM and l OuM final concentration. Cultures were induced for 4h at 37°C with shaking, cells were harvested with centrifugation and flash frozen. Each IL pellet was resuspended in 20mL fresh AlkB lysis buffer (50 mM HEPES pH8.0, lOmM Fe(II) sulfate, 300mM NaCl and 5mM imidazole). Cells were lysed by sonication and addition of lysozyme (Sigma). Lysates were clarified with a 12,000 x g spin for 30min at 4°C. Lysates were filtered through a 0.2uM filter and loaded onto a HisTrap 5mL nickel column (Cytiva). Unbound protein and RNA were removed with extensive washing with lysis buffer, and crude alkB protein was eluted from the Ni column using AlkB Lysis buffer with 250mM imidazole. AlkB protein containing fractions were pooled and desalted using a Zeba spin desalting column (Thermo). Desalted protein was purified away from bound RNA using a MonoS column (Cytiva) with a lOOmM-lM NaCl gradient. AlkB protein containing fractions were pooled and concentrated using Amicon Ultra- 15 10KMWCO filters (Milipore). Concentrated AlkB protein was fractionated over a HiLoad 16/60 Superdex S200 column (Cytiva). S200 fractions containing AlkB were again concentrated using Amicon Ultra- 15 10KMWCO filters (Milipore), diluted to 50% glycerol, and flash frozen.
Total RNA Isolation
A549 cells were harvested by pelleting and resuspending fresh or frozen (-80°C) pellets in ImL of QIAzol (Qiagen). Total RNA was harvested according to the manufacturer’s protocol. tRNA sequencing
Total RNA from A549 cells was resuspended in lOOmM NaOAc/HOAc pH 4.8. 3pL IM NaICU (50mM FC) was added and the mixture was incubated at 22°C. After 30 minutes, 6.65pL IM glucose was added. Total RNA was then recovered by EtOH precipitation. Briefly, lOpL 3M NaOAc, ImL EtOH were added, incubated at -20°C for 15min, and then spun at 4°C and maximum speed (22,500 x g) for 30 min. The RNA pellet was washed with 70% EtOH and spund again for 5min. The pellet was resuspended in 50pL of Na Borate pH 9.5 and incubated at 45°C for 90 minutes. Large RNAs were depleted from the total RNA with Qiagen miRNeasy spin colums using manufactures recommendations. Small RNAs were demethylated with AlkB and AlkB D135S in AlkB buffer (50 mM HEPES KOH, pH 8, 75 pM ferrous ammonium sulfate pH 5, 1 mM a-ketoglutarate, 2 mM sodium ascorbate, 50 pg/ml BSA) with with 4* molar ratio of wtAlkB and 4* molar ratio of D135S at 37°C for 100 minutes. RNA was recovered with denaturing SILANE bead cleanup. RNA was then 3’ end healed using T4 PNK (NEB) and CIP (NEB), and recovered again with denaturing SILANE bead cleanup. A 3’ adapter was ligated onto the small RNA using T4Rnl2. 5pL of RNA was incubated with 1.5pL DMSO and 0.5pL 80uM preadenylated 3’ adapter. This mixture was incubated at 65°C for 2min, and placed on ice for Imin. ligations were incubated overnight at 16°C with 3.5pL water, 2pL 10X NEB ligase 50 buffer, 5pL 50% PEG 8000, IpL SUPERASIN (Thermo), and 2pL RNA ligase (NEB). RNA was recovered again with denaturing SILANE bead cleanup. RNA was reverse transcribed using superscript III. 8pL of RNA was annealed to RT primer at 65°C for 5 min, and 10 min cooling to RT on benchtop. RT was performed following manufacturer’s instructions. RNA was removed from cDNA by adding 1 pL IM NaOH to the RT reaction, incubating 5m 95°C and adding 1 pL IM HC1. cDNA was recovered with denaturing SILANE bead cleanup. A 5’ linker was ligated to the cDNA using T4 RNA ligase. cDNA was mixed with ,8pL 80uM 5’ adapter, and IpL DMSO. This mixture was incubated at 75°C for 2min and placed on ice for Imin. to this was added 4.6pL water, 2pL 10X NEB RNA ligase buffer, 0.2pL 0. IM ATP, 5pL 50% PEG 8000, and 2pL RNA ligase. This mixture was incubated at RT overnight with shaking. Linker ligated cDNA was recovered with denaturing SILANE bead cleanup. Final library PCR was performed with Phusion DNA polymerase according to manufacturer’s recommendation. tRNA rescue experiment
