Methods for Nucleic Acid Cleavage

Abstract

The present invention provides a method for cleaving a target nucleic acid molecule. The method comprises treating the target nucleic acid molecule with a bifunctional molecule of formula (I), CLB, where -C is one to three C 1-6 and -B is a cleavage group which is an imidazole optionally substituted with an alkyl group, -L- is a linker, and -B is a non-covalent linking group for non-covalently linking the bifunctional molecule to a target nucleic acid molecule, and allowing the bifunctional molecule to cleave the target nucleic acid molecule bound thereto. The present invention also provides methods for identifying secondary or tertiary structure within a target nucleic acid, as well as bifunctional molecules and bifunctional molecules for use in therapeutic methods.

Description

[Technical field]

[0001] The project leading to this application has received financial support from the European Union's Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement No. 889922.

[0002] Related Applications This application claims the benefit of, and priority to, UK Patent Application No. 2115540.3, filed on 28 October 2021 (28 / 10 / 2021), the contents of which are incorporated by reference in their entirety.

[0003] The present application relates to methods for the non-enzymatic cleavage of target nucleic acids, for example for use in epigenomic and epitranscriptomic mapping, and for use in therapeutics, such as antimicrobial and antiviral therapy. [Background technology]

[0004] Advances in nucleic acid manipulation and editing techniques have revolutionized the way biological research is conducted. RNA interference and shRNA expression systems have proven invaluable for target validation and elucidating the role that specific genes play in molecular disease (Zamore, et al., 2000). More recently, CRISPR-based technologies have enhanced our ability to manipulate DNA (Gasiunas, et al., 2012; Jinek, et al., 2012) and RNA (Cox, et al., 2017), further enhancing simpler systems such as gene knockout cells and enabling large-scale sophisticated CRISPR-Cas9-based genetic screening approaches (Tzelepis, et al., 2016). However, the most commonly used nucleic acid manipulation techniques are genetic, and applying them to more complex biological systems such as rare populations, animal models, and whole tissues is difficult, if not impossible, and developing therapeutics based on these techniques is even more difficult (Bobbin, et al., 2016).Therefore, there is a need to develop novel small molecule-based techniques that allow the manipulation of nucleic acids in unexplored settings.

[0005] Mikutis et al., 2020, describe small molecule "click degraders" that can be covalently attached to RNA species via click chemistry, thus cleaving the bound RNA molecule. The authors report that 6 -Methyladenosine (m 6We describe a methylation CLICK degradation sequencing method (meCLICK-Seq) to identify the presence of methylated transcripts (A) that are methylated by RNA methyltransferases. This method deactivates an RNA methyltransferase to introduce an alkyne moiety into the RNA in place of a methyl group. A subsequent copper(I)-catalyzed azide-alkyne cycloaddition reaction incorporates the click degrader molecule, resulting in RNA cleavage. The method identifies methylated transcripts, determines the specificity of the RNA methylase, and reliably maps modification sites in intronic and intergenic regions.

[0006] Because the Click degrader molecules are covalently incorporated into the target RNA, they can only be used to degrade RNA species that can be edited to bear the appropriate Click reactive group (typically an alkyne). Furthermore, the need for RNA editing limits the application of this technology to therapeutics. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Zamore, et al., Cell, 2000, Vol. 101, pp. 25-33 [Non-Patent Document 2] Gasiunas, et al., Proc. Natl. Acad. Sci. USA, 2012, Vol. 109, E2579-E2586 [Non-Patent Document 3] Jinek, et al., Science, 2012, Vol. 337, pp. 816-821 [Non-Patent Document 4] Cox, et al., Science, 2017, Vol. 358, pp. 1019-1027 [Non-Patent Document 5] Tzelepis, et al., Cell Reports, 2016, Vol. 17, pp. 1193-1205 [Non-Patent Document 6] Bobbin, et al., Annual Review of Pharmacology and Toxicology, 2016, Vol. 56, pp. 103-122 [Non-Patent Document 7] Mikutis et al., ACS Cent. Sci., 2020, Vol. 6, pp. 2196-2208 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention has been devised in light of the above concerns. [Means for solving the problem]

[0009] The present invention relates to the discovery that the bifunctional molecules described herein as degrading agents can be used as catalytic agents for non-covalently binding and cleaving target nucleic acid molecules. The degrading agents disclosed herein bind to target nucleic acids through non-covalent interactions. Surprisingly, the inventors have found that non-covalent binding is sufficient to allow selective degradation of target nucleic acids. Thus, the degrading agents do not require the incorporation of click-reactive groups into the target nucleic acid. The selective cleavage of target nucleic acid molecules using the degrading agents described herein can be useful in epigenetic and epitranscriptomic analysis, bifunctional mapping, and therapeutics, such as antibacterial and antiviral therapy, and even anticancer.

[0010] Thus, in a first aspect of the invention there is provided a method for cleaving a target nucleic acid molecule comprising the steps of: The target nucleic acid molecule is treated with a bifunctional molecule of formula (I): CLB(I) (Wherein, -C represents 1 to 3 C 1-6-L- is a linker; and -B is a non-covalent linking group for non-covalently linking the bifunctional molecule to a target nucleic acid molecule. and Allowing the bifunctional molecule to cleave the target nucleic acid molecule bound thereto. The method includes:

[0011] Preferably, the non-covalent linking group (-B) is not a polynucleotide group.

[0012] Preferably, the non-covalent binding group (-B) has a molecular weight of 1000 kDa or less, more preferably 800 kDa or less.

[0013] Preferably, the non-covalent binding group (-B) is attached to a secondary or tertiary structure within the target nucleic acid, such as a quadruplex, a pseudoknot, a triplex, a tetraloop, a step loop, or a hairpin loop. More preferably, the non-covalent binding group (-B) is attached to a quadruplex or a pseudoknot.

[0014] Preferably, the bifunctional molecule has a dissociation constant (k) of 10,000 nM or less as determined, for example, by SPR, or alternatively by microscale thermophoresis (MST), or by fluorescence quenching assays. D Preferably, the bifunctional molecule has a k of 1000 nM or less, more preferably 500 nM or less, more preferably 200 nM or less, and most preferably 100 nM or less. D It binds to secondary or tertiary structures.

[0015] Additionally, or alternatively, the bifunctional molecule may have a dissociation constant (k) of 10 mM or less, as determined, for example, by microscale thermophoresis (MST). DPreferably, the bifunctional molecule has a k of 8 mM or less, more preferably 7 mM or less, more preferably 6 mM or less, and even more preferably 5 mM or less. D It binds to secondary or tertiary structures.

[0016] Preferably, the non-covalent bonding group (-B) is selected from the formulae (BI) to (B-III) shown below.

[0017] Preferably, the target nucleic acid molecule is an RNA molecule, such as a viral RNA or a bacterial ribozyme.

[0018] Preferably, the target nucleic acid molecule is contacted with the bifunctional molecule intracellularly.

[0019] In a second aspect of the invention there is provided a method for identifying a secondary or tertiary structure in a target nucleic acid molecule comprising the steps of: providing a first and a second population of nucleic acid molecules, each of said populations comprising said target nucleic acid molecule; A first population of nucleic acid molecules is provided with a bifunctional molecule of formula (I): CLB(I) (Wherein, -C represents 1 to 3 C 1-6 -L- is a linker; and -B is a non-covalent linking group for non-covalently linking the bifunctional molecule to a target nucleic acid molecule. Introducing allowing the bifunctional molecule to cleave the target nucleic acid molecules present in the first population; and Identifying nucleic acid molecules that are present in less abundance in the first population than in the second population. A method is provided that includes:

[0020] The preferred embodiments of the bifunctional molecule of formula (I) given for the first aspect also apply to the second aspect.

[0021] In a third aspect of the present invention, there is provided a bifunctional molecule of formula (I) for use in a method of treatment: CLB(I) (Wherein, -C represents 1 to 3 C 1-6 -L- is a linker; and -B is a non-covalent linking group. to provide.

[0022] The preferred embodiments of the bifunctional molecule of formula (I) given for the first aspect also apply to the third aspect.

[0023] Preferably, the treatment is treatment of a bacterial or viral infection.

[0024] Preferably, the viral infection is an infection with an RNA virus, more preferably a (+)ssRNA virus, more preferably a coronavirus.

[0025] Preferably, the treatment is treatment of a respiratory tract infection, a urinary tract infection, or gastroenteritis.

[0026] Additionally or alternatively, the treatment is treatment of cancer.

[0027] In a fourth aspect of the present invention, there is provided a bifunctional molecule of formula (I): CLB(I) (Wherein, -C represents 1 to 3 C 1-6 -L- is a linker; and -B is a non-covalent group that binds to a nucleic acid molecule. to provide.

[0028] The preferred embodiments of the bifunctional molecule of formula (I) given for the first aspect also apply to the fourth aspect.

[0029] Preferably, the bifunctional molecule is selected from the compounds Deg-I to Deg-V.

[0030] These and other aspects and embodiments of the invention are described in further detail below. [Brief description of the drawings]

[0031] The present invention will now be described with reference to the drawings listed below. [Figure 1] 1 is a schematic diagram of the mode of action of the degrading agent. FIG. 1 shows the use of the degrading agent in a coronavirus pseudoknot degradation strategy. The pseudoknot degrading agent binds and then directly degrades the coronavirus region that has the pseudoknot without the need for other drugs. [Diagram 2] Figure 1: rG4 degraders cleave rG4-containing oligomers and the SARS-CoV-2 genome in vitro. (a) Shows the effect of rG4 degraders on rG4-competent oligomers. The degraders cleave oligomers under conditions that promote rG4 formation. n=3. (b) Shows the effect of rG4 degraders on oligomers that do not form rG4. No degradation was observed. n=3. (c) Shows nanopore sequencing data showing extensive degradation of the SARS-CoV-2 genome upon treatment with the rG4 degrader PDS-deg6(9A) in its ORF1b. *p<0.05, **p<0.01, ***p<0.005, ns not significant. [Diagram 3] Figure 1 shows preliminary in vitro results of anti-SARS-CoV-2 activity of G4 breakers. (a) Inhibition of plaque forming units (PFU) in samples treated with 50 μM PDS-deg4 (9B), PDS-deg6 (9A), and PDS-DegALK (8). (b) PCR measurements of viral RNA. Results show inhibition of viral replication by 5 μM and 50 μM PDS-deg6 (9A). As shown, PDS-Imi6 (9A) appears to inhibit viral growth more than PDS-deg4 (9B). (c) Cell viability after 24 h incubation with increasing concentrations of G4 breakers. None of the compounds showed cytotoxicity up to 50 μM. [Figure 4]Figure 1. Anti-SARS-CoV-2 activity of G4 degraders in vivo. (a) Mice treated with PDS-deg4(9B) (purple, triangular symbols) showed a 10% weight loss on day 1 post-infection, which remained stable between days 1 and 3 and then dropped again, reaching a 75% threshold on day 5, as seen in vehicle (0.1% DMSO in water) treated animals (grey, square symbols). Uninfected mice treated with vehicle (0.1% DMSO in water), as a control (circular symbols), did not show any weight loss. (b) Quantification of lung viral load on day 5 by plaque assay showed lower load in PDS-deg4(9B) treated animals (purple, right) compared to the vehicle control group (grey, left). *p<0.01. [Diagram 5] Figure 16 shows that MTDB degrader (16a) cleaves coronavirus pseudoknots in vitro. (a) Synthetic design of MTDB-deg (16a). (b) Structure of control molecule TDB-deg (16b) featuring a weak pseudoknot binder and imidazole cleavage moiety. (c) LC-MS data showing the disruption of pseudoknots in the presence of the degrader compared to the control. n=3. (d) Gel image confirming the activity of the pseudoknot degrader. (e) LCMS data showing the efficiency of the pseudoknot degrader weakened when one of the pseudoknot systems is mutated to disrupt the secondary structure of the pseudoknot. n=3. (f) Gel image showing the degradation of native RNA extracted from SARS-CoV-2 by MTDB-deg (16a) compared to the control. ns not significant. [Figure 6]Figure 1: Direct RNA nanopore sequencing reveals genomic locations resolved by MTDB-deg. (a) Distribution and abundance of aligned reads flanking pseudoknot regions for control (0.1% DMSO in water) or MTDB-deg treated SARS-CoV-2 RNA based on alignments in minimap2. b. Distribution and abundance of aligned reads exclusively mapping to the S sgRNA region for control or MTDB-deg treated SARS-CoV-2 RNA based on alignments in minimap2. [Figure 7] Figure 2: MTDB-deg treatment has no effect on subgenomic SARS-CoV-2 RNA. Distribution and abundance of aligned reads mapped exclusively to the indicated sgRNA regions for control (0.1% DMSO in water) or MTDB-deg treated SARS-CoV-2 RNA based on alignments in minimap2. [Figure 8]Figure 1: MTDB degraders inhibit SARS-CoV-2 replication in cells. (a), (b) Percentage of inhibition of viral replication normalized to vehicle control (dashed line) after incubation with increasing concentrations of pseudoknot degrader (MTDB-deg(16a)) and control molecules (MTDB and TDB-deg(16b)). Viral replication was assessed based on RNA levels of the E gene and pseudoknot region after 24 hours of infection (multiplicity of infection (MOI) 0.05). Antiviral activity of MTDB-deg(16a) was seen both before (a) and after (b) infection with SARS-CoV-2 at 0.05 MOI. Means ± SD of triplicate sets are shown, with differences between means of p<0.01 indicated. *p<0.05, **p<0.01, paired two-tailed t-test. (c) The IC50 of the pseudoknot disintegrator MTDB-deg (16a) is shown to decrease when the drug is added post-infection. (d) Photograph of cell monolayers after 4 days of incubation with supernatants from virus cultures treated with 6 uM MTDB-deg (16a), MTDB, and TDB-deg (16b) for 24 h. Treatment with 6 mM MTDB-deg (16a) for 24 h showed a reduction in virus plaque numbers compared to vehicle control, both when added pre-infection or post-infection. The control molecule MTDB only showed a reduction in virus plaque numbers when added pre-infection, whereas TDB-deg (16b) showed no reduction. (e) Cell viability assay showing that none of the compounds showed cytotoxicity in VeroCCL81 cells after 24 h. (f) Percentage of virus replication compared to vehicle control 24 h after removal of the media containing the disintegrators. Treatment with MTDB-deg(16a) for 24 hours reduced the ability of the virus to survive drug exposure. [Figure 9]Dose response curves of MTDB-deg, MTDB, and TDB-deg. (a), (b) Pre- and post-infection IC50 values ​​of the pseudoknot disintegrator MTDB-deg (16a). Control molecules (MTDB and TDB-deg (16b)) did not inhibit viral replication and therefore IC50 values ​​were not determined. (c) Dose response curve (determined by PCR targeting the E gene) including a higher concentration of 18 μM showing an increased IC50. [Figure 10] Figure 1 shows virus survival and virucidal activity after exposure to MTDB degraders. (a) Viral viability after 24 hours of incubation with MTDB-deg (16a) and control molecules MTDB and TDB-deg (16b) as determined by qPCR targeting the pseudoknot region. Viral survival was reduced in samples treated with MTDB-deg (16b) but not in samples treated with control molecules MTDB and TDB-deg (16b). (b) Virucidal activity was assessed by incubating 1000 PFU of SARS-CoV-2 with 6 μM of compound for 1 hour at 37 °C, after which the infectivity of the remaining virus was determined by plaque assay. MTDB-deg(16a), MTDB, and TDB-deg(16b) showed no virucidal effect against cell-free virions, suggesting that the antiviral activity of MTDB-deg(16a) is mediated by inhibition of viral replication within host cells rather than by inactivation of cell-free virions. [Figure 11] Agarose gel analysis of ribosome degradation assay. Left: Ethyl linker, no degradation seen. Center left: Diethylene glycol linker, degradation seen at 15 mM concentration. Center right: Hexaethylene glycol linker, no degradation seen. Right: Chloramphenicol control, no degradation. B=Blank (no degradation agent), Degrader concentrations: 1a / 1b=15 mM, 2a / 2b=7.5 mM, 3a / 3b=3.75 mM, 4a / 4b=1.88 mM, 5a / 5b=0.94 mM, 6a / 6b=0.47 mM. [Figure 12]Figure 1. In vivo activity of MTDB degraders against SARS-CoV-2 infection in K18-hACE2 mice. (a) 8-12 week old female K18-hACE2-transgenic mice were intranasally infected with 104 plaque forming units (PFU) of SARS-CoV-2 and treated intranasally 1 h before and 3 h after infection with MTDB-deg 16a (25 mg / kg) (n=6), MTDB (10 mg / kg, the maximum dose that could be administered at the given limiting solubility) (n=3), TDB-deg 16b (25 mg / kg) (n=5), and vehicle control (n=6). (b) Administration of MTDB-deg 16a reduces lung viral load in SARS-CoV-2 infected K18-hACE2 mice. No difference was found between lung viral loads between vehicle control and MTDB- and TDB-deg 16b-treated mice. Mean ± SD is shown. *p<0.05, unpaired t-test. (c) Western blot analysis of phospho-p38 in lung extracts of transgenic K18-hACE2 mice treated with three doses of 10 mg / kg vehicle (V1, V2) or MTDB-deg 16a (D1, D2) 1 h before and 1 and 2 days after infection (n=2). [Figure 13] RT-qPCR validation of degradation specificity in a cellular system. SARS-CoV-2 infected Vero CCL-81 cells were treated with either 6 μM MTDB-deg or vehicle (HO) control for 24 h (n=3). Student's t-test. Means + SD of three independent replicates are shown. *p<0.05, **p<0.01. [Figure 14] Figure 1 shows the binding affinity of PDS family molecules and MTDB family molecules to their targets. (a) Binding affinity of PDS family ligands to G4 forming NRAS oligonucleotides as determined via fluorescence quenching assay. (b) Binding affinity of MTDB family ligands to pseudoknot oligonucleotides as measured via MST. (c) Binding affinity of MTDB family ligands to disrupted pseudoknot oligonucleotides as measured via MST. n=3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present invention relates to the discovery that bifunctional molecules, also described herein as degrading agents, can be used as catalytic agents for non-covalently binding and cleaving target nucleic acid molecules. The degrading agents disclosed herein bind to target nucleic acids through non-covalent interactions. Thus, the degrading agents do not require the incorporation of click-reactive groups into the target nucleic acid. The selective cleavage of target nucleic acid molecules using the degrading agents described herein can be useful in epigenetic and epitranscriptomic analysis, bifunctional mapping, and therapeutics, such as antibacterial and antiviral therapy, and even anticancer.

