Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

• the present inveto ntion relates to methods for the non-enzymatic cleavage of target nucleic acids, for example for use in epigenomic and epitranscriptomic mapping, as well as in therapy, for example anti-microbial and anti-viral therapy.
• RNA interference and shRNA expression systems have proven invaluable for target validation as well as elucidation of the role particular genes play in molecular diseases (Zamore et al., 2000). More recently, CRISPR-based technologies have enhanced the ability to manipulate DNA (Gasiunas et al., 2012; Jinek et al., 2012) and RNA (Cox et al. 2017) even further, empowering simpler systems such as gene knock-out cells and enabling large and elaborate CRISPR-Cas9-based genetic screening approaches (Tzelepis et al., 2016).
• nucleic acid manipulation technologies are genetic and it is challenging if not outright impossible to apply them to more complex biological systems such as rare populations, animal models and whole tissues, and yet even harder to develop therapeutics based on these technologies (Bobbin et al, 2016). Therefore, there is a need to develop new small molecule-based technologies, which would enable manipulation of nucleic acids in yet unexplored contexts.
• Mikutis et al., 2020 describes a small molecule “click degrader” that can be covalently attached to an RNA species through click-chemistry and can then cleave the attached RNA molecule.
• the authors describe a methylation CLICK degradation sequencing method (meCLICK-Seq) for identifying the presence of N 6 -methyladenosine (m 6 A) in an RNA sequence.
• the method hijacks an RNA methyltransferase to introduce an alkyne moiety, instead of a methyl group, on RNA.
• a subsequent copper(l)-catalyzed azide-alkyne cycloaddition reaction incorporates the click-degrader molecule, leading to RNA cleavage.
• the method identifies methylated transcripts, determines RNA methylase specificity, and reliably maps modification sites in intronic and intergenic regions.
• the click degrader molecules are covalently incorporated into the target RNA, they can only be used to degrade RNA species which can be edited to contain a suitable click-reactive group (typically an alkyne).
• a suitable click-reactive group typically an alkyne.
• the required editing of the RNA limits the application of the technology to therapeutics.
• the present invention has been devised in light of the above considerations.
• the present invention relates to the finding that a bifunctional molecule, known herein as a degrader, can be used as a catalytic agent to non-covalently bind to and cleave a target nucleic acid molecule.
• the degraders disclosed herein bind to a target nucleic acid through non-covalent interactions. Surprisingly, the inventors have found that non-covalent binding is sufficient to enable the selective degradation of the target nucleic acid. Accordingly, the degraders do not require incorporation of a click-reactive group into the target nucleic acid.
• the selective cleavage of target nucleic acid molecules using the degraders described herein may be useful in epigenetic and epitranscriptomic analysis, bifunctional mapping, as well as in therapy, for example anti-bacterial and anti-viral therapy, and additionally anti-cancer.
• a method for cleaving a target nucleic acid molecule comprising: contacting the target nucleic acid molecule with a bifunctional molecule of formula (I): C-L-B (I) where -C is a cleavage group that is imidazole, optionally substituted with 1 to 3 C1-6 alkyl groups, -L- is a linker and -B is a non-covalent binding group, such that the bifunctional molecule non-covalently binds to the target nucleic acid molecule, and; allowing the bifunctional molecule to cleave the target nucleic acid molecule bound thereto.
• the non-covalent binding group (-B) is not a polynucleotide group.
• the non-covalent binding group (-B) has molecular weight of 1 ,000 kDa or less, more preferably 800 kDa or less.
• the non-covalent binding group (-B) binds to a secondary or tertiary structure within the target nucleic acid, such as a quadruplexes, a pseudoknot, a triplex, a tetraloop, a step-loop or a hairpin loop. More preferably, the non-covalent binding group (-B) binds to a quadruplex or pseudoknot.
• the bifunctional molecule binds to a secondary or tertiary structure with a dissociation constant (ko) of 10,000 nM or less, such as determined by SPR, or alternatively by microscale thermophoresis (MST) or by a fluorescence quenching assay.
• ko dissociation constant
• the bifunctional molecule binds to the secondary or tertiary structure with a ko of 1 ,000 nM or less, more preferably 500 nM or less, even more preferably 200 nM or less, and most preferably 100 nM or less.
• the bifunctional molecule binds to a secondary or tertiary structure with a dissociation constant (ko) of 10 mM or less, such as determined by microscale thermophoresis (MST).
• ko dissociation constant
• MST microscale thermophoresis
• the bifunctional molecule binds to the secondary or tertiary structure with a ko 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.
• the non-covalent binding group (-B) is selected from formulae (B-l) to (B-lll) set out below.
• the target nucleic acid molecule is an RNA molecule, such as viral RNA or a bacterial ribozyme.