T7 template DNA was constructed using PCR to fuse the T7 promoter sequence to the tRNA CysGCA sequence with CCA tail added. tRNA CysGCA RNA was prepared by run off transcription with T7 RNAP at 37°C for 8 hours followed by template removal with DNAsel (Ambion) at 37°C for 30 minutes. Full length tRNA CysGCA was purified on an 8% denaturing urea-PAGE gel, eluted overnight, precipitated with ethanol, and resuspended water. For rescue experiments, 2pg tRNACysGCA and 2pg translational reporter plasmid were co-transfected into cells using TransIT-X2 (Mirus). Forty-eight (48) hours after transfection, cells were harvested in 500 pL IX passive lysis buffer (Promega) and flash frozen. Lysates were freeze thawed 2x and 75pL of lysate was used to measure firefly and renilla luciferase activity with the dual-luciferase reporter assay system (Promega) according to manufacturer’s instructions. DUS2 rescue experiment
Full length DUS2 was cloned into pcDNA3.1 (CMV promoter, C-terminal FLAG tag) and 2pg of DUS2 plasmid was transfected into cells using TransIT-X2 (Mirus). Forty-eight (48) hours after transfection, cells were split into 6 well plates and allowed to recover for 24 hours. At 40-50% confluency, cells were treated with indicated concentrations of ferroptosis inducing compounds for 12 hours. Cells then stained with Annexin/PI as below. tRNAseq data analysis
Demultiplexed reads were adapter trimmed using BBTools bbduk.sh. Adapter trimmed reads were then PCR-duplicate collapsed based on unique molecular identifier (UMI) using dedupe, sh. The UMI was then force trimmed with a second round of trimming. Adapter trimmed and duplicate collapsed reads were then aligned to a single copy of each isodecoder pseudogenome using bbmap.sh. tRNA expression was quantified by counting the number of uniquely mapping reads that mapped to a tRNA, and differential expression analysis was performed using limma-voom. tRNAs with less than 100 uniquely mapping reads were not considered during expression analysis. tRNA charging ratio was determined using custom python scripts ratioing the number of reads terminating with CC-3’ or CCA-3’.
35 S Met total protein synthesis
Equal amounts of DUS2 KO and wt cells were seeded into 6 well plates. Cells were allowed to grow to -80% confluency, and media was switched to DMEM -Met for 20m. lOpL lOOuCi/mL 35S Met was added to each well, and incubated at 37°C for 30m. To harvest, cells were washed in IX PBS 2x, and harvested in 200pL RIPA with lx PMSF and lx cOmplete. Lysates were freeze thawed 2x, and spun at 4°C at 22,500 x g for 15min to pellet cellular debris. Equal amounts of whole cell lysate, as determined by BCA assay were loaded on a 4-20% SDS- PAGE gel, dried for 2hrs and exposed overnight on a storage phosphor screen.
Dual luciferase assay pCMV:codonarray:P2A:Fluc:IRES:Rluc or pCMV:metallothionein:P2A:Fluc:IRES:Rluc constructs were constructed by gibson assembly into pTwist CMV Hygro, and successful assembly was confirmed by sanger sequencing. 2pg of each plasmid was transfected into cells using TransIT-X2 (Minis). Forty-eight (48) hours after transfection, cells were harvested in IX passive lysis buffer (Promega), and flash frozen. Lysates were freeze thawed 2x, and then 75 pL of lysate was used to measure firefly and renilla luciferase activity with the dual -luciferase reporter assay system (Promega) according to manufacturer’s instructions.