[0033] The decomposition agent has the formula (I): CLB(I) where C is a cleavage group, -L- is a linker, and -B is a non-covalent linking group. has.

[0034] Cleavage group (-C) The cleaving group is imidazole (1,3-diazole), which may be substituted as described herein.

[0035] The imidazole cleavage group of the degrading agent can react with and cleave the target nucleic acid molecule. Typically, imidazole can remove a proton from the 2' hydroxyl group of the ribose sugar. In some cases, imidazole can bind copper to induce copper-mediated RNA degradation (Li, Zhong-Rui, et al. Nat Chem 11.10 (2019):880-889; Wong, K, et al. Can J Biochem 52.11 (1974): 950-958; Subramaniam, Siddharth, et al. F1000Research 4 (2015)).

[0036] Imidazole is a basic group, i.e., it can accept a hydrogen cation (H+). Imidazole can also donate an electron pair.

[0037] The basicity of a group can be quantitatively evaluated using the pKa of the associated conjugate acid. That is, the basicity of a basic group [Ba] is the ratio of the pKa of the conjugate acid [BaH] to the pKa of the associated conjugate acid [BaH]. + The pKa of the conjugate acid may be known or may be determined using standard techniques such as acid-base titration. Without wishing to be bound by theory, the inventors believe that basic residues with conjugate acids whose pKa values ​​are above a certain threshold, e.g., pKa of 6.5 or greater, e.g., 6.8 or greater, may deprotonate the hydroxyl group at the 2' position of the ribose sugar, allowing cleavage of the phosphodiester backbone within the target nucleic acid. Imidazole has a pKa close to 7.

[0038] Imidazole contains a nitrogen atom with a lone pair of electrons. The group containing the nitrogen atom with a lone pair of electrons may typically be coordinated with copper. Imidazole has been shown to chelate copper.

[0039] The imidazole may be unsubstituted or may have one, two or three C 1-6 It may be substituted with an alkyl group.

[0040] An alkyl group is a monovalent saturated hydrocarbon group. 1-6 Alkyl groups, such as C 1-4 In this situation, the prefix (e.g., C 1-6 ) indicates the number of carbon atoms in the hydrocarbon backbone. Alkyl groups can be straight or branched.

[0041] C 1-6 Examples of linear alkyl groups include methyl (-Me), ethyl (-Et), n-propyl (-nPr), n-butyl (-nBu), n-pentyl (-Amyl), and n-hexyl.1-6 Examples of branched alkyl groups include isopropyl (-iPr), isobutyl (-iBu), sec-butyl (-sBu), tert-butyl (-tBu), isopentyl, neopentyl, isohexyl, and neohexyl.

[0042] In some preferred embodiments, the imidazole is represented by formula (CI)-(C-III): [ka] (In the formula, R 1 , R 2 , and R 3 are each independently a hydrogen atom or C 1-6 represents an alkyl group, R N is a hydrogen atom or C 1-6 represents an alkyl group, and * represents the point of attachment to the remainder of the molecule (typically a linker unit L) The compound is selected from the group represented by

[0043] In such cases, the degrader may be described as an imidazole degrader.

[0044] Preferably, R 1 , R 2 , R 3 , and R N are each independently a hydrogen atom or C 1-4 Represents an alkyl group.

[0045] More preferably, the cleaving group is a group represented by formula (CI).

[0046] More preferably, R 1 , R 2 , and R 3 each independently represents hydrogen. In such a case, the cleaving group is an unsubstituted imidazole group. That is, the cleaving group is represented by formula (C-IV): [ka] (In the formula, * represents the bonding position with the remaining part of the decomposition agent (typically a linker unit L) It is represented by:

[0047] When non-covalently bound to a target nucleic acid molecule through a linker and a linking group, the imidazole group reacts with the target nucleic acid molecule to cleave one or more phosphodiester bonds, thereby causing degradation of the target nucleic acid molecule. For example, the imidazole group of the bound degrading agent can remove a proton from the 2'OH position on the nucleic acid molecule, causing cleavage of the phosphodiester bond in the target nucleic acid molecule. Furthermore, the imidazole group can form a copper complex that cleaves the phosphodiester bond in the target nucleic acid molecule.

[0048] Linker (-L-) The linker L of the degrading agent comprises a group for attaching (i.e. covalently attaching) the cleavable group (C) to the non-covalent linking group (B). Suitable linkers are well known in the art.

[0049] Typically, the linker comprises a divalent group, one of the free valences forming part of a single bond to the cleavage group (C) and the remaining free valence forming part of a single bond to the non-covalent linking group (B).

[0050] Preferably, the linker is a stable linker, i.e., the linker comprises a group that is not substantially cleaved or degraded in vivo. A stable linker is typically non-reactive at physiological pH and is not substantially degraded by enzymatic action in vivo.

[0051] Typically the linker is a flexible linker, i.e., the linker allows the cleavage group (C) and the binding group (B) to move with a high degree of freedom relative to each other.

[0052] Exemplary linkers include groups selected from alkylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, and heteroarylene. Mixed linkers containing different covalently linked groups, such as alkylene-arylene (aralkylene) and heteroalkylene-arylene, are also possible.

[0053] An alkylene (alkanediyl) group is a divalent saturated hydrocarbon group in which each of the two free valences forms part of a single bond to an adjacent atom. 1-6 Alkylene groups, such as C 1-4 , C 1-3 , or C 1-2 In this context, the prefix (e.g., C 1-6 ) indicates the number of atoms in the hydrocarbon backbone. Alkylene groups can be straight-chained or branched. Examples of straight-chained alkylene groups include methanediyl (methylene bridged), ethane-1,2-diyl (ethylene bridged), propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyl, and hexane-1,6-diyl. Examples of branched alkylene groups include ethane-1,1-diyl and propane-1,2-diyl.

[0054] A heteroalkylene group is an alkylene group in which one or more carbon atoms are replaced with a heteroatom such as N, O, or S. A heteroalkylene group is any group including, but not limited to, C 1-6 Heteroalkylene groups, such as C 1-4 , C 1-3 , or C 1-2 In this context, the prefix (e.g., C 1-6) indicates the number of atoms in the heteroalkylene backbone, either carbon atoms or heteroatoms. Heteroalkylene groups may be linear or branched. Examples of linear heteroalkylene groups include those derived from oxymethylene (e.g., polyoxymethylene (POM)), ethylene glycol (e.g., polyethylene glycol (PEG)), ethyleneimine (e.g., linear polyethyleneimine (PEI), polyaziridine), and tetramethylene glycol (e.g., polytetramethylene glycol (PTMEG), polytetrahydrofuran). Examples of branched heteroalkylene groups include those derived from propylene glycol (e.g., polypropylene glycol (PPG)). When a nitrogen atom is present in a heteroalkylene group, the nitrogen atom may be unsubstituted (NH) or C 1-4 If a sulfur atom is present in a heteroalkyl group, the sulfur atom may be substituted with S, S(O), or S(O) 2 It could be.

[0055] A cycloalkylene group is a divalent saturated hydrocarbon group containing one ring in which all of the ring atoms are carbon atoms and in which the two free valences each form part of a single bond to an adjacent atom. 5-6 In this context, the prefix (e.g., C 5-6 ) indicates the number or range of ring atoms. A cycloalkylene group can be monocyclic. Examples of monocyclic cycloalkylene groups include 1,3-cyclopentylene and 1,4-cyclohexylene.

[0056] A heterocycloalkylene (heterocyclene) group is a cycloalkylene group in which one or more of the carbon atoms is replaced with a heteroatom, such as N, O, and S, or one or more of the carbon atoms has an oxo substituent (=O). 5-6 In this context, the prefix (e.g., C 5-6 ) indicates the number or range of ring atoms, either carbon atoms or heteroatoms. Heterocycloalkylene groups can be single ring. If a nitrogen atom is present in a heteroalkylene group, it may be unsubstituted (NH) or C 1-4 If a sulfur atom is present in a heteroalkyl group, the sulfur atom may be substituted with S, S(O), or S(O) 2 It could be.

[0057] An arylene group is a divalent hydrocarbon group containing one aromatic ring in which all of the ring atoms are carbon atoms and in which the two free valences each form part of a single bond to an adjacent atom. 6-10 In this situation, the prefix (e.g., C 6-10 ) indicates the number or range of ring atoms. Arylene groups may be monocyclic or contain two or more rings. Examples of monocyclic arylene groups include 1,4-phenylene. Examples of bicyclic arylene groups include 2,6-naphthylene.

[0058] A heteroarylene group is an arylene group that contains one aromatic ring in which one or more of the ring atoms are heteroatoms, such as N, O, and S, or one or more of the carbon atoms have an oxo substituent (=O). 6-10 In this context, the prefix (e.g., C 6-10 ) indicates the number or range of ring atoms, either carbon or heteroatoms. Heteroarylene groups can be a single ring or contain two or more rings. Examples of single ring heteroarylene groups include pyrrolylene and pyridylene.

[0059] Preferred linkers include groups selected from alkylene and heteroalkylene. More preferred linkers include heteroalkylene groups. More preferred linkers include alkylene ether groups. Most preferred linkers include ethylene oxide groups (e.g., derived from polyethylene glycol, PEG).

[0060] In a preferred embodiment, the linker has the formula (LI): [ka] (In the formula, L 1 is a covalent bond or C 1-2 is an alkylene group, L 2 is C 1-6 Alkylene group or C 1-6 is a heteroalkene group, L 3 is C 1-6 is an alkylene group, n is 1 to 8; * is the point of attachment to the non-covalent linking group (-B), and ** is the point of attachment to the cleavable group (-C) The compound is a group represented by or includes a group represented by:

[0061] Appropriate C 1-2 Alkylene groups include methylene (methanediyl) and ethylene (ethane-1,2-diyl).

[0062] Appropriate C 1-6 Alkylene groups include methylene (methanediyl), ethylene (ethane-1,2-diyl), propylene (propane-1,3-diyl), butylene (butane-1,4-diyl), pentylene (pentane-1,5-diyl), and hexylene (hexane-1,6-diyl).

[0063] Preferably, L1 is a covalent bond or methylene.

[0064] Preferably, L 3 is C 1-4 More preferably, L 3 is ethylene.

[0065] Preferably, n is an integer of 2 to 5.

[0066] Appropriate C 1-6 Heteroalkene groups include alkylene ether groups, such as ethylene oxide (-CH 2 CH 2 O-), propylene oxide (-CH 2 CH 2 CH 2 O-), and tetramethylene oxide (-CH 2 CH 2 CH 2 CH 2 O-) is included.

[0067] Preferably, L 2 is ethylene oxide. In such a case, the linker is represented by formula (L-II): [ka] (In the formula, L 1 , L 3 ,n, * , and ** is as described for formula (LI) and the same preferences apply. The compound is a group represented by or includes a group represented by:

[0068] In an alternative embodiment, the linker has formula (L-III): [ka] (In the formula, L 4 is C 1-6 is an alkylene group, L5 is C 1-6 Alkylene group or C 1-6 is a heteroalkene group, L 6 is a covalent bond or C 1-2 is an alkylene group, m is 1 to 8; * is the point of attachment to the non-covalent linking group (-B), and ** is the point of attachment to the cleavable group (-C) The compound is a group represented by or includes a group represented by:

[0069] Preferably, L 4 is C 1-4 More preferably, L 3 is ethylene.

[0070] Preferably, L 6 is methylene or ethylene.

[0071] Preferably, m is an integer of 2-5.

[0072] Preferably, L 5 is ethylene oxide. In such a case, the linker is represented by formula (L-IV): [ka] (In the formula, L 4 , L 6 , m, * , and ** is as described in formula (L-III) and the same preferences apply. The compound is a group represented by or includes a group represented by:

[0073] Non-covalent group (-B) The binding group of the degradative agent comprises a group capable of binding to a target nucleic acid molecule. The binding group binds to the target nucleic acid molecule via a non-covalent bond.

[0074] Certain small molecule ligands have been shown to bind non-covalently to nucleic acids and therefore can form the basis of non-covalent binding groups.

[0075] A non-covalently linked group is not a polynucleotide (e.g., nucleic acid) group. A covalently linked group is not and does not include nucleotides.

[0076] The non-covalent binding group is not an antibody.

[0077] Typically, the non-covalently attached group has a molecular weight of 1000 kDa or less. Preferably, the non-covalently attached group has a molecular weight of 800 kDa or less.

[0078] Typically, the non-covalent binding group binds to a secondary or tertiary structure in the target nucleic acid. Suitable secondary or tertiary structures include quadruplexes, pseudoknots, triplexes, tetraloops, step loops, and hairpin loops. Preferably, the non-covalent binding group binds to quadruplexes or pseudoknots.

[0079] Preferably, the non-covalent binding group preferentially binds to a secondary or tertiary structure within the target nucleic acid. In such cases, the non-covalent binding group preferentially binds to a secondary or tertiary structure within the target nucleic acid over linear or unstructured nucleic acid. Preferably, the non-covalent binding group preferentially binds to a quadruplex or pseudoknot.

[0080] Preferably, the non-covalent binding group selectively binds to ribonucleic acid (RNA) and therefore may be known as a non-covalent RNA binding group.

[0081] Non-covalent binding groups can bind to target nucleic acids via electrostatic interactions, such as ionic interactions, hydrogen bonds, and halogen bonds; van der Waals interactions, such as persistent dipole-dipole interactions, dipole-induced dipole interactions, and induced dipole-induced dipole interactions; and π effects, such as π-π interactions, π-cation interactions, and polar-π interactions.

[0082] The non-covalent binding group may be based on the following small nucleic acid binding molecules: [Table 1]

[0083] The non-covalent linking group can be attached to the linker at any suitable position. Typically, the non-covalent linking group is attached to the linker via a heteroatom (such as O or NH) or adjacent to a carbonyl group (C=O).

[0084] Preferably, the linking group is selected from formulae (BI) to (B-III).

[0085] Dynamic characteristics The interaction between the cleavage agent and the target nucleic acid is governed by the dissociation constant (k D ) can be used to quantify the dissociation constant between a degrading agent containing a given non-covalent group and a nucleic acid. The dissociation constant between a degrading agent containing a given non-covalent group and a nucleic acid can be known, or the constant can be determined using standard techniques such as surface plasmon resonance (SPR), e.g., Biacore (Santos et al., 2021). Suitable systems for measuring the dissociation constant include the Biacore T200.

[0086] Typically, the degraders have a dissociation constant (k) of 10,000 nM or less, as determined, for example, by SPR. D Preferably, the degrading agent binds to the target nucleic acid with a k of 1000 nM or less, more preferably 500 nM or less, more preferably 200 nM or less, and most preferably 100 nM or less. D The target nucleic acid is bound to the

[0087] As described above, the non-covalent binding groups of the degrading agents typically bind to secondary or tertiary structures within the target nucleic acid. Thus, the degrading agents typically have a dissociation constant (k DPreferably, the degrading agent binds to the secondary or tertiary structure with a k of 1000 nM or less, more preferably 500 nM or less, more preferably 200 nM or less, and most preferably 100 nM or less. D It binds to secondary or tertiary structures.

[0088] In some embodiments, the degrading agent has a dissociation constant (k D In such cases, the degrading agent preferably binds to the quadruplex with a k of 1000 nM or less, more preferably 500 nM or less, more preferably 200 nM or less, and most preferably 100 nM or less. D It binds to the quadruplex.

[0089] In some embodiments, the degrading agent has a dissociation constant (k D In such cases, the decomposition agent preferably has a k of 1000 nM or less, more preferably 500 nM or less, more preferably 200 nM or less, and most preferably 100 nM or less. D It joins to a pseudoknot.

[0090] In further or alternative embodiments, the dissociation agent has a dissociation constant (k ) of 100 mM or less, as determined, for example, by microscale thermophoresis (MST). D ) to bind to the target nucleic acid. In such a case, k D may be 50 mM or less, such as 25 mM or less, for example 20 mM or less, such as 15 mM or less, for example 10 mM or less, such as 9 mM or less, for example 8 mM or less, such as 7 mM or less, for example 6 mM or less, such as 5 mM or less, for example 4 mM or less, such as 3 mM or less, for example 2 mM or less. Preferably, the degrading agent has a dissociation constant (k D ) binds to the target nucleic acid.

[0091] k Dcan be determined using standard techniques, for example by microscale thermophoresis (MST), for example as described in the examples below. Measurements can be performed using fluorescently tagged nucleic acids, for example FAM functionalized nucleic acids, incubated with the dissociation agent. The nucleic acid can be at a concentration of 50 nM and may be provided in a buffer, for example HEPES buffer at pH 7.4. The dissociation agent can be tested in serial dilutions, at a maximum concentration of up to 8 mM, for example up to 250 μM. Measurements can be performed at a temperature of 25° C. MST measurements can be performed at an MST power of 30%. Suitable systems for measuring dissociation constants include the NanoTemper Monolith NT.115.