• the target nucleic acid molecule is contacted with the bifunctional molecule within a cell.
• a method for identifying a secondary or tertiary structure within a target nucleic acid molecule comprising: providing first and second populations of nucleic acid molecules, each population comprising the target nucleic acid molecule; introducing into the first population of nucleic acid molecules a bifunctional molecule of formula (I):
• C-L-B (I) where -C is a cleavage group that is imidazole, optionally substituted with 1 to 3 C1-6 alkyl groups , -L- is a linker and -B is a non-covalent binding group, such that the bifunctional molecule non-covalently binds to the target nucleic acid molecule; allowing the bifunctional molecule to cleave the target nucleic acid molecule present in the first population; and identifying nucleic acid molecules which are present in a reduced amount in the first population relative to the second population.
• Preferred embodiments of the bifunctional molecule of formula (I) set out for the first aspect also apply to the second aspect.
• the treatment is treatment of a bacterial or viral infection.
• the viral infection is an infection with an RNA virus, more preferably a (+)ssRNA virus, even more preferably a coronavirus.
• the treatment is treatment of a respiratory tract infection, a urinary tract infection, or gastroenteritis.
• the treatment is treatment of cancer.
• Preferred embodiments of the bifunctional molecule of formula (I) set out for the first aspect also apply to the fourth aspect.
• the bifunctional molecule is selected from compounds Deg-I to Deg-V.
• Figure 1 Schematic diagram of the mode of action of degraders.
• Figure 1 demonstrate the use of the degraders in a coronaviral pseudoknot degradation strategy.
• the pseudoknotdegrader binds and then directly degrades the coronaviral region that contains the pseudoknot without a need for other agents.
• Figure 2 rG4 degraders cleave rG4-containing oligomers and SARS-CoV-2 genome in vitro.
• (c) Shows nanopore sequencing data indicating widespread degradation on SARS-CoV-2 genome upon treatment with rG4 degrader PDS-deg6 (9A) on its ORF1 b.
• Figure 3 In vitro preliminary findings of G4-degraders anti-SARS-CoV-2 activity, (a) Shows the inhibition of plaque forming units (PFU) on samples treated at 50 pM with PDS-deg4 (9B), PDS-deg6 (9A) and PDS-DegALK (8). (b) Provides PCR measurements of viral RNA. The results show the inhibition of viral replication by PDS-deg6 (9A) at 5 pM and 50 pM. As shown, PDS-lmi6 (9A) appears to inhibit viral growth to a greater extent than PDS-deg4 (9B). (c) Shows the viability of cells after 24 hours of incubation with increasing concentrations of the G4-degrader. None of the compounds showed cytotoxicity up to 50 pM.
• Figure 4 In vivo anti-SARS-CoV-2 activity of G4-degrader.
• Mice administered with PDS- deg4 (9B) (purple, triangular markers) showed 10% loss of body weight in the first day after infection, which stabilized between Day 1 and Day 3 before decreasing again to reach the 75% threshold on Day 5, as observed in vehicle (0.1 % DMSO in water) treated animals (grey, square markers).
• Non-infected mice treated with vehicle (0.1% DMSO in water) as a control as showed no decrease in body-weight
• (b) Quantification of lung viral load on Day 5 by plaque assay showed decreased loads in animals treated with PDS-deg4 (9B) (purple, right) in comparison to vehicle control group (grey, left). * p ⁇ 0.01.
• MTDB-degrader (16a) cuts coronaviral pseudoknots in vitro
• (a) Shows the synthetic design of MTDB-deg (16a).
• (b) Shows the structure of control molecule TDB-deg (16b) that features a weak pseudoknot binder and the imidazole cleavage portion
• (d) Shows gel photographs validating the activity of the pseudoknot degraders
• (f) Are gels photographs showing the degradation of native RNA extracted from SARS-CoV-2 by MTDB-deg (16a) against control, n.s. - not significant.
• Figure 6 Direct RNA Nanopore sequencing reveals the genomic loci which get degraded with MTDB-deg.
• b Distribution and abundance of aligned reads mapped exclusively on the S sgRNA region for control- or MTDB-deg-treated SARS-CoV-2 RNA, based on alignments with minimap2
• FIG. 7 Treatment with MTDB-deg has no effect on the subgenomic SARS-CoV-2 RNAs. Distribution and abundance of aligned reads mapped exclusively on the indicated sgRNA regions for control- (0.1% DMSO in water) or MTDB-deg-treated SARS-CoV-2 RNA, based on alignments with minimap2.