SILAC proteomics
The SILAC experiment was configured as a two channel experiment: Cells were grown in either 1 :1 DMEM:F12 with dialyzed FBS (Gibco) supplemented with either un-labeled Arg and Lys (Invitrogen) or 13C6 ,15N4 Arg and 13C6 ,15N2 Lys (Invitrogen). Cells were maintained in isotopically labeled medium for 10 doublings, and then were harvested in RIPA supplemented with ImM PMSF and IX HALT phosphatase/protease cocktail (Pierce). Lysates were clarified at 4°C and 22,500 x g for 10 minutes. Total protein was quantified using a BCA assay, and 120pg total protein was submitted to the Yale MS & Proteomics Resource where they were processed and analyzed. Total protein samples were filtered through a 3-kDa Amicon Ultra filter, and the retentate was SpeedVac dried and used for downstream proteomics preparation. Dried protein pellets were reduced with DTT, alkylated with iodoacetamide, enzymatically digested with trypsin, and desalted using C18 RP microspin column. High-resolution liquid chromatography mass spectrometry MS/MS data were collected on an Orbitrap Fusion mass spectrometer coupled to aNanoACQUITY UPLC. All MS/MS samples were analyzed using Mascot (Matrix Science, Mascot version 2.7.0) For peptide identification, Mascot was set up to search SwissProt assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.020 Da and a parent ion tolerance of 10.0 PPM. Scaffold (version 4.11.1, Proteome Software)) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm.
RNAseq Total RNA was isolated for three replicates of A549 and both DUS2 KO cell lines as described above. Stranded poly(A)+ selected mRNA-seq libraries were prepared by Genewiz and sequenced on a HiSeq X 10 with paired end 150-bp reads. qRT-PCR
Total RNA was isolated as described above. For siRNA knockdown experiments, cells were seeded into 6-well plates, and transfected with siGIGFY2 or siNT siRNAs using TransIT- X2 (Mirus) for 48 hours. Total RNA was DNAse treated using TURBO DNAse (Thermo) according to manufacture instructions. One-step qRT-PCR was performed with gene specific forward and reverse primers using Luna Universal One-Step RT-qPCR (NEB) reagents on a CFX96 Real-Time PCR instrument (Bio-Rad). Fold change was calculated using the Pfaffl method, with GAPDH as the housekeeping gene. For qPCR experiments from tissues and tumors, cells were disassociated, pelleted and resuspended in TRIzol (Invitrogen). RNA was then extracted following manufacturer’s instructions.
Cell death measurements by Annexin V/Propidium Iodide Staining
Cells were counted and seeded into 6-well plates (Corning). At 40-50% confluency, cells were treated with ferroptosis inducing or inhibiting compounds (RSL3, Cayman Chemical, ML 162 Cayman Chemical, Trolox, Sigma, ZVAD-FMK, Promega, ZnC12, Sigma)for 12 hours. Cells were harvested by trypsinization and centrifugation, washed once with IX Hanks Buffered Salt Solution (HBSS), and resuspended in IX annexin-binding buffer (Thermo) and stained with Annexin V/Propidium Iodide according to manufacturer’s instructions. Cells were fdtered through 70 micron filters and analyzed on a BD LSR II FACS analyzer using FITC and Propidium Iodide filter sets.
Quantification of lipid oxidation using Cl 1-BODIPY staining
Cells were counted and seeded into 6-well plates (Corning). At 40-50% confluency, cells were treated with indicated concentration of ferroptosis inducing compound for 12 hours followed by treatment with luM Cl 1-BODIPY for 30 minutes. Cells were then harvested by trypsinization and centrifugation, washed once with IX HBSS, and resuspended in IX Dulbecco's phosphate-buffered saline (DPBS). Cells were filtered through 70 micron filters and analyzed on a BD LSR II FACS analyzer using FITC (reduced Cl 1 -BODIPY) or PE (oxidized Cl 1-BODIPY) filter sets.
Cellular Glutathione Concentration Measurements
Cells were seeded into black 96 well cell culture treated plates (Coming). At 80% confluency, media was removed and cells were washed once with IX DPBS. Glutathione levels were measured using GSH-Glo reagents (Promega) according to manufacturer instructions.
Subcutaneous Mouse Xenografts
All animal protocols were reviewed by the Yale University IACUC and approved under protocol 2020-20303. A549 and A549 DUS2 KO cells were washed with and resuspended in IX PBS and combined 1 : 1 with Matrigel (Corning) to a concentration of 5,000,000 cells per mb. Mice were randomized before injection. 500,000 A549 cells (100 pL) were subcutaneously injected into both flanks of six female nude mice. 500,000 A549 DUS2 KO cells (100 pL) were subcutaneously injected into both flanks of six female nude mice. Mice were anesthetized with isoflurane twice weekly, during which time mice were weighed and tumor volumes were measured. Tumors were measured in two dimensions with calipers and tumor volumes were calculated with the formula Volume = 0.5 * Li * L22, where Li > L2.