[0092] Preferably, the degrading agent has a k of 20 nM or less, more preferably 15 nM or less, more preferably 10 mM or less, more preferably 5 mM or less. D The nucleic acid molecule binds to a secondary or tertiary structure within the target nucleic acid.

[0093] In some embodiments, the degrading agent has a dissociation constant (k ) of 20 mM or less, more preferably 15 mM or less, more preferably 10 mM or less, more preferably 5 mM or less, and most preferably 2 mM or less. D ) to form a pseudoknot.

[0094] Additionally, or alternatively, the interaction between the degrading agent and the target nucleic acid may be determined by a 50% maximum effectiveness constant (EC 50 ) can be used to quantify the concentration of 50 is the dissociation constant (k D ) or EC 50 may be different. EC 50 The preference for the dissociation constant (k D ) is as described above.

[0095] The 50% maximum effective constant (EC) between a degrading agent containing a given non-covalent group and a nucleic acid. 50) is known or can be determined experimentally, for example using a fluorescence quenching assay (see Di Antonio et al., 2012, and the methods described in the Examples below). EC 50 Suitable systems for measuring include systems for measuring fluorescence, such as plate readers, such as BMG CLARIOstar. The nucleic acid can be a fluorescently tagged nucleic acid, which can be treated with a degrading agent, for example, for a time of 40 minutes, with incubation at a temperature of, for example, 4°C. Measurements can be performed at a temperature of 25°C. The concentration of the nucleic acid can be 50 nM, and in some cases, the nucleic acid can be in a buffer, such as a HEPES buffer at pH 7.4. The concentration of the degrading agent can be up to 10 μM, such as treating the degrading agent with a serial dilution from approximately 2 nM to approximately 10 μM.

[0096] Typically, degraders have a median maximal efficacy constant (EC ) of 10,000 nM or less, as determined, for example, by a fluorescence quenching assay. 50 Preferably, the degrading agent binds to the target nucleic acid with an EC of 1000 nM or less, more preferably 500 nM or less, more preferably 200 nM or less, and most preferably 100 nM or less. 50 The target nucleic acid is bound to the

[0097] As described above, the non-covalent binding groups of the degrading agents typically bind to secondary or tertiary structures within the target nucleic acid. Thus, the degrading agents typically have a 50% maximum effectiveness constant (EC 50 Preferably, the degrading agent binds to the secondary or tertiary structure with an EC of 1000 nM or less, more preferably 500 nM or less, more preferably 200 nM or less, and most preferably 100 nM or less. 50 It binds to secondary or tertiary structures.

[0098] In some embodiments, the degraders have a median maximal efficacy constant (EC ) of 10,000 nM or less, as determined, for example, by a fluorescence quenching assay described herein. 50In such cases, the degrading agent preferably binds to the quadruplex with an EC of 1000 nM or less, more preferably 500 nM or less, even more preferably 200 nM or less, and most preferably 100 nM or less. 50 It binds to the quadruplex.

[0099] In some embodiments, the degraders have a median maximal efficacy constant (EC ) of 10,000 nM or less, as determined, for example, by a fluorescence quenching assay described herein. 50 In such cases, the disruptor preferably has an EC of 1000 nM or less, more preferably 500 nM or less, more preferably 200 nM or less, and most preferably 100 nM or less. 50 It joins to a pseudoknot.

[0100] In some embodiments the degradant has a 50% maximum effectiveness constant (EC) of 100 mM or less, such as 50 mM or less, for example 25 mM or less, such as 20 mM or less, for example 15 mM or less, such as 10 mM or less, for example 9 mM or less, such as 8 mM or less, for example 7 mM or less, such as 6 mM or less, for example 5 mM or less, such as 4 mM or less, for example 3 mM or less, such as 2 mM or less. 50 Preferably, the degrading agent binds to the target nucleic acid with a median maximal effectiveness constant (EC) of 10 mM or less, more preferably 5 mM or less. 50 ) binds to the target nucleic acid.

[0101] Preferably, the degrading agent has a median maximal effectiveness constant (EC ) of 20 nM or less, more preferably 15 nM or less, more preferably 10 mM or less, and more preferably 5 mM or less. 50 ) binds to secondary or tertiary structures within the target nucleic acid.

[0102] In some embodiments, the degrading agent has a 50% maximum effective constant (EC) of 20 mM or less, such as 15 mM or less, more preferably 10 mM or less, more preferably 5 mM or less, and most preferably 2 mM or less. 50 ) to form a pseudoknot.

[0103] As mentioned above, the non-covalent binding group of the degrading agent preferably selectively binds to a secondary or tertiary structure in the target nucleic acid. Binding selectivity can be quantified using the ratio of the dissociation constant of binding to a given secondary or tertiary structure to the dissociation constant of binding to a linear or unstructured nucleic acid, such as a linear or unstructured RNA. Typically, a comparative linear or unstructured nucleic acid is prepared by mutating one or more residues in the desired secondary or tertiary structure to prevent the secondary or tertiary structure from forming while maintaining the rest of the sequence. For example, the selectivity of binding to a G-quadruplex of an RNA can be evaluated by using a comparative RNA in which one or more GGG motifs are changed to AUC motifs.

[0104] Typically, the binding selectivity between a given secondary or tertiary structure and linear or unstructured nucleic acid is 5:1 or greater. Preferably, the selectivity between a given secondary or tertiary structure and linear or unstructured nucleic acid is 10:1 or greater, more preferably 20:1 or greater, more preferably 50:1 or greater, and most preferably 100:1 or greater.

[0105] In one embodiment, the binding selectivity between the quadruplex and linear or unstructured nucleic acid is 5:1 or greater. Preferably, the selectivity between the quadruplex and linear or unstructured nucleic acid is 10:1 or greater, more preferably 20:1 or greater, more preferably 50:1 or greater, and most preferably 100:1 or greater.

[0106] In one embodiment, the binding selectivity between the quadruplex and linear or unstructured nucleic acid is 5:1 or greater. Preferably, the selectivity between the quadruplex and linear or unstructured nucleic acid is 10:1 or greater, more preferably 20:1 or greater, more preferably 50:1 or greater, and most preferably 100:1 or greater.

[0107] Preferred Embodiments In a preferred embodiment, the decomposition agent is selected from compounds Deg-I to Deg-V. [Table 2]

[0108] Salts and solvates The degrading agents of formula (I) may be provided in free base form.

[0109] The degrading agent of formula (I) may be provided in the form of a salt, preferably a pharma- ceutically acceptable salt.

[0110] In some embodiments, the degradants disclosed herein may be provided as salts in protonated form with an appropriate counter anion.

[0111] Suitable counterions include both organic and inorganic anions. An example of an inorganic anion is chloride (Cl - ), bromide (Br - ), iodine (I - ), sulfate (SO 4 2- ), sulfite (SO 3 2- ), nitrate (NO 3 - ), Nitrite (NO 2 - ), phosphate (PO 4 3- ), and phosphite (PO 3 3-Examples of organic anions include those derived from inorganic acids, including 2-acetoxybenzoate, acetate, ascorbate, aspartate, benzoate, camphorsulfonate, cinnamate, citrate, edetate, ethanedisulfonate, ethanesulfonate, formate, fumarate, gluconate, glutamate, glycolate, hydroxymalate, carboxylate, Examples of suitable polymeric organic anions include lactate, laurate, lactate, maleate, malate, methanesulfonate, oleate, oxalate, palmitate, phenylacetate, phenylsulfonate, propionate, pyruvate, salicylate, stearate, succinate, sulfanilate, tartarate, toluenesulfonate, and valerate. Examples of suitable polymeric organic anions include those derived from tannic acid and carboxymethylcellulose.

[0112] In some embodiments, the degradants disclosed herein may be provided as salts in deprotonated form with an appropriate counter cation.

[0113] Suitable counterions include both inorganic and organic cations. Examples of suitable inorganic cations include Na + and K + Alkali metal ions such as Ca 2+ and Mg 2+ Alkaline earth cations such as Al 3+ Examples of suitable organic cations include the ammonium ion (i.e., NH 4 + ) and substituted ammonium ions (e.g., NH 3 R + , N.H. 2 R 2 + , N.H.R. 3 + , N.R. 4 + ). Examples of substituted ammonium ions include those derived from ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. Additional or alternative examples of substituted ammonium ions include those derived from putrescine and spermidine, or polyvalent amines such as tetramethylethylenediamine (TEMED). An example of a common quaternary ammonium ion is N(CH 3 ) 4 + It is.

[0114] The decomposition agent of formula (I) may be provided in the form of a solvate (a complex of a solute (e.g., a compound, a salt of a compound) and a solvent). Examples of solvates include hydrates, such as monohydrates, dihydrates, and trihydrates.

[0115] The decomposition agent of formula (I) may be provided in a desolvated form, such as a dehydrated form.

[0116] Methods for cleaving at a target nucleic acid The present invention provides a method for cleaving a target nucleic acid molecule, the method comprising: The target nucleic acid molecule is treated with a degrading agent of formula (I): CLB(I) (Wherein, -C represents 1 to 3 C 1-6 -L- is a linker; and -B is a non-covalent linking group for non-covalently linking the degrading agent to a target nucleic acid molecule. and Allowing the degradative agent to cleave the target nucleic acid molecule bound thereto. Includes.

[0117] Preferred embodiments of the decomposition agent of formula (I) are given above.

[0118] In some embodiments, the target nucleic acid molecule may be contacted with the degradative agent in solution.

[0119] More preferably, the target nucleic acid molecule may be contacted with the degradative agent inside a cell (i.e., intracellularly). The cell may be in vitro and may be an isolated cell, for example, an isolated cell or cell line isolated from an individual (from a tissue sample such as a biopsy).

[0120] Suitable cells may include mammalian cells, preferably human cells. The cells may include somatic and germline cells, and may be at any stage of development, including fully or partially differentiated or undifferentiated or pluripotent cells, including stem cells, such as adult or somatic stem cells, fetal or embryonic stem cells. For example, the cells may include neural cells, including neurons and glial cells, contractile muscle cells, smooth muscle cells, hepatocytes, hormone synthesizing cells, sebaceous gland cells, pancreatic islet cells, adrenal cortex cells, fibroblasts, keratinocytes, endothelial and urothelial cells, bone cells, and chondrocytes. In some embodiments, the cells may be associated with a disease state, for example, cancer cells, such as carcinoma cells, sarcoma cells, lymphoma cells, blastoma cells, or germline tumor cells, and cells with the genotype of a genetic disorder, such as Huntington's disease, cystic fibrosis, sickle cell disease, phenylketonuria, Down's syndrome, or Marfan syndrome.

[0121] The target nucleic acid molecule may be an endogenous nucleic acid present in a cell. The degradative agent may be an exogenous molecule. The method may include introducing the degradative agent into the cell and allowing the degradative agent to bind to the target nucleic acid molecule.

[0122] The target nucleic acid molecule may be a DNA molecule or an RNA molecule. Suitable target RNA molecules may include mRNA and long non-coding RNA (lncRNA). RNA molecules may include intronic and intergenic regions.

[0123] The target nucleic acid molecule may comprise a secondary or tertiary structure. Suitable secondary and tertiary structures include a quadruplex, a pseudoknot, a tetraloop, a step loop, and a hairpin loop. Preferably, the target nucleic acid molecule comprises a quadruplex or a pseudoknot.

[0124] For example, a method for cleaving a target nucleic acid containing a secondary or tertiary structure includes the steps of: The target nucleic acid molecule is treated with a degrading agent of formula (I): CLB(I) (Wherein, -C represents 1 to 3 C1-6 -L- is a linker; and -B is a non-covalent linking group that interacts with a secondary or tertiary structure to non-covalently link the degradative agent to the target nucleic acid molecule. contacting the Allowing the degradative agent to cleave the target nucleic acid molecule bound thereto. may include.

[0125] In a preferred embodiment, the secondary or tertiary structure is a quadruplex. In such a case, the method comprises: The target nucleic acid molecule comprising the quadruplex is subjected to a degradation agent of formula (I): CLB(I) (Wherein, -C represents 1 to 3 C 1-6 -L- is a linker; and -B is a non-covalent linking group that interacts with the quadruplex to non-covalently link the degrading agent to the target nucleic acid molecule. and Allowing the degradative agent to cleave the target nucleic acid molecule bound thereto. may include.

[0126] In a preferred embodiment, the secondary or tertiary structure is a pseudoknot. In such a case, the method comprises: The target nucleic acid molecule containing a pseudoknot is treated with a degradation agent of formula (I): CLB(I) (Wherein, -C represents 1 to 3 C 1-6 -L- is a linker; and -B is a non-covalent linking group that interacts with the pseudoknot to non-covalently link the degradative agent to the target nucleic acid molecule. and Allowing the degradative agent to cleave the target nucleic acid molecule bound thereto. may include.

[0127] Upon non-covalent binding to a target nucleic acid molecule, the degradative agent cleaves the target nucleic acid. Typically, the degradative agent cleaves one or more phosphodiester bonds in the target nucleic acid.

[0128] When non-covalently bound to a target nucleic acid molecule, the cleaving group can remove the protein from the 2'OH of a nucleotide in the target nucleic acid. Cleavage of the phosphodiester backbone can occur by intramolecular attack on the 3' phosphate group.

[0129] When non-covalently bound to the target nucleic acid molecule, the cleaving group may bind to one or more transition metals (e.g., copper). The degradative agent is capable of cleaving the target nucleic acid through copper-mediated nucleolysis.

[0130] Binding of the degradative agent to the target nucleic acid may proceed via an intermediate species, i.e., the target nucleic acid molecule is reacted with the degradative agent to form a compound of the formula: CLB~NA (Wherein, -C is 1 to 3 of C 1-6 is a cleavage group which is imidazole optionally substituted with an alkyl group; -L- is a linker; -B is a non-covalent linking group; ~ is a non-covalent interaction; and NA is a target nucleic acid. and forming an intermediate having the formula: Allowing the degradative agent to cleave the target nucleic acid molecule. The cleavage may be performed as described herein by a method comprising:

[0131] Preferred embodiments of the decomposition agent of formula (I) are given above.

[0132] Methods for identifying secondary or tertiary structure After selective cleavage of the target nucleic acid molecule by the degradative agent, the method may include a step of identifying the target nucleic acid molecule, which may be useful, for example, in mapping sites containing secondary or tertiary structure within the nucleic acid.

[0133] The method may also include determining the abundance or amount of one or more nucleic acid molecules in the nucleic acid population. A decrease in the abundance or amount of the nucleic acid molecule in the population compared to a control indicates that the nucleic acid molecule is a target nucleic acid molecule that has been selectively cleaved by a degradative agent. A suitable control may be a population of nucleic acids that has not been treated with a degradative agent.

[0134] Accordingly, the present invention provides a method for identifying secondary or tertiary structure within a target nucleic acid molecule, comprising the steps of: providing a first and a second population of nucleic acid molecules, each of said populations comprising said target nucleic acid molecule; A first population of nucleic acid molecules is provided with a degradation agent of formula (I): CLB(I) (Wherein, -C represents 1 to 3 C 1-6 -L- is a linker; and -B is a non-covalent linking group for non-covalently linking the degrading agent to a target nucleic acid molecule. Introducing allowing a degradative agent to cleave the target nucleic acid molecules present in the first population; and Identifying nucleic acid molecules that are present in less abundance in the first population than in the second population. The present invention provides a method comprising:

[0135] Preferred embodiments of the decomposition agent of formula (I) are given above.

[0136] The non-covalent binding group may bind to a secondary or tertiary structure in the target nucleic acid molecule. Suitable secondary or tertiary structures include quadruplexes, pseudoknots, tetraloops, step loops, and hairpin loops. Preferably, the secondary or tertiary structure is a quadruplex or a pseudoknot.

[0137] The first and second populations of nucleic acid molecules can independently be isolated (ex vivo) populations of nucleic acid molecules, or one or more of the populations of nucleic acid molecules can be present within a cell.

[0138] The method may include extracting total nucleic acid, such as total DNA or total RNA, from a cell. The nucleic acid may be further analyzed, for example, to determine the abundance or amount of one or more nucleic acid molecules. For example, the extracted total nucleic acid may be sequenced and the sequence reads may be analyzed.

[0139] Suitable methods for determining the abundance or amount of nucleic acid molecules in a cell are well known in the art and include RT-qPCR, RNA-sequencing, next generation sequencing (NGS), nanopore sequencing, and other sequencing techniques such as Sanger sequencing, Tracking Indels by DEcomposition (TIDE) (Brinkman et al Nucleic Acids Res. 2014 Dec 16; 42(22): e168), and PCR analysis. In some embodiments, the method may include extracting nucleic acid molecules from a cell, sequencing the extracted nucleic acid molecules, and determining the number of sequence reads (i.e., read count) for each extracted nucleic acid molecule to determine the abundance or amount of each nucleic acid molecule in the cell. In some embodiments, raw read counts may be normalized and expressed as RPKM (reads per kilobase of exon model per million reads) or FPKM (fragments per kilobase of exon model per million reads mapped). Suitable methods of sequencing and sequence analysis are well established in the art.

[0140] Use in medicine Selective cleavage of a target nucleic acid molecule by such a degradative agent can alter downstream effects of the target nucleic acid molecule, which can be useful, for example, in the treatment or prevention of a disease mediated by the target nucleic acid molecule.

[0141] Thus, the present invention provides a degrading agent of formula (I) for use in a method of treatment of the human or animal body by therapy, for example for use in a method of treatment of a disorder (eg disease).

[0142] Another aspect of the invention relates to a method of treatment, e.g., a method of treating a disorder (e.g., a disease), comprising the step of administering a therapeutically effective amount of a degrading agent of formula (I) to a subject in need of treatment.