• FIG. 8 MTDB-degrader inhibits SARS-CoV-2 replication in cells, (a), (b) Show the percentage of inhibition of viral replication normalised to vehicle control (dashed line) after incubation with increasing concentrations of the pseudoknot degrader (MTDB-deg (16a)) and control molecules (MTDB and TDB-deg (16b)). Viral replication was assessed after 24 h of infection (multiplicity of infection (MOI) of 0.05) based on E gene and Pseudoknot region RNA levels. Antiviral activity of the MTDB-deg (16a) was observed both before (a), and after infection (b), with SARS-CoV-2 at a 0.05 MOI.
• MOI multiplicity of infection
• Control molecule MTDB only showed decreased number of viral plaques when added before infection, and TDB-deg (16b) showed no decrease,
• FIG. 9 Dose-response curves of MTDB-deg, MTDB and TDB-deg. (a), (b) Show the 50% inhibitory concentration (IC50) values for pseudoknot degrader MTDB-deg (16a) before and after infection. Control molecules (MTDB and TDB-deg (16b) did not inhibit viral replication and thus IC50 values could not be determined, (c) Is a dose-response curve (as determined by PCR targeting the E gene), including a higher concentration of 18 pM showing increased IC50.
• FIG. 10 Viral recovery and virucidal activity after MTDB-degrader exposure, (a) Virus ability to recover after 24 h incubation with MTDB-deg (16a) and control molecules MTDB and TDB-deg (16b), as determined by qPCR targeting the pseudoknot region. Viral recovery was impaired in MTDB-deg (16b) treated samples, but not in samples treated with the control molecules MTDB and TDB-deg (16b). (b) Virucidal activity was assessed by incubating 1000 PFU of SARS-CoV-2 with compounds at 6 pM, for 1 h at 37 °C after which residual viral infectivity was determined by plaque assay.
• MTDB-deg (16a), MTDB and TDB-deg (16b) showed no virucidal effect on cell free virions, suggesting that the MTDB-deg (16a) antiviral activity is mediated by inhibiting virus replication in host cells and not by inactivation of cell free virions.
• PFU plaque-forming units
• FIG. 13 RT-qPCR validation of degradation specificity in a cellular system.
• the present invention relates to the finding that a bifunctional molecule, also known herein as a degrader, can be used as a catalytic agent to non-covalently bind to and cleave a target nucleic acid molecule.
• the degrader disclosed herein bind to a target nucleic acid through non-covalent interactions. Accordingly, the degrader does not require incorporation of a click- reactive group into the target nucleic acid.
• the selective cleavage of target nucleic acid molecules using the degraders described herein may be useful in epigenetic and epitranscriptomic analysis, bifunctional mapping, as well as in therapy, for example antibacterial and anti-viral therapy, and additionally anti-cancer.
• the degrader has formula (I):
• the cleavage group is imidazole (1 ,3-diazole), which may optionally be substituted as described herein.
• the imidazole cleavage group of the degrader is capable of reacting with a target nucleic acid molecule to cleave the target nucleic acid molecule.
• the imidazole is capable of abstracting a proton from the hydroxyl group at the 2’ position of a ribose sugar.
• the imidazole is capable of binding 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)).
• Imidazole is a basic group. That is, imidazole is capable of accepting a hydrogen cation (H+). The imidazole is also capable of donating an electron pair.
• the basicity of a group may be quantitatively assessed using the pKa of the associated conjugate acid. That is, the basicity of basic group [Ba] may be assessed using the pKa of the conjugate acid [BaH] + .
• the pKa of the conjugate acid may be known or it may be determined using standard techniques, such as acid-base titration.
• the inventors believe the basic residues having a conjugate acid with a pKa value above a certain threshold, such as a pKa of 6.5 or greater, for example 6.8 or greater, are capable of deprotonating the hydroxyl group at the 2’ position of a ribose sugar in order to permit cleavage of the phosphodiester backbone within a target nucleic acid.
• Imidazole has a pKa close to 7.
• Imidazole comprises a nitrogen atom having a lone electron pair. Groups comprising a nitrogen atom having a lone electron pair are typically capable of coordinating copper. Imidazole is known to chelate copper.
• the imidazole may be unsubstituted, or it may be substituted by one, two or three C1-6 alkyl groups, which may be the same or different.
• alkyl group is monovalent saturated hydrocarbon group.
• the alkyl group may be a C1-6 alkyl group, for example CM alkyl group.
• the prefix e.g. Ci-e
• the alkyl group may be linear or branched.
• C1-6 linear alkyl groups include methyl (-Me), ethyl (-Et), n-propyl (-nPr), n-butyl (-nBu), n-pentyl (-Amyl) and n-hexyl.
• C1-6 branched alkyl groups include iso-propyl (-iPr), iso-butyl (-iBu), sec-butyl (-sBu), tert-butyl (-tBu), iso-pentyl, neo-pentyl, iso-hexyl and neo-hexyl.