Each mouse began dosing once either tumor was at least 5 mm long in at least one dimension. Mice within each group were randomized before dosing. Mice were given 10 mg/mL JKE-1674 (MedChemExpress) (10% 100 mg/mL JKE-1674 dissolved in DMSO, 90% 2O%- - cyclodextran in IX PBS) to a concentration of 50 mg JKE-1674 per kg body weight, or vehicle solution (10% DMSO, 90% 20%-P-cyclodextran in IX PBS) by oral gavage. Three of the six mice injected with only A549 or A549 DUS2 KO cells were dosed with JKE-1674, and the remaining mice were dosed with vehicle solution. Mice were dosed twice weekly. Survival endpoints were defined by death (either naturally or as required by veterinary technicians based on the health of each mouse), a 15% decrease in body weight, or a tumor reaching 2 cm in length in any dimension.
Example 3: In the study described in Example 3, the effects of a non-limiting example of the RNA based DUS/PUS inhibitors herein on various types of cells were studied.
In Example 3, HepG2 cells were maintained in DMEM plus 10% FBS. A549 cells were maintained in 50:50 DMEM:F12 plus 10% FBS. CLB-001 tRNA was transfected using Lipofectamine RNAiMAX. Cell viability was measured using Cell TiterGlo 2.0.
Example 3-1: RNA-based inhibitor for tRNA modifying enzymes kills cancer cells but not non-transformed cells
Referring to Figs. 14A and 14B, the non-limiting example of the RNA-based inhibitors for DUS enzymes described in Example 1 (the tRNACys having the sequence set forth in SEQ ID NO: 1, in which the all the uridine residues are replaced with the non-natural modified nucleoside 5-flurouradine (5-FU), referred to as CLB-001 in Example 3 section), was found to be able to kill two different types of cancer cells with strong IC50.
Referring to Fig. 14A, the cultured hepatocellular carcinoma cell line, HepG2, was treated with various concentrations of CLB-001. At higher concentrations, CLB-001 was found to be able to almost complete kill the hepatocellular carcinoma cells. The IC50 was calculated to be around 5 nM.
Referring to Fig. 14B, the cultured non-small cell lung cancer cell line, A549, was treated with various concentrations of CLB-001. At higher concentrations, CLB-001 was found to be able to almost complete kill the non-small cell lung cancer cells. The IC50 was calculated to be around 10 nM.
Referring to Fig. 19, the cultured hepatocellular carcinoma cell lines, PLC/PRF/5 and SNU-387, were treated with various concentrations of CLB-001. At higher concentrations, CLB- 001 was found to be able to almost complete kill the Hepatocellular Carcinoma cells. The IC50 was calculated to be between 3 nM and 30 nM.
Notably, CLB-001 did not show significant toxicity toward non-cancer cells. Referring to Fig. 15, when the non-transformed hepatocyte cell line, AML12, was subjected to various concentrations of CLB-001, no statistically significant cell death caused by the DUS inhibitor was observed. This is true even at the relatively high 1000 nM CLB-001 concentration.
Example 3-2: CLB-001 was vastly more potent than 5-fluorouracil in killing cancer cells The present study discovered that CLB-001 is vastly more potent than 5FU in killing HepG2 cancer cells.
Referring to Fig. 16, HepG2 cells were separated into two groups. The first group was subjected to various concentrations of 5-fluorouracil (5FU), and the second group was subjected to various concentrations of CLB-001, in which 5FU was incorporated into the tRNACys molecule. The experiment demonstrates that CLB-001 is more than 7000 times more potent than 5FU in killing the HepG2 cancer cells.
Example 3-3: High tRNA modifying enzymes levels are associated with hepatocellular carcinoma and worse outcomes in hepatocellular carcinoma
Referring to Figs. 17A-17C, various modifying enzymes (PUS1, PUSL1, PUS7, RPUSD1, RPUSD2, TRMT2A, TRMT2B, DUS IL, DUS2, DUS3L, and DUS4L) are upregulated in hepatocellular carcinoma tumors, as assayed by the mRNA levels of these enzymes.