[0143] Another aspect of the invention relates to the use of a degradant of formula (I) in the manufacture of a medicament for use in the treatment of a disorder (e.g., a disease). Typically, the medicament comprises a degradant of formula (I).

[0144] Disorders to be treated In one embodiment (eg, of use in methods of treatment, of use in the manufacture of medicaments, of methods of treatment), the treatment is treatment of a bacterial infection or a viral infection.

[0145] Preferably, the viral infection is an infection with an RNA virus (e.g., a virus whose viral genome has single-stranded or double-stranded RNA). Many pathogenic viruses utilize -1 ribosomal frameshifting as a mechanism for accurate translation of proteins, and this phenomenon is made possible by secondary RNA structures such as stem-loops and pseudoknots. Therefore, by targeting these secondary RNA structures with the degrading agent of formula (I), viral RNA can be cleaved and inactivated, and viral infection can be treated.

[0146] Examples of RNA viruses include (+)ssRNA viruses, such as coronaviruses, picornaviruses, and togaviruses; (-)ssRNA viruses, such as orthomyxoviruses and rhabdoviruses; and dsRNA viruses, such as reoviruses.

[0147] Preferably, the virus is a (+)ssRNA virus, more preferably a coronavirus. Examples of coronaviruses include alphacoronaviruses such as transmissible gastroenteritis virus, feline coronavirus, and canine coronavirus; betacoronaviruses such as Middle East Respiratory Syndrome-related coronavirus (MERS-CoV), murine coronavirus (M-CoV), and severe acute respiratory syndrome-related coronavirus (SARS-CoV-2); gammacoronaviruses such as avian coronavirus; and deltacoronaviruses such as brown-eared bulbul coronavirus HKU11 and porcine coronavirus HKU15.

[0148] The bacterial infection can be an infection with Gram-negative bacteria or Gram-positive bacteria. Both classes of bacteria have bacterial ribosomes, which are ribozymes that contain both protein and RNA units. Therefore, targeting the RNA unit with the degrading agent of formula (I) can cleave and inactivate bacterial ribosomes, and can treat bacterial infection.

[0149] Examples of medically relevant Gram-negative bacteria include Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, and Pseudomonas aeruginosa (which are primarily associated with respiratory problems); Escherichia coli and Enterobacter cloacae (which are primarily associated with urinary problems); and Helicobacter pylori and Salmonella enterica (which are primarily associated with gastrointestinal problems); Neisseria meningitidis (which is primarily associated with meningitis).

[0150] Thus, in one embodiment, the gram-negative bacterial species is selected from the group consisting of Escherichia coli, Enterobacter cloacae, Helicobacter pylori, Salmonella enterica, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, and Neisseria meningitidis.

[0151] Examples of medically relevant gram-positive bacteria include actinomyces, bacillus, clostridium, corynebacterium (e.g., Corynebacterium diphtheriae), enterococcus, erysipelothrix, listerial (e.g., Listeria monocytogenes), nocardia, staphylococcal, and streptococcal (e.g., Staphylococcus aureus).

[0152] Thus, in one embodiment, the gram-negative bacterial genus is selected from the group consisting of Actinomyces, Bacillus, Clostridium, Corynebacterium, Enterococcus, Erysipelothricus, Listeria, Nocardia, Staphylococcus, and Streptococcus.

[0153] In one embodiment (eg, of use in methods of treatment, of use in the manufacture of medicaments, of methods of treatment), the treatment is treatment of a respiratory tract infection, a urinary tract infection, or gastroenteritis.

[0154] In some additional or alternative embodiments (e.g., of use in methods of treatment, of use in the manufacture of medicaments, of methods of treatment), the treatment is treatment of cancer. The cancer may be one for which the associated cancer gene is known, or for which the associated cancer gene is predicted to form or is predicted to be suitable for forming a secondary or tertiary structure, or for which the associated cancer gene expresses a nucleic acid having a secondary or tertiary structure, such as a quadruplex or pseudoknot structure as described herein.

[0155] The disease to be treated, such as cancer, may be associated with altered expression or regulation (e.g., upregulation) of neuroblastoma RAS (NRAS), metastasis associated lung adenocarcinoma transcript 1 (MALAT1), EWS RNA Binding Protein 1 (EWSR1), telomeric repeat containing RNA (TERRA), B-cell lymphoma-extra large (BCL-XL), fibroblast growth factor receptor (FGFR), and MicroRNA 21 (MIR21), among others.

[0156] Patients to be treated In one embodiment (eg, of use in methods of treatment, of use in the manufacture of medicaments, of methods of treatment), the treatment is administered to a subject in need of treatment.

[0157] The subject (patient) in need of treatment may be a chordate, vertebrate, mammal, placental mammal, marsupial (e.g., kangaroo, wombat), rodent (e.g., guinea pig, hamster, rat, mouse), murine (e.g., mouse), lagomorph (e.g., rabbit), avian (e.g., bird), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), porcine (e.g., pig), ovine (e.g., sheep), bovine (e.g., cow), primate, ape (e.g., monkey or ape), monkey (e.g., marmoset, baboon), ape (e.g., gorilla, chimpanzee, orangutan, gibbon), or human.

[0158] The subject in need of treatment may be an adult or a juvenile.

[0159] Preferably, the subject in need of treatment is a human, more preferably an adult human.

[0160] Alternatively, the subject in need of treatment is a non-human animal used in experimental studies. Preferably, the non-human animal is a rodent (e.g., guinea pig, hamster, rat, mouse).

[0161] Route of administration In one embodiment (e.g., of use in methods of treatment, of use in the manufacture of medicaments, of methods of treatment), the treatment is administered systemically / peripherally or locally (i.e., at the site of the desired effect) by any convenient route of administration.

[0162] Routes of administration may be oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by patches, salves, etc.); transmucosal (including, e.g., by patches, salves, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eye drops); pulmonary (e.g., via aerosol, e.g., through the mouth or nose, e.g., by inhalation or insufflation therapy); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal injection; parenteral, for example, by implantation of a depot or reservoir subcutaneously or intramuscularly.

[0163] formulation In one embodiment (e.g., of use in a method of treatment, of use in the manufacture of a medicament, of a method of treatment), the degrading agent of formula (I) is administered alone. Typically, however, in a pharmaceutical formulation (e.g., composition, preparation, medicament) comprising at least one degrading agent described herein, the degrading agent is preferably present together with one or more other pharma- ceutically acceptable components well known to those skilled in the art, including, but not limited to, pharma-ceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents, and sweeteners. The formulation may further comprise other active agents, such as other therapeutic or prophylactic agents.

[0164] Thus, the present invention further provides a pharmaceutical composition and a method of making the pharmaceutical composition comprising the step of mixing at least one disintegrant described herein with one or more other pharma- ceutically acceptable ingredients well known to those skilled in the art, such as carriers, diluents, excipients, etc. When formulated as discrete units (e.g., tablets), each unit contains a predetermined amount (dose) of the compound.

[0165] The term "pharmacologically acceptable" as used herein refers to compounds, ingredients, materials, compositions, dosage forms, and the like, that are suitable for use in contact with the tissues of the subject (e.g., human) in question without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0166] Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts, such as Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990, and Handbook of Pharmaceutical Excipients, 5th edition, 25 2005.

[0167] The formulation can be prepared by any method known in the field of pharmacy. Such a method includes the step of combining the disintegrant with the carrier which constitutes one or more accessory ingredients. In general, the formulation can be prepared by uniformly mixing the disintegrant with the carrier (e.g., liquid carrier, finely divided solid carrier, etc.), and then shaping the product as necessary.

[0168] The formulations may be prepared to provide fast or slow release; immediate, delayed, extended, or sustained release; or combinations thereof.

[0169] The formulations may suitably be in the form of a liquid, a solution (e.g., aqueous, non-aqueous), a suspension (e.g., aqueous, non-aqueous), an emulsion (e.g., oil-in-water, water-in-oil), an elixir, syrup, lozenge, mouthwash, drops, a tablet (including, for example, coated tablets), granules, powders, lozenges, pastilles, capsules (including, for example, hard and soft gelatin capsules), cachets, pills, ampoules, boluses, suppositories, pessaries, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mist, or aerosols.

[0170] The formulations may suitably be provided as a patch, adhesive plaster, bandage, dressing, or the like impregnated with one or more compounds and, optionally, one or more other pharma- ceutically acceptable ingredients including, for example, penetration enhancers, permeation enhancers, and absorption enhancers. The formulations may also suitably be provided in the form of a depot or reservoir.

[0171] The degradant may be dissolved, suspended or mixed with one or more other pharma- ceutically acceptable components. The compound may be present within a liposome or other microparticle which is designed to target the compound, for example, to blood components or one or more organs.

[0172] Dosage In one embodiment (e.g., of use in a method of treatment, of use in the manufacture of a medicament, of a method of treatment), the treatment comprises administering a therapeutically effective amount of a degrading agent of formula (I) to a subject in need of treatment.

[0173] Those skilled in the art will understand that the appropriate dosage of the degrading agent and composition comprising the degrading agent described herein may vary from patient to patient. Determining the optimal dosage generally involves balancing the level of therapeutic benefit against any risk or adverse side effects. The selected dosage level will depend on a variety of factors, including, but not limited to, the activity of the particular degrading agent, the route of administration, the time of administration, the rate of excretion of the degrading agent, the duration of treatment, other drugs, compounds, and / or materials used in combination, the severity of the disorder, and the patient's species, sex, age, weight, condition, overall health, and previous medical history. The amount and route of administration of the degrading agent are ultimately at the discretion of the physician, veterinarian, or clinician, but generally, the dosage is selected to achieve a local concentration at the site of action that achieves the desired effect without causing significant adverse or toxic side effects.

[0174] Administration can be in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosages of administration are well known to those of skill in the art and will vary with the formulation used for treatment, the purpose of the treatment, the target cell being treated, and the subject of the treatment. Single or multiple administrations can be made, with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.

[0175] Typically, suitable dosages of the degradant range from about 10 μg to about 250 mg (more typically about 100 μg to about 25 mg) per kilogram of subject body weight per day. Where the compound is a salt, ester, amide, prodrug, or the like, the amount administered is calculated based on the parent compound and therefore the actual weight used will be increased proportionately.

[0176] Other Preferences Each and every compatible combination of the above embodiments is expressly disclosed herein as if each and every combination was individually and explicitly set forth.

[0177] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in light of the present disclosure.

[0178] "And / or," as used herein, refers to the specific disclosure of each of two specified features or components, with or without the other. For example, "A and / or B" refers to the specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually set forth herein.

[0179] Unless the context indicates otherwise, the feature descriptions and definitions set forth above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0180] [Example] Certain aspects and embodiments of the invention will now be described, by way of example, and with reference to the above-mentioned drawings.

[0181] General Experimental Protocol In vitro pseudoknot oligolysis reaction RNA oligos (20 μM) were added to HEPES (20 mM) buffer at pH 7.5 supplemented with KCl (50 mM) and EDTA (10 mM). The mixture was incubated at 37° C. for 30 min. MTDB-deg 16a, MTDB, or TDB-deg 16b (1 mM) was then added. The reaction mixture was incubated at 37° C. for 3 h and then kept at 4° C. The reaction mixture was analyzed by LC-MS or gel electrophoresis.

[0182] LC-MS analysis of oligonucleotides. Oligonucleotides were analyzed by LC-MS according to the method of Mikutis et al., 2020.

[0183] Oligomers were analyzed using a Xevo G2-S TOF mass spectrometer coupled to an Acquity UPLC system using an Acquity UPLC BEH C18 1.7 μm column. The system utilizes electrospray ionisation (ESI). Two mobile phases were used: 16.3 mM TEA, 400 mM HFIP in H2O and 16.3 mM TEA, 400 mM HFIP in 80:20 v / v MeCN and H2O, at a flow rate of 0.200 mL / min. Calibration curves of RNA species were based either on A260 or on the intensity of specific negative m / z signals. Intensities of incorporated peaks were calculated using a native module of the KNIME software platform (33). Total mass spectra were reconstructed from ion series using the MaxEnt algorithm pre-installed in the MassLynx software (Waters version 4.1) according to the manufacturer's instructions. To obtain the negative ion series described, oligomeric peaks in the chromatograms were selected for incorporation and further analysis.

[0184] RNA Degradation Gel Electrophoresis Gel electrophoresis was performed according to the method of Mikutis et al., 2020.

[0185] In vitro RNA degradation reactions were performed as described above. The quenched reaction mixture was mixed with loading buffer (95% formamide, 0.025% SDS, 0.025% bromophenol blue (BPB), 0.025% xylene cyanol FF, 0.025% ethidium bromide, 0.5 mM EDTA) in a 1:1 ratio, heated to 70°C for 5 min, and cooled to 0°C. PAGE was performed on Novex™ TBEUrea Gels containing 15% polyacrylamide in 1×TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA) at 180V for 60 min. Gel staining was performed using SYBR Green II RNA Gel Stain (Invitrogen) in 1×TBE buffer. Stained RNA was visualized using a ChemiDoc MP (Bio-Rad, UK).

[0186] Virus stock The SARS-CoV-2 stock used to infect Vero CCL-81 cells was approximately 1.7 × 10 6 The titer, in PFU / mL, was established from passage 4 of SARS-CoV-2 isolated from a Portuguese patient (internal reference: 606_IMM ID_5452). The titer of the stock was calculated by plaque assay. Briefly, it was approximately 8 × 10 5CCL-81 cells / well were seeded in 6-well plates and grown for 24 h until confluence. The medium was removed and 500 μL of 10-fold serial dilutions of virus-containing supernatants were adsorbed in duplicate for 1 h at 37°C. The plates were rocked by hand every 15 min to redistribute the inoculum. The cells were overlaid with 1.25% carboxymethylcellulose (CMC) in supplemented DMEM and incubated at 37°C for 4 days. After incubation, the CMC overlay was removed and the cells were fixed with 4% formaldehyde / PBS and stained with 0.1% toluidine blue. After inactivation by fixation, the plates were sealed with parafilm, disinfected, and then removed from the BSC and BSL3. Viral plaques were counted to determine the infectious titer (PFU (plaque forming units) / mL).

[0187] Vero CCL-81 virus infection Vero CCL-81 cells at 80% confluency were incubated with the SARS-CoV-2 inoculum for 1 h at 37° C. After incubation, the inoculum was removed and DMEM medium supplemented with 2.5% FCS was added for 24 h or until samples were taken.

[0188] Gel electrophoresis analysis 500 ng of SARS-CoV-2 RNA was incubated with or without 100 μM MTDB-deg 16a in 1× HEPES buffer with gentle agitation for 2 hours at 37° C. Samples were then analyzed on a 1.5% agarose gel.

[0189] Nanopore Sequencing 500ng of SARS-CoV-2 RNA was incubated with or without 100μM MTDB-deg 16a in 1x HEPES buffer at 37°C for 2 hours with gentle agitation. Samples were then prepared for sequencing according to the manufacturer's protocol for Direct RNA Sequencing (SQK-RNA002, ONT). Prepared libraries were loaded onto a FLO-MIN 106D flow cell (ONT) and sequenced on a MinION Mk1C device (ONT).

[0190] The genome sequence (GenBank: MN908947.3) and genome annotation (NC_45512.2) of the Wuhan-hu1 strain of SARS-CoV-2 were downloaded from the NCBI database. Sequence reads were aligned against the Wuhan-hu1 genome using minimap2 with parameters "-ax splice-N32-un-k13" (Li et al., 2018). CIGAR strings of the alignments were processed by a customized script. Reads were flagged as leaders if a splice junction within the read started between the first 60–120 bp of the genome. Reads were assigned to individual transcripts if they covered >90% of the annotated transcript or >90% of the read sequence was present within the transcript.

[0191] Drug assay to determine 50% inhibitory concentration Increasing concentrations of MTDB-deg 16a (ranging from 0.07 to 25 μM) were tested to determine the 50% inhibitory concentration (IC 50 ) was determined. 2O) Controls and control molecules were included in parallel. Cells were seeded in 96-well plates at approximately 40% confluency and infected 24 hours later. MTDB-deg 16a, MTDB, or TDB-deg 16b were added 1 hour before or 1 hour after infection. Frozen stocks of SARS-CoV-2 were thawed at room temperature and used to infect cells at a 0.05 multiplicity of infection (MOI). Cells were harvested 24 hours after infection to measure the inhibition of viral growth. Viral growth was assessed by measuring the viral load by PCR targeting the E gene and the pseudoknot region.

[0192] Detection of viral plaque-forming units by plaque assay Approximately 8 x 10 5 CCL-81 cells / well were seeded in 6-well plates and grown for 24 h until 80% confluence. Supernatants of compound-treated cultures were diluted in DMEM medium supplemented with 2.5% FCS, added to the wells of pre-seeded 6-well plates, and incubated at 37°C for 1 h. Plates were rocked by hand every 15 min to redistribute the inoculum. Cells were overlaid with 1.25% CMC in supplemented DMEM and incubated at 37°C for 4 days. After incubation, the CMC overlay was removed and cells were fixed with 4% formaldehyde / PBS and stained with 0.1% toluidine blue. After inactivation by fixation, plates were sealed with parafilm, disinfected, and then removed from the BSC and BSL3. Viral plaques were counted to determine infectious titers (PFU (plaque forming units) / mL).

[0193] Quantification of viral load by PCR The cell pellet was taken up in 300 μL of lysis buffer. Viral RNA was extracted using the NZY Viral RNA Isolation Kit (NZYtech) and cDNA was synthesized using the NZY First-Strand cDNA Synthesis Kit (NZYtech) according to the manufacturer's instructions. Quantitative RT-PCR (RT-qPCR) was then performed by using PowerUp SYBR Green Master Mix (BIO-RAD) set up by an Applied Biosystems RT-PCR 7500Fast instrument with the default SYBR green program.

[0194] The primers used for detection of SARS-CoV-2 were as follows: E gene: 5'-ACAGGTACGTTAATAGTTAATAGCGT-3' (forward), 5'-ATATTGCAGCAGTACGCACACA-3' (reverse); N gene: 5'-GACCCCAAAATCAGCGAAAT-3' (forward), 5'-TCTGGTTACTGCCAGTTGAATCTG-3' (reverse); Pseudoknot: 5'-CCGCGAACCCATGCTTCAGTCA-3' (forward), 5'-CACGGTGTAAGACGGGCTGCAC-3'(reverse); 18S: 5'-GTAACCCGTTGAACCCCATT-3' (forward), 5'-CCATCCAATCGGTAGTAGCG-3' (reverse).

[0195] Viral survival assay Two sets of samples were prepared for survival assays, in which cells at 80% confluency were infected with SARS-CoV-2 cryopreserved stock at MOI of 0.05 for 2 h. The inoculum was then removed and the infected cells were incubated with 6 μM MTDB-deg 16a, MTDB, and TDB-deg 16b at 37 °C and 5% CO. 2 After 24 hours, cells in one set of samples were harvested in lysis buffer for PCR analysis of viral growth. In the other set of samples, compounds in the supernatant were removed and replaced with drug-free medium and incubated at 37°C and 5% CO. 2 The cells were incubated for an additional 24 hours at RT. After 24 hours of incubation (corresponding to the 48 hour time point), the cells were harvested in lysis buffer and viral growth was measured by PCR targeting the E gene and the pseudoknot region. The percentage of viral survival was normalized to the vehicle control.

[0196] Cytotoxicity assay To determine whether the compound was toxic to the cells, 1 x 10 cells were added per well. 4 Vero E6 cells were seeded in 96-well plates. After 24 h, cells were incubated with increasing concentrations of MTDB-deg 16a, MTDB, or TDB-deg 16b (ranging from 0.05 μM to 25 μM). Cell viability after 24 h of incubation with compounds was assessed by CellTiter Blue viability assay (Promega) according to the manufacturer's protocol. Briefly, Cell titer blue stock solution was diluted 1:20. A volume of 80 μL of diluted Cell titer blue was added to each well and incubated for 2 h at 37 °C.

[0197] Dynamic Light Scattering (DLS) Stock solutions (10 mM) of each screening molecule were prepared in neat DMSO and serially diluted in water to final concentrations of 25 or 12.5 μM. Data were collected on a Zetasizer Nano S (Malvern) at 25° C.

[0198] Antiviral activity in animal models of SARS-CoV-2 infection Specific pathogen-free mice, 10-12 weeks of age, hemizygous for Tg(K18-ACE2)2Prlmn (strain B6.Cg-Tg(K18-ACE2)2Prlmn / J, Jackson laboratory strain 034860) were used in this study. Mice were cultured at 1 × 10 4 Mice were infected intranasally with PFU of SARS-CoV-2. Compounds were administered intranasally 1 hour before and 3 hours after infection. Mice were treated with vehicle (n=6), 25 mg / kg MTDB degrader 16a (n=6), 10 mg / kg MTDB (n=3), or 25 mg / kg TDB degrader 16b (n=5). Five days after SARS-CoV-2 infection, animals were humanely euthanized and the left lung was harvested for virus quantification by plaque assay and the right lung was harvested for histopathological analysis.

[0199] Western blot analysis. For in vitro experiments, samples were incubated in a medium (H 2O) or MTDB-deg 16a (6 mM) for 24 h. Cells were then lysed using whole cell lysis buffer (50 mM Tris-HCl, pH=8.0, 450 mM NaCl, 0.1% NP-40, 1 mM EDTA) supplemented with 1 mM DTT, protease inhibitors (Sigma), and phosphatase inhibitors (Sigma). For in vivo experiments, whole left lungs of mice were homogenized in 3 mL DMEM and 750 μL was transferred to an equal volume of whole cell lysis buffer supplemented as above. Protein concentration was determined using a Bradford assay (BioRad). Before loading, samples were supplemented with LDS loading buffer (Life technologies) and Sample Reducing Agent (Life technologies). 40 μg of protein was separated on SDS-PAGE gels and blotted onto polyvinylidene difluoride (PVDF) membranes (GE Healthcare). Western blot experiments were performed using the following antibodies: anti-beta actin (Abcam, ab8224), anti-phospho-MAPKAPK-2 (Thr334) (27B7) (Cell Signalling, 3007), anti-phospho-p38 MAPK (Thr180 / Tyr182) (D3F9) XP® (Cell Signalling, 4511), goat anti-mouse IgG H&L (HRP) (Abcam, ab205719), and goat anti-rabbit HRP (Abcam, ab6721).

[0200] Synthesis of azido-imidazole [ka] Scheme 1: Synthesis of azido-imidazoles

[0201] Synthesis of hexaethylene glycol di(p-toluenesulfonate) (1) Hexaethylene glycol (1.0 mmol) was dissolved in DCM (10 mL) and p-toluenesulfonyl chloride (2.2 mmol), and KOH (10 mmol) was added at 0° C. The reaction mixture was stirred at room temperature for 6 h, then filtered and washed with water. MgSO 4 After drying on the stove, the solvent was evaporated under reduced pressure. No further purification was necessary. Yield: 95% (colorless oil).

[0202] 1 H NMR (400 MHz, CDCl 3 ):δ H 7.78(d,4H), 7.33(d,4H), 4.14(t,4H), 3.67(br tr,4H,3.60(br s,8H), 3.57(br s,8H), 2.43(br s,6H).MS:C 26 H 39 O 11 S 2 m / z:591.19.

[0203] The physical and spectroscopic data were consistent with those described in the literature (Mikutis et al., 2020).

[0204] Synthesis of hexaethylene glycol p-toluenesulfonate azide (2a) Hexaethylene glycol di(p-toluenesulfonate) 1 (1.0 mmol) was dissolved in DMF (10 mL) and sodium azide (1.0 mmol) was added. The reaction mixture was stirred at 60 °C for 6 h, then cooled to room temperature and stirred overnight. The mixture was washed with brine and MgSO 4 The mixture was dried over ice. Toluene was added to remove DMF, and the solvent was evaporated under reduced pressure. The crude product was purified via column chromatography (EtOAc:Hexane, 1:1). Yield: 54% (colorless oil).

[0205] 1 H NMR (400 MHz, CDCl 3 ) δ H7.82(d,2H), 7.36(d,2H), 4.18(t,2H), 3.59~3.73(20H,PEG), 3.41(t,2H), 2.47(s,3H). MS:C 19 H 31 N 3 NaO 8 S m / z: 484.2.

[0206] The physical and spectroscopic data were consistent with those described in the literature (Mikutis et al., 2020).

[0207] Synthesis of tetraethylene glycol p-toluenesulfonate azide (2b) Tetraethylene glycol di(p-toluenesulfonate) (2.7 g, 5.4 mmol) was dissolved in anhydrous DMF (10 ml). Sodium azide (355 mg, 5.4 mmol) was added and the mixture was purified by N 2 Placed down and stirred at 55° C. for 18 hours. The solvent was removed in vacuo and the product was purified via flash column chromatography (3:1 Pet. Ether:AcOEt to 1:1 Pet. Ether:AcOEt). The product was obtained as a colorless oil (798 mg, 2.1 mmol, 39%).

[0208] 1 H NMR (400MHz, CDCl3) δ7.82(d,2H), 7.37(d,2H), 4.19(t,1H), 3.60~3.73(12H,PEG), 3.40(t,2H), 2.47(s,3H). MS:C 15 H 23 N 3 NaO 6 S m / z 396.1207.

[0209] Synthesis of diethylene glycol p-toluenesulfonate azide (2c) Diethylene glycol di(p-toluenesulfonate) (1.0 mmol) was dissolved in DMF (10 mL) and sodium azide (1.0 mmol) was added. The reaction mixture was stirred at 60° C. for 6 h, then cooled to room temperature and stirred overnight. The mixture was washed with brine and MgSO 4 The mixture was dried over ice. Toluene was added to remove DMF, and the solvent was evaporated under reduced pressure. The crude product was purified via column chromatography (EtOAc:Hexane, 1:1). Yield: 59% (colorless oil).

[0210] 1 H NMR (400 MHz, CDCl 3 ) δ H 7.80(d,2H), 7.35(d,2H), 4.17(t,2), 3.70(t,2H), 3.61(t,2H), 3.32(t,2H), 2.45(s,3H). MS:C 11 H 15 N 3 NaO 4 S m / z: 308.068.

[0211] The physical and spectroscopic data were consistent with those described in the literature (Mikutis et al., 2020).

[0212] Synthesis of hexaethylene glycol imidazolate azide (3a) Imidazole (1.0 mmol) was dissolved in dry DMF (15 mL) under inert conditions and sodium hydride (60% dispersion in mineral oil, 1.2 mmol) was added. After stirring at 0 °C for 30 min, 2a (1.0 mmol) was added. The reaction mixture was stirred at 60 °C overnight and after cooling to room temperature, the mixture was quenched with water (20 mL). Subsequent extraction with EtOAc and DCM, MgSO 4 Drying on a stover and evaporation of the solvent under reduced pressure gave the crude product, which was then purified via column chromatography (EtOAc:MeOH, 3:1). Yield: 35% (colorless oil).

[0213] 1H NMR (400 MHz, CDCl 3 ) δ H 7.52(s,1H), 7.02(s,1H), 6.98(s,1H), 4.09(t,2H), 3.72(t,2H), 3.55~3.78(18H,PEG), 3.36(t,2H). MS:C 15 H 28 N 5 O 5 m / z 358.21.

[0214] The physical and spectroscopic data were consistent with those described in the literature (Mikutis et al., 2020).

[0215] Synthesis of tetraethylene glycol imidazolate azide (3b) Imidazole (18 mg, 0.27 mmol) and NaH (60% dispersion in mineral oil, 12 mg, 0.27 mmol) were suspended in anhydrous DMF (1 ml) at 0° C. The mixture was cooled to 5° C. with 5% CO. 2 Place under atmosphere, warm to room temperature, and stir for 30 min. 2b (100 mg, 0.27 mmol) was dissolved in anhydrous DMF (1 ml) and the resulting solution was added to the first mixture. This was then stirred at 55° C. for 20 h. The solvent was then removed in vacuo and the resulting residue was purified via flash chromatography (dry loading, gradient EtOAC to 9:1 EtOAc:MeOH). The product was obtained as a colorless oil (54 mg, 0.20 mmol, 74%).

[0216] 1 H NMR (400 MHz, CDCl 3 )δ7.55(s,1H), 7.05(s,1H), 7.05(s,1H), 4.12(t,2H), 3.75(t,2H), 3.60~3.71(10H,PEG), 3.39(t,2H). 13 C NMR (100 MHz, CDCl 3 ) δ C 137.6, 129.2, 119.4, 70.5-70.7 (multiple PEG peaks), 70.0, 50.7, 47.1. MS:C 11 H 19 N5 O 3 m / z 270.1582.

[0217] Synthesis of diethylene glycol imidazolate azide (3c) Imidazole (1.0 mmol) was dissolved in dry DMF (15 mL) under inert conditions and sodium hydride (60% dispersion in mineral oil, 1.2 mmol) was added. After stirring at 0 °C for 30 min, 2c (1.0 mmol) was added. The reaction mixture was stirred at 60 °C overnight and then cooled to room temperature. The mixture was quenched with water (20 mL). Extraction with EtOAc and DCM, MgSO 4 After drying on a kettle and evaporating the solvent under reduced pressure, the crude product was obtained. The crude product was then purified via column chromatography (EtOAc:MeOH, 3:1). Yield: 38% (colorless oil).

[0218] 1 H NMR (400 MHz, CDCl 3 ) δ H 7.53(s,1H), 7.06(s,1H), 6.99(s,1H), 4.14(t,2H), 3.75(t,2H), 3.60(t,2H), 3.36(t,2H). MS:C 11 H 19 N 5 O 3 m / z 182.1

[0219] The physical and spectroscopic data were consistent with those described in the literature (Mikutis et al., 2020).

[0220] Synthesis of azido-ethylimidazole (4) [ka] Scheme 2: Synthesis of ethyl azido-imidazole 4

[0221] Hydroxyethylimidazole (1.0 mmol) was dissolved in DCM (10 mL) at 0° C., KOH (10 mmol) and p-toluenesulfonyl chloride (1.2 mmol) were added. The reaction mixture was stirred at room temperature for 6 h, then filtered and the solvent was removed under reduced pressure. The crude product was dissolved in DMF and sodium azide (1.0 mmol) was added. It was stirred at 60° C. for 6 h, then cooled to room temperature and stirred overnight. Toluene was added and the solvent was removed under reduced pressure. Purification was performed via column chromatography (EtOAC:MeOH, 3:1). Yield: 26% (white solid).

[0222] 1 H NMR (400 MHz, CDCl 3 )δ7.51(s,1H), 7.09(s,1H), 6.96(s,1H), 4.09(t,J=5.7Hz,1H), 3.62(t,J=5.7Hz,1H).

[0223] Synthesis of pyridostatin decomposition agents [ka] Scheme 3: Synthesis of pyridostatin derivatives.

[0224] Synthesis of chelidamic acid dimethyl ester (5) Chelidamic acid hydrate (2.0 g, 11 mmol) was suspended in 20 ml MeOH. Thionyl chloride (500 μL, 6.9 mmol) was added dropwise to the suspension at -10°C. A color change from white to brown was observed. The solution was warmed to RT and stirred overnight. The brown solution was refluxed for 2 h and the solvent was removed in vacuo. The brown crude product was then recrystallized from EtOH to give chelidamic acid dimethyl ester 5 (864 mg, 3.9 mmol, 36%) as a beige solid.

[0225] 1 H NMR (400MHz, DMSO) δ11.77(brs,1H), 7.61(s,2H), 3.88(s,6H). 13C NMR (101MHz, DMSO) δ165.97, 164.88, 149.37, 115.33, 52.68. HRMS(ES)C 9 H 10 NO 5 Calculated value of ([M+H] + ) m / z: 212.0559, measured value 212.0567.

[0226] Synthesis of propargylchelidamic acid (6) Chelidamic acid dimethyl ester 5 (0.82 g, 3.8 mmol), propargyl alcohol (0.33 mL, 5.7 mmol), and polymer-bound triphenylphosphine (3.47 g, 1.5 mmol loading / g, 5.2 mmol) were suspended in 55 mL of freshly distilled THF. The solution was degassed using freeze-pump-thaw cycling, cooled to 0° C., and DIAD (1.0 mL, 5.1 mmol) was added dropwise under argon. The solution was warmed to RT and stirred for 3 days. The solution was filtered and the solvent was removed in vacuum. Chelidamic acid dimethyl ester was obtained via column chromatography (50% EtOAc, 50% pet. ether). It was then dissolved in 50 mL of MeOH, followed by the addition of 50 mL of aqueous NaOH (0.33 g, 7.5 mmol) solution. The resulting mixture was stirred for 5 min and deprotection was confirmed by TLC. The organic solvent was removed in vacuo. 5% formic acid was added to make it acidic, and then it was extracted with 3×100 mL of EtOAC. The organic layer was then washed with MgSO 4 The mixture was dried at 40° C., filtered, and the solvent was removed in vacuo to give propargyl chelidamic acid 6 (0.17 g, 0.77 mmol, 20%) as an off-white solid.

[0227] 1 H NMR (400MHz, MeOD) δ7.93 (s, 2H), 5.02 (d, J = 2.4Hz, 2H), 3.15 (t, J = 2.5Hz, 1H). 13C NMR (100MHz, MeOD) δ168.12, 166.98, 150.54, 150.43, 115.73, 78.97, 77.90, 77.88, 57.74, 57.68. HRMS(ES)C 10 H 8 NO 5 Calculated value of ([M+H] + ) m / z: 222.0397, actual value 222.0391.

[0228] Synthesis of O-(ethyl-2-N-boc-amine)-2-aminoquinolinone (7) 2-Aminoquinolinone (1.0 g, 6.2 mmol), N-boc ethanolamine (1.4 mL, 9.1 mmol), and triphenylphosphine (3.3 g, 13 mmol) were dissolved in 10 mL of freshly distilled THF. The solution was degassed using freeze-pump-thaw cycles, cooled to 0° C., and DIAD (1.8 mL, 9.2 mmol) was added dropwise under argon. The solution was allowed to warm to RT and stirred for 3 days. The solvent was then removed in vacuo. The product was purified by gradient column chromatography from 100% EtOAc to 90% EtOAc, 10% MeOH. The solvent was removed in vacuo to give an off-white solid 7 (552 mg, 1.82 mmol, 29%).

[0229] 1 H NMR (400 MHz, CDCl 3 )δ7.98(dd,J=8.0,1.0Hz,2H), 7.60(dd,J=8.4,1.2Hz,2H), 7.55(ddd,J=8.5,6.7,1.6Hz,2H), 7.23(ddd,J=8.1,6.6,1.3Hz,2H), 6.04(s,1H), 5.01(br s,1H), 4.69(br s,2H), 4.18(t,J=5.1Hz,4H), 3.68(q,J=5.5Hz,4H), 1.46(s,9H). 13 C NMR (100 MHz, CDCl 3)δ162.32, 158.13, 155.90, 148.55, 130.25, 125.63, 121.97, 121.60, 117.52, 90.09, 79.83, 67.52, 39.82, 28.38. HRMS(ES)C 16 H 22 N 3 O 3 Calculated value of ([M+H] + ) m / z: 304.1661, measured value 304.1649.