Referring to Figs. 18A-18G the upregulations of various modifying enzymes generally predict worse outcomes in hepatocellular carcinoma.
Enumerated Embodiments
In some aspects, the present invention is directed to the following non-limiting embodiments:
Embodiment 1: An RNA molecule comprising at least one nucleotide capable of forming a covalent bond with a tRNA modifying enzyme, wherein formation of the covalent bond inhibits the tRNA modifying enzyme.
Embodiment 2: The RNA molecule of Embodiment 1, wherein the RNA molecule is a tRNA molecule.
Embodiment 3: The RNA molecule of Embodiment 1 or 2, wherein the at least one nucleotide comprises a non-natural base.
Embodiment 4: The RNA molecule of Embodiment 3, wherein the at least one nucleotide is 5-halouridine (5-haloU), 5-halocytidine (5-haloC), 5-aza-cytidine (5-azaC), 8-haloadenosine (8-haloA), 8-azanebularine, 8-aza-adenosine (8-azaA), or 8-haloguanosine (8-haloG). Embodiment 5: The RNA molecule of any one of Embodiment 1-4, wherein the tRNA modifying enzyme is a dihydrouridine synthase (DUS) or a pseudouridine synthase (PUS).
Embodiment 6: The RNA molecule of any one of Embodiment 1-5, wherein the tRNA modifying enzyme is dihydrouridine synthase 1 (DUS1), dihydrouridine synthase 2 (DUS2), dihydrouridine synthase 3 (DUS3), dihydrouridine synthase 4 (DUS4), dihydrouridine synthase 1 -like (DUS1L), dihydrouridine synthase 3 like (DUS3L), dihydrouridine synthase 4 like (DUS4L), pseudouridine synthase 1 (PUS1), pseudouridine synthase like l(PUSLl), pseudouridine synthase 3 (PUS3), TruB pseudouridine synthase family member 1 (TRUB1), TruB pseudouridine synthase family member 2 (TRUB2), dyskerin pseudouridine synthase 1 (DKC1), pseudouridine synthase 7 (PUS7), pseudouridine synthase 7 like (PUS7L), RNA pseudouridylate synthase domain containing 1 (RPUSD1), RNA pseudouridylate synthase domain containing 2 (RPUSD2), RNA pseudouridylate synthase domain containing 4 (RPUSD4), pseudouridine synthase 10 (PUS10), tRNA methyltransferase 2 homolog A (TRMT2A), tRNA methyltransferase 2 homolog B (TRMT2B), NOP2/Sun RNA methyltransferase 2 (NSUN2), NOP2/Sun RNA methyltransferase 3 (NSUN3), NOP2/Sun RNA methyltransferase 6 (NSUN6), DNA methyltransferase 2 (DNMT2), Methyltransferase-Like Protein 1 (METTL1), WD repeat domain 4 (WDR4), adenosine deaminase TRNA specific 1 (ADAT1), adenosine deaminase TRNA specific 2 (ADAT2), adenosine deaminase TRNA specific 2 (ADAT3), or ISCU.
Embodiment 7: The RNA molecule of any one of Embodiment 1-6, wherein the non-natural base is 5-haloU, and the tRNA modifying enzyme is DUS2, DUS IL, DUS3L, DUS4L, ISCU, PUS1, PUS3, PUS7, PUS10, PUSL1, PUS7L, RPUSD1, RPUSD2, RPUSD4, TRMT2A, TRMT2B, TRUB1, or TRUB2; the nucleotide comprising the non-natural base is 5-azaC, and the tRNA modifying enzyme is DNMT2, NSUN2, NSUN3, or NSUN6; the nucleotide comprising the non-natural base is 8-halo-G, and the tRNA modifying enzyme is METTL1 or WDR4; or the nucleotide comprising the non-natural base is 8-azaA, and the tRNA modifying enzyme is ADAT1, ADAT2, or ADAT3. Embodiment 8: The RNA molecule of any one of Embodiments 3-7, wherein the at least one nucleotide is at a position corresponding to a natural position of a natural nucleotide of a natural tRNA that is modified by the tRNA modifying enzyme.