[0230] Synthesis of alkyne-pyridostatin (8) Propargyl chelidamic acid 6 (0.12 g, 0.54 mol) was dissolved in 1.2 mL of DCM. Ghosez's reagent (170 μL, 1.3 mmol) was then added dropwise at 0° C. The orange solution was then stirred at RT for 2 h. Chlorination was confirmed by TLC. Triethylamine (0.18 mL, 1.3 mmol) was added dropwise at 0° C. The solution was then stirred at RT for 1 h. 7 (0.37 g, 1.2 mmol) was suspended in 1.2 mL of DCM and then added dropwise to the mixture. The mixture turned red-brown and was stirred overnight under argon. The crude protected product 8a (not shown) was precipitated as a red solid from hot MeCN. The red solid 8a was then dissolved in DCM. A 2:1 DCM:TFA mixture was added to acidify the solution and remove the N-boc protection. The solvent was removed in vacuo and the product was purified by HPLC (100% H 2 0, 0.1% FA to 100% MeCN, 0.1% FA) and lyophilization afforded alkyne-pyridostatin 8 (51 mg, 86 μmol, 16%) as an off-white solid.

[0231] HRMS(ES) C 32 H 3 0N 7 O 5 Calculated value of ([M+H] + ) m / z: 592.2308, actual value 592.2327.

[0232] Synthesis of pyridostatin decomposition agents (9A-9C) Alkyne-pyridostatin 8 (15 mg, 25 μmol) was dissolved in 2.5 mL of 2:1 H 2 The solution was dissolved in a 20:300:tBuOH mixture. A copper(II) sulfate pentahydrate solution (250 μL, 100 mM, 25 μmol) was added followed by sodium ascorbate solution (1.3 mL, 100 mM, 130 μmol). The cloudy yellow solution was degassed and stirred for 10 min. A solution (3.8 mL, 10 mM) of the appropriate azido-imidazole (3a, 3b, or 3-azidopropionic acid) was then added. The solution was stirred under argon for 2 h. The organic solvent was removed in vacuuo. The product was then purified via HPLC (100% H 2 0, 0.1% FA, gradient to 100% MeCN, 0.1% FA) The product was obtained as a white or off-white solid.

[0233] 9A (PDS-deg6). Yield 48% (11.3 mg, 12 μmol). HRMS (ES) calculated for C47H52N12O10 ([M+H]+) m / z: 949.4321, found 949.4344. 9B (PDS-deg4). Yield 69% (14.8 mg, 17 μmol). HRMS (ES) calculated for C43H49N12O8 ([M+H]+) m / z: 861.3796, found 861.376. 9C (PDS-CBX). Yield 28% (4.9 mg, 6.9 μmol). HRMS (ES) calculated for C35H34N10O7 ([M+H]+) m / z: 707.2690, found: 707.2684.

[0234] Synthesis of pseudoknot breakers [ka] Scheme 4: Synthesis of pseudoknot breakers

[0235] Synthesis of compound 11a 2-Methylthiazole-4-carbaldehyde (10.0 g, 78.6 mmol, 1.0 equiv.) in DCM (100 mL) was added to compound 10 (16.5 g, 82.6 mmol, 16.2 mL, 1.1 equiv.) at 25 °C and N 2 The mixture was stirred at 25° C. for 3 h. NaBH(OAc) was added to the mixture. 3 (25.0 g, 118 mmol, 1.5 equiv) was added and stirred for 10 h. The residue was poured into water (50 mL) and stirred for 10 min. The aqueous phase was extracted with DCM (3×20 mL). The combined organic phase was washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C. and filtered. The solvent was removed in vacuo. The product was purified via column chromatography (petroleum ether to 10 / 1 petroleum ether / ethyl acetate gradient) to give compound 11a (13.5 g, 43.4 mmol, 55% yield) as a yellow oil.

[0236] LCMS [+scan]: Calculated m / z C 20 H 26 N 3 O 3 S 388.2; measured value 388.1.

[0237] Synthesis of compound 12a TFA (40.0 g, 351 mmol, 26 mL, 8.4 equiv.) was added to compound 11a (13.0 g, 41.7 mmol, 1.0 equiv.) in DCM (130 mL) at 25 °C and N 2 The mixture was stirred for 12 h. The solvent was removed in vacuo to give the TFA salt of compound 12a (23.0 g, crude product) as a red oil.

[0238] LCMS [+scan]: Calculated m / z C 10 H 18 N 3 S 212.1; measured value 212.0.

[0239] Synthesis of MTDB (compound 13a) To a solution of compound 12a (20.0 g, 45.5 mmol, 1.0 equiv) in DCM (200 mL), TEA (9.21 g, 91.0 mmol, 12.7 mL, 2.0 equiv) was added at 20 °C and N 2 C. to 100.degree. C. under reduced pressure. Ethyl 2-isocyanatobenzoate (8.70 g, 45.5 mmol, 1.0 equiv.) was then added to the mixture at 0.degree. C. The mixture was stirred at 20.degree. C. for 12 h. The solvent was removed in vacuo. The residue was purified by column chromatography (petroleum ether / ethyl acetate gradient from 100 / 1 to 20 / 1) to give MTDB (compound 13a, 5.62 g, 14.0 mmol, 31% yield) as an off-white solid.

[0240] 1 H NMR (400MHz, CD 3 OD): δ8.42(br d,J=8.4Hz,1H), 8.08(br d,J=8.0Hz,1H), 7.62(s,1H), 7.52~7.59(m,1H), 7.09(br t,J=7.6Hz,1H), 4.34~4.46(m,4H), 3.93(br s,2H), 3.77(br t,J=6.0Hz,2H), 3.49(br s,4H), 3.28~3.31(m,1H), 2.74(s,3H), 2.31(br d,J=4.8Hz,2H), 1.43(t,J=7.2Hz,3H). LCMS[+scan]: Calculated value m / z C 20 H 27 N 4 O 3 S 403.2; measured value 403.1.

[0241] Synthesis of compound 14a MTDB (compound 13a 5.60 g, 13.9 mmol, 1.0 equiv.) in EtOH (120 mL) and H 2 LiOH monohydrate (2.34 g, 55.7 mmol, 4.0 equiv.) was added to a mixture of 200 mL of 2M HO at 25 °C and N 2 The mixture was stirred at 25° C. for 12 h. The mixture was adjusted to pH 6 with 1 M HCl and the aqueous phase was extracted with ethyl acetate (3×40 mL). The organic phase was then washed with anhydrous Na 2 SO4 Drying at 40° C., filtration and concentration in vacuo afforded compound 14a (2.60 g, 6.94 mmol, 50%) as a yellow solid.

[0242] LCMS [+scan]: Calculated m / z C 18 H 23 N 4 O 3 S 375.1; measured value 375.1.

[0243] Synthesis of compound 15a To a mixture of compound 14a (2.60 g, 6.94 mmol, 1.0 equiv) and propargylamine (1.15 g, 20.8 mmol, 1.33 mL, 3.0 equiv) in DMF (200 mL), DIEA (4.49 g, 34.7 mmol, 6.05 mL, 5.00 equiv) was added at 25 °C and N 2 The mixture was added under 3 P (4.42 g, 13.9 mmol, 4.13 mL, 2.0 equiv) was added and stirred at 50° C. for 12 h. The mixture was poured into water (200 mL) and the aqueous phase was extracted with ethyl acetate (3×70 mL). The organic phase was then washed with brine (60 mL) and the organic phase was washed with anhydrous Na 2 SO 4 It was dried at 40° C., filtered, and the solvent was removed in vacuo. The residue was purified by column chromatography (100 / 1 petroleum ether / ethyl acetate, gradient to ethyl acetate) to give 15a (1.20 g, 2.80 mmol, 40% yield).

[0244] 1 H NMR (400 MHz, CDCl 3):δ10.54(br s,1H), 8.46(d,J=8.2Hz,1H), 7.43~7.51(m,2H), 7.11~7.11(m,1H), 6.99(q,J=7.6Hz,2H), 6.49(br s,1H), 4.22(dd,J=5.2,2.6Hz,2H), 3.72~3.83(m,4H), 3.64~3.69(m,2H), 2.77~2.96(m,4H), 2.72(s,3H), 2.32(t,J=2.6Hz,1H), 2.04(br d,J=15.2Hz,2H). LCMS[+scan]: Calculated value m / z C 21 H 26 N 5 O 2 S 412.2; measured value 412.0.

[0245] Synthesis of MTDB-deg (compound 16a) A mixture of azido-imidazole 3a (200 mg, 280 μmol, 1.0 equiv.), compound 15a (115 mg, 280 μmol, 1.0 equiv.), and CuSO in DCM (5 mL) was added. 4 (22.3 mg, 140 μmol, 21.5 uL, 0.5 equiv.), MeOH (5 mL), and H 2 The mixture was stirred at 20° C. for 0.5 h. Then, NaAsc (11.1 mg, 55.9 μmol, 0.2 equiv.) was added to the mixture and stirred at 20° C. for 4.5 h. The mixture was diluted with H 2 The mixture was diluted with 2×O (10 mL) and then extracted with DCM (3×10 mL). The combined organic phase was washed with anhydrous Na 2 SO 4 The mixture was dried over 1000 ml, filtered and concentrated in vacuo. The residue was purified by HPLC (column: Phenomenex Gemini-NX, 80×40 mm×3 um, mobile phase: [water (10 mM NH 4 HCO 3 )-ACN]; B%: 15% to 35%, 8 min) to afford MTDB-deg 16a (28.0 mg, 34.9 μmol, 13% yield) as a pale yellow oil.

[0246] 1 H NMR (400MHz, DMSO-d 6):δ11.03(s,1H), 9.29(br t,J=5.2Hz,1H), 8.37(d,J=8.4Hz,1H), 7.96(s,1H), 7.74(br d,J=7.2Hz,1H), 7.63(br s,1H), 7.43(t,J=7.60Hz,1H), 7.15~7.31(m,2H), 6.99(t,J=7.6Hz,1H), 6.89(br s,1H), 4.46~4.53(m,4H), 4.10(br t,J=5.2Hz,2H), 3.77~3.81(m,2H), 3.71(br s,2H), 3.66(br t,J=5.07Hz,2H), 3.44~3.57(m,20H), 3.34(s,25H), 2.73(br s,1H), 2.62(br s,6H), 1.84(br s,2H). HRMS[+scan]: Calculated value m / z C 36 H 53 N 10 O 7 S 769.3819;Actual value 769.3830.

[0247] Synthesis of control decomposers for pseudoknot experiments [ka] Scheme 5: Synthesis of pseudoknot-controlling decomposer (TBD-deg)

[0248] Synthesis of compound 11b To thiophene-3-carbaldehyde (2.00 g, 17.8 mmol, 1.63 mL, 1.0 equiv.) in DCM (80 mL) was added compound 10 (3.93 g, 19.6 mmol, 3.85 mL, 1.1 equiv.) at 20 °C and N 2 The mixture was stirred at 20° C. for 3 h. Then, NaBH(OAc) 3 (5.67 g, 26.8 mmol, 1.5 equiv) was added to the mixture at 0° C. and stirred at 20° C. for 10 h. The reaction mixture was quenched by adding water (60 mL) and extracted with DCM (2×20 mL). The combined organic layers were washed with brine (30 mL) and anhydrous Na 2 SO 4The mixture was dried over 100 ml, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate=100 / 1 to 20 / 1 gradient) to give compound 11b (1.70 g, 5.73 mmol, 32% yield) as a red oil.

[0249] 1 H NMR (400 MHz, CDCl 3 ):δ7.21~7.31(m,1H), 7.03~7.15(m,2H), 3.64(s,2H), 3.37~3.54(m,4H), 2.55~2.68(m,4H), 1.81(br dd,J=10.8,4.9Hz,2H), 1.40~1.52(m,9H).

[0250] Synthesis of compound 12b To a solution of compound 11b (1.70 g, 5.73 mmol, 1.0 equiv) in DCM (20 mL), TFA (6.16 g, 54.0 mmol, 4.00 mL, 9.4 equiv) was added at 20 °C and N 2 The mixture was stirred at 20° C. for 8 hours. The solvent was removed in vacuo to give the TFA salt of compound 12b (3.00 g, crude) as a red oil, which was used in the next step without further purification.

[0251] Synthesis of TDB (compound 13b) To a mixture of compound 12b (3.00 g, 9.67 mmol, 1.0 equiv) in DCM (40 mL), TEA (1.96 g, 19.3 mmol, 2.69 mL, 2.0 equiv) was added at 20 °C and N 2 The mixture was stirred at 20° C. for 12 h. The solvent was removed in vacuo. The residue was purified by column chromatography (petroleum ether / ethyl acetate=100 / 1 to 20 / 1 gradient) to give TDB 13b (3.30 g, 8.35 mmol, 86% yield) as an off-white solid.

[0252] 1 H NMR (400 MHz, CDCl3 ): δ10.59(s,1H), 8.52(d,J=8.4Hz,1H), 7.94(dd,J=8.0,1.53Hz,1H), 7.42(t,J=7.6Hz,1H), 7.14~7. 24(m,1H), 6.96~7.07(m,2H), 6.88(t,J=7.2Hz,1H), 4.28(q,J=7.2Hz,2H), 3.55~3.67(m,6H), 2.70(br s,2H), 2.53~2.65(m,2H), 1.91(br s,2H), 1.33(t,J=7.2Hz,3H). LCMS[+scan]: Calculated value m / z C 20 H 26 N 3 O 3 S 388.2; measured value 388.1.

[0253] Synthesis of compound 14b TDB21 (100 mg, 0.26 mmol, 1.0 equiv) in EtOH (1.2 mL) and H 2 LiOH monohydrate (65 g, 1.5 mmol, 6.0 equiv.) was added to a mixture of 1.2 mL of 2H2O at 25 °C and N 2 The mixture was stirred at 25 °C for 16 h, after which time additional LiOH monohydrate (130 g, 3.1 mmol, 12.0 equiv) was added. After 2 h, the mixture was adjusted to pH 6 with 1 M HCl and the aqueous phase was extracted with DCM (3 × 10 mL). The organic phase was then washed with anhydrous MgSO 4 Drying at 40° C., filtration and concentration in vacuo afforded compound 14b (70 mg, 0.19 mmol, 76%) as a yellow oily solid.

[0254] 1 H NMR (400MHz, CD 3 O.D.):δ H 8.41(d,J=8.5Hz,1H), 8.08(d,J=8.5Hz,1H), 7.76(br s,1H), 7.64(m,1H), 7.53(t,J=7.5Hz,1H), 7.32(d,J=4.5Hz,1H), 7.07(t,J=7.5Hz,1H), 4.46(br s,2H), 3.95(m,2H), 3.75(tr,J=6.0Hz,2H), 3.47(m,4H), 2.34(m,2H).13 C NMR (100 MHz, CD 3 O.D.)δ C 170.9, 155.4, 142.7, 133.7, 131.2, 129.7, 129.3, 128.8, 127.6, 121.2, 119.0, 115.3, 55.2, 54.9, 53.3, 44.5, 40.2, 24.0. HRMS [+scan]: calculated m / z C 18 H 22 N 3 O 3 S 360.1382;Actual value 360.1386.

[0255] Synthesis of compound 15b To a mixture of compound 14b (70 mg, 195 μmol, 1.0 equiv.) and propargylamine (11.2 mg, 200 μmol, 13.1 μL, 1.0 equiv.) in DMF (3 mL), TEA (75.5 mg, 74.6 μmol, 104 μL, 4.0 equiv.) was added at 25 °C and N 2 The mixture was added with 50% T in DMF (238 mg, 370 μmol, 1.9 equiv.). 3 P was added and stirred at 25° C. for 16 h. The solvent was removed in vacuo and the title compound was purified on a column (gradient from DCM to 9:1 DCM:MeOH). To remove residual DMF, the compound was dissolved in DCM (10 mL) and diluted with H 2 The organic phase was washed with 20 mL of O (10 mL) and 1% aqueous NaOH solution (10 mL). 4 and the solvent was removed in vacuo to give 15b (26 mg, 66 μmol, 34% yield, 4:1 ratio).

[0256] 1 H NMR (400 MHz, CDCl 3, reported for the main diastereomer): δ 10.42 (br s, 1H), 8.35 (d, J = 8.9 Hz, 1H), 7.36-7.41 (m, 2H), 7.26 (m, 1H), 7.11 (m, 1H), 7.06 (m, 1H), 6.87-6.97 (m, 2H), 6.49 (br s, 1H), 4.16 (dd, J = 5.2, 2.5 Hz, 2H), 3.59-3.71 (m, 6H), 2.75 (br s, 2H), 2.65 (t, J = 5.5 Hz, 2H), 2.27 (t, J = 2.5 Hz, 1H), 1.96 (br s, 2H). 13 C NMR (100 MHz, CDCl 3 ) δ C 169.4, 155.4, 141.5, 140.0, 132.5, 128.4, 126.8, 125.5, 122.7, 121.1, 120.9, 118.9, 79.2, 71.8, 57.4, 55.0, 46.0, 29.6. HRMS [+scan]: calculated m / z C 36 H 52 N 9 O 7 S 397.1698; measured value 397.1716.

[0257] Synthesis of TDB-deg (compound 16b) Compound 15b (9.9 mg, 25 μmol, 1.0 equiv.) was dissolved in H 2 The solution was dissolved in a mixture of 2H2O (1.7 mL) and tBuOH (0.8 mL). 4A solution of 3a (250 μL, 100 mM, 25 μmol, 1.0 equiv.) was added followed by aqueous NaAsc solution (1.3 mL, 100 mM, 130 μmol, 5.2 equiv.). The resulting cloudy yellow mixture was placed under an argon atmosphere. An aqueous solution of azido-imidazole 3a (3.8 mL, 10 mM, 38 μmol, 1.5 equiv.) was then added. The reaction was stirred at room temperature for 1 h, after which the reaction mixture turned clear yellow. The reaction was quenched with disodium EDTA dihydrate (9.3 mg, 25 μmol, 1 equiv.), the organic solvent was removed in vacuo, and the mixture was purified via HPLC. The fractions containing the product were lyophilized to give the resulting TDB-deg (15b) as a yellow to brown oily solid (10.2 mg, 14 μmol, 54% yield).