Embodiment 9: The RNA molecule of any one of Embodiments 3-8, wherein the RNA molecule is a tRNA molecule, and wherein the non-natural modified base is a 5-halouracil in the D-loop, the t-psi-c loop, the anticodon loop, or a stem of the tRNA molecule.
Embodiment 10: The RNA molecule of any one of Embodiments 1-9, wherein the RNA molecule comprises the sequence of any one of SEQ ID NOs: 1-53; or comprises at least about 80% sequence identity to the sequence of any one of SEQ ID NOs: 1-53.
Embodiment 11 : The RNA molecule of any one of Embodiments 1-10, wherein the RNA molecule is an isolated tRNA molecule.
Embodiment 12: The RNA molecule of any one of Embodiments 1-11, wherein the RNA molecule comprises two or more non-natural bases, and wherein the two or more non-natural bases inhibit two or more different tRNA modifying enzymes.
Embodiment 13: A composition comprising the RNA molecule of any one of Embodiments 1-12.
Embodiment 14: The composition of Embodiment 13, further comprising a pharmaceutically acceptable carrier, wherein the composition is a pharmaceutical composition.
Embodiment 15: A method for killing a cell, the method comprising: contacting the RNA molecule of any one of Embodiments 1-12 with the tRNA modifying enzyme in the cell, wherein formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cell.
Embodiment 16: The method of Embodiment 15, wherein the cell is a brain cancer cell, a digestive tract cancer cell, a kidney cancer cell, a liver cancer cell, or a lung cancer cell.
Embodiment 17: The method of any one of Embodiments 15-16, wherein the cell is a cancer cell in a culture.
Embodiment 18: A method for treating a cancer in a subject in need thereof, the method comprising: administering to the subject an effective amount of the pharmaceutical composition of Embodiment 14, wherein the RNA molecule contacts the tRNA modifying enzyme in a cancer cell of the cancer, wherein formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cancer cell.
Embodiment 19: The method of Embodiment 18, wherein the cancer is a lung cancer, a brain cancer, a digestive tract cancer, a kidney cancer, or a liver cancer.
Embodiment 20: The method of Embodiment 18, wherein the cancer is a bladder cancer, a breast cancer, a cervix cancer, a bile duct cancer, a colon cancer, an esophageal cancer, a head/neck cancer, a kidney clear cancer, a kidney papillary cancer, a liver cancer, a lung non small cell cancer, a lung small cell cancer, a prostate cancer, a rectum cancer, a sarcoma cancer, a stomach cancer, a uterine cancer, or a liquid tumor.
Embodiment 21 : The method of any one of Embodiments 18-20, further comprising administering a chemotherapy to the subject.
Other Embodiments
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:
1. An RNA molecule comprising at least one nucleotide capable of forming a covalent bond with a tRNA modifying enzyme, wherein formation of the covalent bond inhibits the tRNA modifying enzyme.
2. The RNA molecule of claim 1, wherein the RNA molecule is a tRNA molecule.
3. The RNA molecule of claim 1 or 2, wherein the at least one nucleotide comprises a nonnatural base.
4. The RNA molecule of claim 3, wherein the at least one nucleotide is 5-halouridine (5-haloU), 5-halocytidine (5-haloC), 5-aza-cytidine (5-azaC), 8-haloadenosine (8-haloA), 8-azanebularine, 8-aza-adenosine (8-azaA), or 8-haloguanosine (8-haloG).
5. The RNA molecule of any one of claims 1-4, wherein the tRNA modifying enzyme is a dihydrouridine synthase (DUS) or a pseudouridine synthase (PUS).
6. The RNA molecule of any one of claims 1-5, wherein the tRNA modifying enzyme is dihydrouridine synthase 1 (DUS1), dihydrouridine synthase 2 (DUS2), dihydrouridine synthase 3 (DUS3), dihydrouridine synthase 4 (DUS4), dihydrouridine synthase 1 -like (DUS1L), dihydrouridine synthase 3 like (DUS3L), dihydrouridine synthase 4 like (DUS4L), pseudouridine synthase 1 (PUS1), pseudouridine synthase like 1(PUSL1), pseudouridine synthase 3 (PUS3), TruB pseudouridine synthase family member 1 (TRUB1), TruB pseudouridine synthase family member 2 (TRUB2), dyskerin pseudouridine synthase 1 (DKC1), pseudouridine synthase 7 (PUS7), pseudouridine synthase 7 like (PUS7L), RNA pseudouridylate synthase domain containing 1 (RPUSD1), RNA pseudouridylate synthase domain containing 2 (RPUSD2), RNA pseudouridylate synthase domain containing 4 (RPUSD4), pseudouridine synthase 10 (PUS 10), tRNA methyltransferase 2 homolog A (TRMT2A), tRNA methyltransferase 2 homolog B (TRMT2B), NOP2/Sun RNA methyltransferase 2 (NSUN2), NOP2/Sun RNA methyltransferase 3 (NSUN3), N0P2/Sun RNA methyltransferase 6 (NSUN6), DNA ethyltransferase 2 (DNMT2), Methyltransferase-Like Protein 1 (METTL1), WD repeat domain 4 (WDR4), adenosine deaminase TRNA specific 1 (ADAT1), adenosine deaminase TRNA specific 2 (ADAT2), adenosine deaminase TRNA specific 2 (ADAT3), or ISCU.
7. The RNA molecule of any one of claims 1-6, wherein the non-natural base is 5-haloU, and the tRNA modifying enzyme is DUS2, DUS IL, DUS3L, DUS4L, ISCU, PUS1, PUS3, PUS7, PUS10, PUSL1, PUS7L, RPUSD1, RPUSD2, RPUSD4, TRMT2A, TRMT2B, TRUB1, or TRUB2; the nucleotide comprising the non-natural base is 5-azaC, and the tRNA modifying enzyme is DNMT2, NSUN2, NSUN3, or NSUN6; the nucleotide comprising the non-natural base is 8-halo-G, and the tRNA modifying enzyme is METTL1 or WDR4; or the nucleotide comprising the non-natural base is 8-azaA, and the tRNA modifying enzyme is ADAT1, ADAT2, or ADAT3.
8. The RNA molecule of any one of claims 3-7, wherein the at least one nucleotide is at a position corresponding to a natural position of a natural nucleotide of a natural tRNA that is modified by the tRNA modifying enzyme.
9. The RNA molecule of any one of claims 3-8, wherein the RNA molecule is a tRNA molecule, and wherein the non-natural modified base is a 5-halouracil in the D-loop, the t-psi-c loop, or the anticodon loop of the tRNA molecule.
10. The RNA molecule of any one of claims 1-9, wherein the RNA molecule comprises the sequence of any one of SEQ ID NOs: 1-53; or comprises at least about 80% sequence identity to the sequence of any one of SEQ ID NOs: 1-53.
11. The RNA molecule of any one of claims 1-10, wherein the RNA molecule is an isolated tRNA molecule.
12. The RNA molecule of any one of claims 1 -11 , wherein the RNA molecule comprises two or more non-natural bases, and wherein the two or more non-natural bases inhibit two or more different tRNA modifying enzymes.
13. A composition comprising the RNA molecule of any one of claims 1-12.
14. The composition of claim 13, further comprising a pharmaceutically acceptable carrier, wherein the composition is a pharmaceutical composition.
15. A method for killing a cell, the method comprising: contacting the RNA molecule of any one of claims 1-12 with the tRNA modifying enzyme in the cell, wherein formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cell .
16. The method of claim 15, wherein the cell is a brain cancer cell, a digestive tract cancer cell, a kidney cancer cell, a liver cancer cell, or a lung cancer cell.
17. The method of any one of claims 15-16, wherein the cell is a cancer cell in a culture.
18. A method for treating a cancer in a subject in need thereof, the method comprising: administering to the subject an effective amount of the pharmaceutical composition of claim 14, wherein the RNA molecule contacts the tRNA modifying enzyme in a cancer cell of the cancer, wherein formation of the covalent bond inhibits the tRNA modifying enzyme, thereby killing the cancer cell.
19. The method of claim 18, wherein the cancer is a lung cancer, a brain cancer, a digestive tract cancer, a kidney cancer, or a liver cancer.