[0258] HRMS [+scan]: Calculated m / z C 36 H 52 N 9 O 7 S 754.3710;Actual value 754.3698.

[0259] Synthesis of a chloramphenicol decomposition agent [ka] Scheme 6: Synthesis of Chloramphenicol-PEG-Imidazole Degrader

[0260] Synthesis of chloramphenicol-6PEG-imidazole degradant (18a) Propiolic acid (1.1 mmol) was dissolved in DMF (20 mL) under inert conditions and cooled to 0° C. HATU (2.0 mmol) and DIPEA (2.0 mmol) were added and the reaction mixture was stirred at 0° C. for 30 min. (1R,2R)-(−)-2-amino-1-(4-nitrophenyl)-1,3-propanediol (1.0 mmol) was added and the reaction mixture was stirred at room temperature for 24 h. After filtration, the crude mixture was used directly in the copper click reaction. Azido-imidazole 3a (1.0 mmol), CuSO 4(5 mol%), sodium ascorbate (0.2 mmol) were added and the reaction mixture was stirred at room temperature for another 24 h. After addition of toluene, the solvent was evaporated under reduced pressure. The crude product was purified via prep-HPLC. Yield: 10% (yellow oil).

[0261] 1 H NMR(400MHz,MeOD)δ8.29(s,1H), 8.14(d,J=8.8Hz,2H), 7.79(s,1H), 7.67(d,J=8.7Hz,2H), 7.44(s,1H), 7.22(s,1H), 5.23(d, J=2.6Hz,1H), 4.61~4.56(m,2H), 4.34(s,1H), 4.22(t,J=4.9Hz,2H), 3.89~3.82(m,2H), 3.79~3.68(m,2H), 3.62~3.50(m,18H). MS:C 27 H 39 N 7 O 10 m / z: 621.3.

[0262] Synthesis of Chloramphenicol-2PEG-Imidazole Degrader (18b) Propiolic acid (1.1 mmol) was dissolved in DMF (20 mL) under inert conditions and cooled to 0° C. HATU (2.0 mmol) and DIPEA (2.0 mmol) were added and the reaction mixture was stirred at 0° C. for 30 min. (1R,2R)-(−)-2-amino-1-(4-nitrophenyl)-1,3-propanediol (1.0 mmol) was added and the reaction mixture was stirred at room temperature for 24 h. After filtration, the crude mixture was used directly in the copper click reaction. Azido-imidazole 3c (1.0 mmol), CuSO 4 (5 mol%), sodium ascorbate (0.2 mmol) were added and the reaction mixture was stirred at room temperature for another 24 h. After addition of toluene, the solvent was evaporated under reduced pressure. The crude product was purified via prep-HPLC. Yield: 10% (white solid).

[0263] 1H NMR(400MHz,MeOD)δ8.19(s,1H), 8.16(d,J=8.8Hz,2H), 7.69(d,J=8.6Hz,2H), 7.55 (s,1H), 7.00(s,1H), 6.88(s,1H), 5.25(d,J=2.8Hz,1H), 4.60(dd,J=5.5,4.5Hz,2H) , 4.38(ddd,J=7.0,5.9,2.9Hz,1H), 4.14(dd,J=5.5,4.4Hz,2H), 3.89(dd,J=11.0,7. 0Hz,1H), 3.84(t,J=5.1Hz,2H), 3.75(dd,J=10.9,5.9Hz,1H), 3.71(t,J=4.9Hz,2H). MS:C 19 H 23 N 7 O 6 m / z: 445.2.

[0264] Synthesis of chloramphenicol-ethyl-imidazole decomposer (18c) Propiolic acid (1.1 mmol) was dissolved in DMF (20 mL) under inert conditions and cooled to 0° C. HATU (2.0 mmol) and DIPEA (2.0 mmol) were added and the reaction mixture was stirred at 0° C. for 30 min. (1R,2R)-(−)-2-amino-1-(4-nitrophenyl)-1,3-propanediol (1.0 mmol) was added and the reaction mixture was stirred at room temperature for 24 h. After filtration, the crude mixture was used directly in the copper click reaction. Azido-imidazole 4 (1.0 mmol), CuSO 4 (5 mol%), sodium ascorbate (0.2 mmol) were added and the reaction mixture was stirred at room temperature for another 24 h. After addition of toluene, the solvent was evaporated under reduced pressure. The crude product was purified via prep-HPLC. Yield: 12% (white solid).

[0265] 1H NMR(400MHz,MeOD)δ8.14(d,J=8.8Hz,2H), 8.03(s,1H), 7.64(d,J=8.3Hz,2H), 7.43(s,1H), 6.97(s,1H), 6.93(s,1H), 5.21(d,J=2.7Hz,1H), 4.82 (dd,J=6.8,5.0Hz,2H), 4.58(dd,J=6.7,4.9Hz,2H), 4.31(ddd,J=7.2,5.9,2.8Hz,1H), 3.84(dd,J=10.9,7.2Hz,1H), 3.69(dd,J=10.9,5.9Hz,1H). MS:C 17 H 19 N 7 O 5 m / z: 401.1.

[0266] G-quadruplex targeting and RNA degradation Two degraders were rationally designed to target RNA G-quadruplexes (rG4s) by tethering pyridostatin, a known G4 binder, with azido-imidazoles of different lengths (9A, 9B). The copper-induced azide-alkyne cycloaddition (CuAAC) used to tether the two components is applicable to a large array of substrates and generates amides and triazoles, bioisosteres of moieties that are well tolerated in biological systems. We used the same strategy to synthesize CBX-PDS, a pyridostatin derivative that has been shown to selectively bind RNA G-quadruplexes but not DNA G-quadruplexes. The two rG4 degraders and binding controls were tested in vitro and in cellular systems.

[0267] In vitro degradation of rG4 oligomers To demonstrate that our degraders can selectively degrade G4 structures, we investigated their activity in the presence of different oligomers and cations. We incubated our degraders with either RNA oligomers corresponding to the rG4 structure in the 5'UTR of NRAS mRNA or mutant forms thereof that were unable to form rG4s. Furthermore, we found that K+ (promoter of rG4 formation) and Li + We tested our molecules in the presence of rG4-competent oligomers, which are known to inhibit rG4 formation. + It is only seen in the presence of Li + (Fig. 2a), suggesting that it is not enough for oligomers to have G-rich sequences that are targeted by these molecules; oligomers must form G-quadruplexes to be degraded. Strikingly, K + Or Li + No degradation was observed for disrupted rG4 incompetent NRAS sequences with either PDS or CBX, again indicating that these degraders act specifically on rG4 structures (Figure 2b). Furthermore, no degradation was observed for the control molecule CBX-PDS, which has the rG4 binder moiety PDS but no degrader, indicating that binding alone is not sufficient to achieve degradation, but that the degrader moiety is required (Figure 2a,b). Interestingly, we observed that PDS-deg6 (9A), a degrader with a linker consisting of six PEG subunits, degraded RNA faster than PDS-deg4 (9B), which has four PEG subunits. This may be a result of the longer linker having a wider reach. Taken together, these experiments indicate that our degraders specifically cleave rG4 species but not unfolded RNA regions.

[0268] In vitro degradation of SARS-CoV-2 genomic material To prove that the rG4 degrader can degrade the genome of SARS-CoV-2 and to learn the mechanism of degradation, we extracted viral RNA from SARS-CoV-2-infected VERO cells, treated it with PDS-deg6(9A), and then analyzed it via direct RNA sequencing. Because the SARS-CoV-2 genome has several putative rG4 sites (Zhao et al., 2021), most of which have been shown to be in close proximity to rG4 sites due to tight packing (Ziv, et al., 2020), we expected our degrader to induce widespread damage. Indeed, we observed substantial degradation across many regions of the genome, indicating that our degrader is potent in damaging the genetic material of SARS-CoV-2 (Figure 2c). This phenomenon suggests that our degrading agent can degrade and therefore inactivate viral RNA within cells.

[0269] Antiviral activity of rG4 degraders in vitro To test the antiviral activity of G4 disintegrators in vitro, cells were incubated with 0.5 μM, 5 μM, and 50 μM of G4 disintegrators (PDS-deg6 (9A), PDS-deg4 (9B), and the control molecule PDS-Alk (8) without disintegrator) and 5 μM of chloroquine as a control (Figure 3a and Figure 3b). Inhibition of viral growth was measured by harvesting both supernatants and cells 24 hours after infection. Viral growth was assessed by measuring the viral load by plaque assay (in the supernatant) and PCR (in the cells). Cell viability after 24 hours of incubation with increasing concentrations of G4 disintegrators (ranging from 0.05 μM to 50 μM) was assessed using a conventional cell viability kit (e.g., CellTiter Blue assay) according to the manufacturer's protocol.

[0270] We observed that the G4 degraders successfully inhibited viral growth at 5 μM and 50 μM (Figure 3). PCR results showed that PDS-deg4 did not appear to inhibit viral replication, while PDS-deg6(9A) inhibited 70% of viral replication at 5 μM compared to the DMSO control (Figure 3b). Importantly, neither compound showed cytotoxicity up to 50 μM (Figure 3c).

[0271] Antiviral activity of rG4 degraders in vivo To evaluate the antiviral activity of G4 disintegrators in vivo, transgenic K18hACE2 mice (expressing hACE2 protein) were intranasally challenged with 25 mg / kg PDS-deg4 (9B) and PDS-deg6 (9A), 40 min before infection, and challenged again at 3 and 18 h postinfection (Figure 4). Mice were intranasally infected with SARS-CoV-2 (2.5–5 × 10 in 50 μl PBS on day 0). 4 Mice were monitored daily for total body mass (PFU / mouse), body weight, morbidity, and mortality (found dead or euthanized immediately prior to death), and clinical signs of infection. On day 5, all mice were sacrificed and the left lung was harvested for quantification of viral load by plaque assay. The right lung, heart, liver, kidney, and spleen were harvested for histopathological analysis.

[0272] Results showed that administration of 25 mg / kg PDS-deg6(9A) was toxic and these treated mice had to be sacrificed on day 0. Organs were harvested for histopathological analysis. Mice administered PDS-deg4(9B) showed a 10% weight loss on the first day post-infection (Figure 4a). However, weight remained stable between days 1 and 3 and then decreased again at the same rate as vehicle controls. Animals treated with PDS-deg4(9B) showed a significant reduction in lung viral load (Figure 4b).

[0273] The study showed that administration of a G4 disruptor reduced the viral load in the lungs of SARS-CoV-2-infected k18hACE2 mice.

[0274] Pseudoknot targeting and RNA degradation A non-covalent degrader molecule, MTDB-deg (16a), was rationally designed to target RNA pseudoknots by conjugating the known pseudoknot binder MTDB with azido-imidazole 3a (Figure 5a). MTDB has an ethyl ester moiety, which was exchanged to an amide to increase stability and to serve as a handle for degrader attachment. We used azido-imidazole 3a, which has a linker consisting of six PEG subunits and which we have previously found to be more effective than its shorter counterpart for RNA degradation of alkynyl-tagged RNA (Mikutis et al., 2020). We chose CuAAC as the reaction to conjugate the binder and degrader fragments because it is robust, easy to perform, highly modular, and allows us to vary the structure of the two fragments without changing the conjugation step.

[0275] Selective disassembly of three-stem coronavirus pseudoknots To validate our strategy, we tested our pseudoknot-disintegrating agent against RNA 69-er, which has a sequence that corresponds to and is therefore predicted to form coronavirus pseudoknots. We incubated 69-er with MTDB-deg (16a) or one of two control molecules, MTDB, the parent binder molecule that cannot be disintegrated, or TDB-deg (16b), a disintegrating agent derived from 2-(4-(thiophen-3-ylmethyl)-[1,4]diazepane-1-carbonyl]-amino)-benzoic acid ethyl ester (TDB), which is closely related to MTDB but has a lower binding affinity for pseudoknots (Figure 5b) (Park et al., 2011). After 3 h of incubation with MTDB-deg(16a), the RNA pseudoknot was significantly degraded, with just 23% remaining intact compared to the untreated sample, whereas TDB-deg(16b) showed poor degradation and MTDB showed no degradation (Figure 5c, 5d). To illustrate that these molecules specifically bind and degrade the pseudoknot, we performed the same experiment using an oligo that resembles the pseudoknot but has a large disruption of the third stem, preventing the proper formation of the pseudoknot. As expected, neither molecule affected oligo stability (Figure 5e). Thus, MTDB-deg(16a) was shown to efficiently and selectively bind and degrade the pseudoknot.

[0276] To demonstrate that MTDB-deg(16a) is functional and can cleave full-length coronavirus RNA, we incubated MTDB-deg(16a) and controls MTDB and TDB-deg(16b) with RNA extracted from SARS-CoV-2. The viral RNA was then analyzed on an agarose gel. We observed degradation only in the lane corresponding to MTDB-deg(16a) (Figure 5f), further supporting that MTDB-deg(16a) indeed degrades native coronavirus pseudoknots, whereas the two control molecules do not, highlighting the specificity of this approach.

[0277] Specificity of coronavirus pseudoknot resolution by MTDB-deg To further prove that MTDB-deg(16a) cleaves viral RNA and to get a more precise picture of where cleavage is occurring, we analyzed the cleaved genomic RNA (gRNA) by direct RNA nanopore sequencing. As expected, the region around the pseudoknot was most affected (Figure 6a). Interestingly, the pseudoknot flanking parts were more degraded than the pseudoknot itself. Indeed, studies of the SARS-Cov-2 RNA interactome revealed that the region around the frameshifting element forms extensive short- and long-range interactions with the neighboring ORF1a and especially ORF1b. The proximity of these elements to the pseudoknot likely enables MTDB-deg(16a) to cleave them efficiently (Figure 6a) (Ziv, et al., 2020). Interestingly, the only other structural element affected by the molecule was the S gene, which has been shown to form long-range interactions with ORF1b (Ziv, et al., 2020) and is therefore expected to be within reach of the degrader's influence (Figure 6b). Strikingly, no other subgenomic regions were affected, providing strong evidence of the specificity of MTDB-deg(16a) (Figure 7). The above results provide strong proof of principle that MTDB-deg(16a) is a fully functional and selective degrader of SARS-Cov-2 pseudoknots and their direct RNA-RNA interactome (Ziv, et al., 2020).

[0278] Efficiency and specificity of pseudoknot resolution in SARS-CoV-2-infected cells Having demonstrated the efficiency of MTDB-deg(16a) against coronavirus pseudoknots in vitro, we investigated whether it could degrade the genome of SARS-CoV-2 in infected cells and thus prevent viral replication. We performed an in vitro drug assay in which we measured the replication of SARS-CoV-2 in Vero CCL-81 cells. We observed that low micromolar concentrations of MTDB-deg(16a) exhibited a significant antiviral effect (Figures 8a-c), with a significant reduction in coronavirus RNA in cells treated either pre-infection (Figures 8a, 9a) or post-infection (Figures 8b, 9b). These results were supported by the results of a plaque assay (Figure 8d). Importantly, the control molecules MTDB and TDB-deg (16b) did not show any antiviral effect, even though MTDB is known to disrupt the frameshift of SARS-CoV-2 (Kelly et al., 2020). Furthermore, we found that none of the compounds were cytotoxic to the host cells, indicating that the observed effects on viral replication were the result of the specific antiviral activity of the compounds (Figure 8e). Curiously, the degraders were less active at concentrations higher than 6 μM (Figure 9c), but no colloidal aggregation was observed in the dynamic light scattering screen that could justify these readouts. Furthermore, the ability of the virus to survive a 24-hour drug exposure was reduced in samples treated with MTDB-deg (16a), but not in samples treated with the control molecules MTDB and TDB-deg (16b) (Figures 8f, 10a). Finally, no virucidal effect was observed when cell-free virus was incubated with MTDB-deg(16a), MTDB, or TDB-deg(16b), suggesting that the antiviral activity of MTDB-deg(16a) is mediated by direct inhibition of viral replication in host cells (Figure 10b).Overall, the antiviral drug assays demonstrate that the MTDB degrader (16a) is an efficient antiviral drug against SARS-CoV-2 and has a specific and irreversible effect on the coronavirus three-stem pseudoknot.

[0279] Antiviral activity of pseudoknot disintegrators in vivo We used a SARS-CoV-2 mouse model of infection (transgenic K18-hACE2 mice) to determine the in vivo antiviral activity of MTDB-deg 16a (Figure 12a). Animals administered MTDB-deg 16a (25 mg / kg) showed a significant reduction in lung viral load compared to the vehicle control group by plaque assay (Figure 12b). Furthermore, we investigated the in vivo antiviral capacity of MTDB-deg 16a or vehicle treatment using proteins extracted from the lungs of K18-hACE2 transgenic mice at days 3 or 6 post-infection. Encouragingly, we observed that at both time points of infection, the MTDB-deg 16a-treated cohort showed a large reduction in the phosphorylated levels of p38, a key biomarker of SARS-CoV-2 infection and replication (Figure 12c).