20. The method of claim 18, wherein the cancer is a bladder cancer, a breast cancer, a cervix cancer, a bile duct cancer, a colon cancer, an esophageal cancer, a head/neck cancer, a kidney clear cancer, a kidney papillary cancer, a liver cancer, a lung non small cell cancer, a lung small cell cancer, a prostate cancer, a rectum cancer, a sarcoma cancer, a stomach cancer, a uterine cancer, or a liquid tumor.
21. The method of any one of claims 18-20, further comprising administering a chemotherapy to the subject.
EP24764660.7A 2023-03-02 2024-03-01 Rna-based inhibitors of trna modifying enzymes Pending EP4673226A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363487980P 2023-03-02 2023-03-02
PCT/US2024/018106 WO2024182719A1 (en) 2023-03-02 2024-03-01 Rna-based inhibitors of trna modifying enzymes

Publications (1)

Publication Number Publication Date
EP4673226A1 true EP4673226A1 (en) 2026-01-07

Family

ID=92590887

Family Applications (1)

Application Number Title Priority Date Filing Date
EP24764660.7A Pending EP4673226A1 (en) 2023-03-02 2024-03-01 Rna-based inhibitors of trna modifying enzymes

Country Status (4)

Country Link
EP (1) EP4673226A1 (en)
JP (1) JP2026508359A (en)
KR (1) KR20260007563A (en)
WO (1) WO2024182719A1 (en)

Also Published As

Publication number Publication date
WO2024182719A1 (en) 2024-09-06
JP2026508359A (en) 2026-03-10
KR20260007563A (en) 2026-01-14

Similar Documents

Publication Publication Date Title
US8283331B2 (en) Methods to regulate miRNA processing by targeting Lin-28
CA2579638C (en) Rna interference targeting non-disease causing single nucleotide polymorphisms within a gene encoding a gain-of-function mutant huntingtin protein
EP2688595B1 (en) Lin28-mediated control of let-7 biogenesis
Wang et al. Suppression of PRMT6-mediated arginine methylation of p16 protein potentiates its ability to arrest A549 cell proliferation
US20230149507A1 (en) Treatment of alt cancers
US20210102200A1 (en) Small molecule targeted recruitment of a nuclease to rna
Guh et al. XPF activates break-induced telomere synthesis
Leclair et al. Antisense oligonucleotide-mediated TRA2β poison exon inclusion induces the expression of a lncRNA with anti-tumor effects
KR20170070022A (en) Methods and compositions for the treatment of cancer
JP2006500916A (en) Methods and compositions for treating neoplasia associated with hnRNPA1 and A2 nucleic acid molecules
CA2937767A1 (en) Asymmetric interfering rna compositions that silence k-ras and methods of uses thereof
EP4673226A1 (en) Rna-based inhibitors of trna modifying enzymes
KR20220166490A (en) Pharmaceutical composition for preventing or treating of prostate disease comprising of a METTL1 inhibitor
US9650633B2 (en) Composition for controlling stem cells pluripotency, containing LIN28A methylation inhibitor, and method for screening for LIN28A methylation inhibitor
US7795233B2 (en) Composition comprising double stranded RNA that inhibits expression of NEU3 and method for treating cancer
US8828964B2 (en) Cancer cell identification marker and cancer cell proliferation inhibitor
WO2026055327A2 (en) RNA-BASED INHIBITORS OF tRNA MODIFYING ENZYMES
US7528117B2 (en) High efficacy antisense RIαPKA poly-DNP oligoribonucleotides
WO2011041784A1 (en) Methods for diagnosing and treating cancer
KR101099705B1 (en) Inhibitor of cancer cell growth and anti-cancer drug sensitivity by regulating CAN1 protein
Arleo R-loops and G4-structures: from DNA damage to innate immune response gene activation
WO2024003350A1 (en) Combination therapy for melanoma
WO2025056690A1 (en) Parp1- and immune-checkpoint inhibitors for the treatment of cancer patients overexpressing ubqln1 and/or ubqln4
WO2025257301A1 (en) Treatment of neurofibromatosis type 2 by inhibitors of g6pd, acsl3 and/or oxsm
TW201717969A (en) Methods and compositions for treating malignant tumors associated with KRAS mutation

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20250930

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC ME MK MT NL NO PL PT RO RS SE SI SK SM TR