[0280] Targeting the bacterial ribosome Three degraders were rationally designed to target bacterial ribosomes by tethering chloramphenicol, a known ribosomal RNA binder, with azido-imidazoles of different lengths (18a, 18b, 18c). The chloramphenicol binder moiety was generated by peptide coupling of propargylic acid to (1R,2R)-(-)-2-amino-1-(4-nitrophenyl)-1,3-propanediol using HATU and DIPEA, followed by a conjugation step using copper click chemistry. The copper-induced azide-alkyne cycloaddition (CuAAC) used to tether the two components is amenable to a large array of substrates and generates triazoles, bioisosteres of amides and moieties that are well tolerated in biological systems. The three ribosomal degraders and binding controls were tested in vitro and in cellular systems.

[0281] In vitro degradation of Escherichia coli ribozymes. The degradation activity was measured in an in vitro assay by targeting E. coli ribosomes. 200 μM of the ribozyme was incubated with different concentrations of the degrader ranging from 15 mM to 0.47 mM at 37°C for 18 hours. Evaluation was performed by agarose gel analysis.

[0282] The best results were obtained using the PEG-2 linker (18b), where ribosome degradation was observed at a concentration of 15 mM (Figure 11).

[0283] Additional Examples Materials and Methods Quantification of viral load by RT-qPCR Cells from viral cultures were resuspended in RLT buffer and extracted using the RNeasy Mini Kit. 500 ng of RNA was converted to cDNA using SuperScript™ VILO™ Master Mix. Levels of genomic and subgenomic SARS-CoV-2 transcripts were analyzed on a QuantStudio™ 5 real-time PCR instrument (Applied Biosystems) using PowerUp™ SYBR™ Green Master Mix (Applied Biosciences) according to the manufacturer's instructions. All samples, including template controls, were assayed in triplicate. Relative quantification of target gene expression was determined using the comparative cycle threshold (C T The primer sequences are listed in Table 1. [Table 3]

[0284] Preparation of oligonucleotides for binding studies To prepare oligonucleotides for binding studies, oligonucleotides were dissolved in an appropriate buffer to a final concentration of 100 nM and then incubated with shaking at 95° C. for 5 minutes. Samples were allowed to cool at room temperature for at least 1 hour before further dilution / processing.

[0285] Fluorescence quenching assay Cy5-tagged oligonucleotides (final concentration 50 nM) corresponding to the G4 structure found in the 5'UTR of NRAS mRNA were incubated in 50 mM KCl (100 mM) and MgCl 2Oligonucleotides were dissolved in 20 mM HEPES buffer, pH 7.4, supplemented with 10 mM riboflavin (10 mM) and seeded into 96-well plates. Oligonucleotides were treated with the indicated PDS family ligands or water vehicle control at various concentrations ranging from 5 nM to 10 μM, followed by incubation at 4 °C for 30 min. Fluorescence corresponding to the Cy5 fluorophore was then measured for each oligonucleotide-small molecule / vehicle control combination at 25.0 °C using a plate reader (CLARIOstar, BMG). For all molecules, 12 serial dilutions with a dilution ratio of 1:1 were used, with the maximum concentration tested being 10 μM. Fluorescence was normalized to the vehicle control, and binding curves for each molecule were obtained via sigmoidal fit by setting the coefficient of Hill slope to 1.

[0286] Microscale thermophoresis (MST) assay FAM-tagged oligonucleotides (final concentration 50 nM) were dissolved in 20 mM HEPES buffer, pH 7.4, supplemented with KCl (100 mM) and EDTA (10 mM). The oligonucleotide solutions were then treated with various concentrations of small molecules and analyzed via MST according to the manufacturer's instructions (NanoTemper Monolith NT.115). For these MST measurements, the following program was used: 5 sec laser off, 30 sec laser on, 5 sec laser off; 20% LED (blue) power, 30% MST power; measurements were performed at 25.0°C. For MTDB-Deg, TDB-Deg, and click degrader 1, 12 serial dilutions were used with a dilution ratio of 5:3, and the maximum concentration tested was 8 mM. For MTDB, 12 serial dilutions were used with a dilution ratio of 3:1, and the maximum concentration tested was 250 μM. Binding curves for each molecule were obtained via sigmoidal fit by setting the Hill slope coefficient to one. [Table 4]

[0287] Resolution of pseudoknots by MTDB-deg This example examines the interaction between MTDB-deg and pseudoknots in a SARS-CoV-2 infection model. SARS-CoV-2-infected Vero cells were treated with either MTDB-deg or vehicle control, after which RNA extraction and qPCR analysis were performed at 14 loci in the SARS-CoV-2 genome to obtain extensive genome coverage and reveal which segments were most affected.

[0288] The results are shown in Figure 13. This is largely consistent with the in vitro experiments, indicating that MTDB-deg affects the flanking parts of the pseudoknot region but not any other regions of the full-length genomic or subgenomic SARS-CoV-2 RNA, demonstrating that MTDB-deg is a functional and selective degrader of the SARS-CoV-2 pseudoknot and its direct RNA-RNA interactome.

[0289] Determination of binding affinity Two series of compounds were prepared and then the effect of functionalization with degraders on binding affinity and selectivity was examined. PDS is a known fluorescence quencher, therefore, a fluorescence quenching assay was used to evaluate compounds derived from this scaffold (Di Antonio et al., Angew. Chem. Int. Edit., 2012, Vol 51, pp. 11073-11078).

[0290] PDS and its derivatives were incubated with Cy5-functionalized oligomers corresponding to the G-quadruplex in the 5'UTR of NRAS, a well-established G4 model. Fluorescence quenching is proximally induced, so the signal corresponding to Cy5 is quenched upon PDS binding. Based on this assay, we found that the parent compound PDS has an EC 50(Figure 14a), we found that PDS bound to the PDS derivative with slightly lower affinity compared to the parent compound. This is likely because underivatized PDS has an additional amine group on the pyridine core that is replaced by a triazole in the derivatized compound. This positively charged amine group can form an additional interaction with the negatively charged phosphate backbone.

[0291] MTDB and derived ligands do not quench fluorescence, therefore, microscale thermophoresis (MST) was utilized to assess the binding affinity of this RNA binder family. In the MST workflow, FAM-functionalized oligomers corresponding to coronavirus pseudoknots are incubated with one of the binders, and the fluorescence of the bound oligomers shows a different temperature dependence compared to the unbound oligomers.

[0292] To investigate the binding selectivity of MTDB, we performed assays with pseudoknots as well as oligonucleotides corresponding to disrupted pseudoknots, where one of the stems has been altered to an identical random sequence in length and is therefore no longer able to form a complete pseudoknot structure.

[0293] MTDB uses MST D Although the pseudoknot was found not to be sufficiently soluble for the determination of β-terminal β-terminal binding, the trend of the curves showed significant binding at concentrations above 187.5 μM (Figure 14b). No significant binding was observed with the mutated (disrupted) pseudoknot (Figure 14c). The other two molecules tested, MTDB-deg and TDB-deg, were found to be more soluble in aqueous medium and therefore concentrations ranging from 29 μM to 8 mM were tested.

[0294] Using the MST approach, MTDB-deg and TDB-deg had K values ​​of 1.86 mM and 2.88 mM, respectively. D When the pseudoknot was broken, K DThe values ​​decreased to 5.13 mM and 6.91 mM, respectively (Figure 14c), indicating that the binder decomposer molecule is a selective but weak binder. Similar to MTDB, the shape of the binding curve for MTDB-deg showed sufficient binding at concentrations above 134 μM, indicating that functionalization with the decomposer did not reduce the binding affinity of this molecule.

[0295] The degrader-linker component 3a (click degrader 1) was also tested for binding to pseudoknotted oligonucleotides. At the concentrations tested, binding was K D The binding intensity was too weak to obtain any value, suggesting that this molecule is a much poorer binder than those derived from MTDB or TDB (FIGS. 14b, c).

[0296] References A number of publications have been cited above in order to more fully describe and disclose the invention and the prior art to which this invention pertains. Full citations for these references are set forth below. Each of these references is incorporated herein in its entirety. 1. Bobbin, et al., Annual Review of Pharmacology and Toxicology, 2016, Vol. 56, pp. 103-122 2. Cox, et al., Science, 2017, Vol. 358, pp. 1019-1027 3. Di Antonio et al., Angew. Chem. Int. Edit., 2012, Vol 51, pp. 11073-11078 4. Gasiunas, et al., Proc. Natl. Acad. Sci. USA, 2012, Vol. 109, E2579-E2586 5. Jinek, et al., Science, 2012, Vol. 337, pp. 816-821 6. Kelly et al., J. Biol. Chem., 2020, Vol. 295, pp. 10741-10748 7. Li. Bioinformatics, 2018, Vol. 34, pp. 3094-3100 8. Mikutis et al., ACS Cent. Sci., 2020, Vol. 6, pp. 2196-2208 9. Park et al., J. Am. Chem. Soc., 2011, Vol. 133, pp. 10094-10100 10. Santos et al., Pharmaceuticals, 2021, Vol. 14, No. 769 11. Tzelepis, et al., Cell Reports, 2016, Vol. 17, pp. 1193-1205 12. Zamore, et al., Cell, 2000, Vol. 101, pp. 25-33 13. Zhao et al., Angew. Chem. Int. Ed., 2021, 60 (1), 432-438 14. Ziv, et al., "Mol. Cell., 2020, Vol. 80, pp. 1067-1077

Claims

1. 1. A method for cleaving a target nucleic acid molecule, comprising: The target nucleic acid molecule is treated with a bifunctional molecule of formula (I) or a salt or solvate thereof: C-L-B(I) (Wherein, -C represents 1 to 3 C １－６ -L- is a linker; and -B is a non-covalent linking group for non-covalently linking the bifunctional molecule to the target nucleic acid molecule. and allowing the bifunctional molecule to cleave the target nucleic acid molecule bound thereto. The method comprising:

2. Non-covalent groups (i) is not a polynucleotide group; (ii) has a molecular weight of 1000 kDa or less; (iii) binds to a secondary or tertiary structure within the target nucleic acid; and / or (iv) selectively binds to secondary or tertiary structures within the target nucleic acid compared to the corresponding linear nucleic acid; The method of claim 1.

3. The method of claim 1 , wherein the non-covalent binding group is attached to the quadruplex or pseudoknot.

4. The bifunctional molecule has a dissociation constant (k) of 10 mM or less, such as 10,000 nM or less. Ｄ ) linked in a quadruplex or pseudoknot; and / or 4. The method of claim 3, wherein the bifunctional molecule binds to the quadruplex or pseudoknot with a 5:1 selectivity.

5. The non-covalent bonding group is represented by formula (BI) to (B-III): 【Chemistry 1】 (In the formula, X is O or NH, and ＊ is the point of attachment to the linker) The method according to any one of claims 1 to 4, wherein the compound is selected from the group consisting of:

6. The cleavage group is represented by the formulas (CI) to (C-III): 【Chemistry 2】 (In the formula, R １ , R ２ , and R ３ are each independently a hydrogen atom or C １－６ represents an alkyl group, R Ｎ is a hydrogen atom or C １－６ represents an alkyl group, and ＊ represents the point of attachment to the rest of the molecule (typically a linker unit L) The method according to any one of claims 1 to 4, wherein the compound is selected from the group represented by

7. the target nucleic acid molecule is an RNA molecule; and / or contacting the target nucleic acid molecule with the bifunctional molecule within the cell; The method according to any one of claims 1 to 4.

8. 1. A method for identifying secondary or tertiary structure within a target nucleic acid molecule, comprising: providing first and second populations of nucleic acid molecules, each of said populations comprising said target nucleic acid molecule; The first population of nucleic acid molecules is administered a bifunctional molecule of formula (I) or a salt or solvate thereof: C-L-B(I) (Wherein, -C represents 1 to 3 C １－６ -L- is a linker; and -B is a non-covalent linking group for non-covalently linking the bifunctional molecule to the target nucleic acid molecule. introducing allowing the bifunctional molecule to cleave the target nucleic acid molecules present in the first population; and identifying nucleic acid molecules that are present in less abundance in the first population than in the second population; The method comprising:

9. Non-covalent groups (i) is not a polynucleotide group; (ii) has a molecular weight of 1000 kDa or less; (iii) binds to a secondary or tertiary structure within the target nucleic acid; and / or (iv) selectively binds to secondary or tertiary structures within the target nucleic acid compared to the corresponding linear nucleic acid; The method of claim 8.

10. 9. The method of claim 8, wherein the non-covalent binding group is attached to the quadruplex or pseudoknot.

11. The bifunctional molecule has a dissociation constant (k) of 10 mM or less, such as 10,000 nM or less. Ｄ ) linked in a quadruplex or pseudoknot; and / or 11. The method of claim 10, wherein the bifunctional molecule binds to the quadruplex or pseudoknot with a 5:1 selectivity.

12. The non-covalent bonding group is represented by formula (BI) to (B-III): 【Transformation 3】 (In the formula, X is O or NH, and ＊ is the point of attachment to the linker) The method according to any one of claims 8 to 11, wherein the method is selected from the group consisting of:

13. The cleavage group is represented by the formulas (CI) to (C-III): 【Chemistry 4】 (In the formula, R １ , R ２ , and R ３ are each independently a hydrogen atom or C １－６ represents an alkyl group, R Ｎ is a hydrogen atom or C １－６ represents an alkyl group, and ＊ represents the point of attachment to the rest of the molecule (typically a linker unit L) The method according to any one of claims 8 to 11, wherein the compound is selected from the group represented by

14. the target nucleic acid molecule is an RNA molecule; and / or contacting the target nucleic acid molecule with the bifunctional molecule within the cell; The method according to any one of claims 8 to 11.

15. A bifunctional molecule of formula (I) or a pharmaceutically acceptable salt or solvate thereof for use in a method of treatment: C-L-B(I) (Wherein, -C represents 1 to 3 C １－６ -L- is a linker; and -B is a non-covalent group that binds to the target nucleic acid molecule.

16. Non-covalent groups (i) is not a polynucleotide group; (ii) has a molecular weight of 1000 kDa or less; (iii) binds to a secondary or tertiary structure within the target nucleic acid; and / or (iv) selectively binds to secondary or tertiary structures within the target nucleic acid compared to the corresponding linear nucleic acid; A bifunctional molecule for use according to claim 15.

17. 16. The bifunctional molecule for use according to claim 15, wherein the non-covalent binding group is attached to the quadruplex or pseudoknot.

18. The bifunctional molecule has a dissociation constant (k) of 10 mM or less, such as 10,000 nM or less. Ｄ ) linked in a quadruplex or pseudoknot; and / or 18. The bifunctional molecule for use according to claim 17, wherein the bifunctional molecule binds to the quadruplex or pseudoknot with a 5:1 selectivity.

19. The non-covalent bonding group is represented by formula (BI) to (B-III): 【Transformation 5】 (In the formula, X is O or NH, and ＊ is the point of attachment to the linker) A bifunctional molecule for use according to any of claims 15 to 18, selected from:

20. The cleavage group is represented by the formulas (CI) to (C-III): 【Transformation 6】 (In the formula, R １ , R ２ , and R ３ are each independently a hydrogen atom or C １－６ represents an alkyl group, R Ｎ is a hydrogen atom or C １－６ represents an alkyl group, and ＊ represents the point of attachment to the rest of the molecule (typically a linker unit L) A bifunctional molecule for use according to any of claims 15 to 18, selected from the group represented by

21. The bifunctional molecule for use according to any one of claims 15 to 18, wherein the target nucleic acid molecule is an RNA molecule.

22. Treatment, (i) the treatment of bacterial or viral infections; (ii) treatment of infection with an RNA virus, optionally wherein the RNA virus is a coronavirus; (iii) for the treatment of respiratory tract infections, urinary tract infections, or gastroenteritis; or (iv) is a treatment for cancer; A bifunctional molecule for use according to any one of claims 15 to 18.

23. A bifunctional molecule of formula (I) or a salt or solvate thereof: C-L-B(I) (Wherein, -C represents 1 to 3 C １－６ -L- is a linker; and -B is a non-covalent group that binds to the nucleic acid molecule.

24. Non-covalent groups (i) is not a polynucleotide group; (ii) has a molecular weight of 1000 kDa or less; and / or (iii) selectively binds to secondary or tertiary structures within the target nucleic acid compared to the corresponding linear nucleic acid; 24. The bifunctional molecule of claim 23.

25. 24. The bifunctional molecule of claim 23, wherein the non-covalent binding group is attached to the quadruplex or pseudoknot.

26. The bifunctional molecule described in claim 25, which binds to a quadruplex or pseudoknot with a dissociation constant (kD) of 10 mM or less, such as 10,000 nM or less.

27. The non-covalent bonding group is represented by formula (BI) to (B-III): 【Transformation 7】 (In the formula, X is O or NH, and ＊ is the point of attachment to the linker) The bifunctional molecule according to any one of claims 23 to 26, selected from:

28. The cleavage group is represented by the formulas (CI) to (C-III): 【Transformation 8】 (In the formula, R １ , R ２ , and R ３ are each independently a hydrogen atom or C １－６ represents an alkyl group, R Ｎ is a hydrogen atom or C １－６ represents an alkyl group, and ＊ represents the point of attachment to the rest of the molecule (typically a linker unit L) The bifunctional molecule according to any one of claims 23 to 26, wherein the bifunctional molecule is selected from the group represented by:

29. The linker has the formula (LI): 【Chemistry 9】 (In the formula, L １ is a covalent bond or C １－２ is an alkylene group, L ２ is C １－６ an alkylene group or a C heteroalkene group, L ３ is C １－６ is an alkylene group, n is 1 to 8; ＊ is the point of attachment to the non-covalent bonding group (-B), and ＊＊ is the point of attachment to the cleavage group (-C) The bifunctional molecule of any one of claims 23 to 26, comprising a group represented by:

30. Formulas Deg-I to Deg-V: 【Chemistry 10】 24. The bifunctional molecule of claim 23, wherein the bifunctional molecule is selected from the group consisting of:

Images