• the imidazole is selected from the groups represented by formula (C-l) to (C-lll): formula (C-l) formula (C-ll) formula (C-lll) where: R 1 , R 2 and R 3 each independently represent a hydrogen atom or a C1-6 alkyl group;
• R N represents a hydrogen atom or a C1-6 alkyl group
• the degrader may be referred to as an imidazole degrader.
• R 1 , R 2 , R 3 and R N each independently represent a hydrogen atom or a CM alkyl group.
• the cleavage group is a group represented by formula (C-l).
• R 1 , R 2 , and R 3 each independently represent hydrogen.
• the cleavage group is an unsubstituted imidazole group. That is, the cleavage group is represented by formula (C-IV): formula (C-IV) where * represents the attachment position with the remainder of the degrader (typically the linker unit L).
• the imidazole group When non-covalently bound to the target nucleic acid molecule through the linker and binding 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.
• the imidazole group of the bound degrader may abstract a proton from the 2’OH position on the nucleic acid molecule leading to cleavage of a phosphodiester bond in the target nucleic acid molecule.
• the imidazole group may form a copper complex which cleaves a phosphodiester bond in the target nucleic acid molecule.
• the linker L of the degrader comprises a group for connection (i.e. covalent connection) of the cleavage group (C) to the non-covalent binding group (B).
• Suitable linkers are well known in the art.
• the linker comprises a divalent group in which one of the free valencies forms part of a single bond to the cleavage group (C) and the remaining free valency forms part of a single bond to the non-covalent binding group (B).
• the linker is a stable linker. That is, the linker comprises a group that is not substantially cleaved or degraded in vivo.
• a stable linker is typically unreactive at physiological pH, and not substantially degraded by enzymatic action in vivo.
• the linker is a flexible linker. That is, the linker permits the cleavage group (C) and binding group (B) to move relative to each other with a large degree of freedom.
• Typical linkers comprise groups selected from alkylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene and heteroarylene.
• Mixed linkers comprising different groups in covalent connection, such as alkylene-arylene (aralkylene) and heteroalkylene-arylene, may be permitted.
• alkylene (alkanediyl) group is a divalent saturated hydrocarbon group in which the two free valencies each form part of a single bond to an adjacent atom.
• the alkylene group may be a C1-6 alkylene group, for example, a CM, C1-3 or a C1-2 alkylene group.
• the prefix e.g. Ci-e
• the alkylene group may be linear or branched.
• linear alkylene groups examples include methanediyl (methylene bridge), ethane-1 ,2-diyl (ethylene bridge), propane-1 ,3-diyl, butan-1 ,4-diyl, pentan-1 ,5-diyl and hexan-1 ,6-diyl.
• branched alkylene groups include ethane- 1 ,1-diyl and propane-1 , 2-diyl.
• a heteroalkylene group is an alkylene group in which one or more carbon atoms is replaced with a heteroatom, for example N, O and S.
• the heteroalkylene group may be a C1-6 heteroalkylene group, for example, a C1-4, C1-3 or a C1-2 heteroalkylene group.
• the prefix e.g. C1-6
• the heteroalkylene group 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), ethylenimine (e.g.
• branched heteroalkylene groups include those derived from propylene glycol (e.g. polypropylene glycol PPG).
• nitrogen atom is present in a heteroalkylene group, that nitrogen atom may be unsubstituted (NH) or optionally substituted with an alkyl group, such as a C1-4 alkyl group.
• sulfur atom is present in a heteroalkyl group, that sulfur atom may be S, S(O) or S(O)2.
• a cycloalkylene group is a divalent saturated hydrocarbon group which comprises a ring in which all of the ring atoms are carbon atoms, and in which the two free valencies each form part of a single bond to an adjacent atom.
• the cycloalkylene group may be a C5-6 cycloalkylene group.
• the prefix e.g. C5-6
• the cycloalkylene group may be monocyclic. Examples of monocylic cycloalkylene groups include 1 ,3-cyclopentylene and 1 ,4-cyclohexylene.
• the heterocycloalkylene group may be a C5-6 heterocycloalkylene group.
• the prefix e.g. C5-6 denotes the number or range of ring atoms, whether carbon atoms or heteroatoms.
• the heterocycloalkylene group may be monocyclic.
• nitrogen atom is present in a heteroalkylene group
• nitrogen atom may be unsubstituted (NH) or optionally substituted with an alkyl group, such as a C1-4 alkyl group.
• sulfur atom is present in a heteroalkyl group, that sulfur atom may be S, S(O) or S(O) 2 .
• An arylene group is a divalent hydrocarbon group comprising an aromatic ring in which all of the ring atoms are carbon atoms, and in which the two free valencies each form part of a single bond to an adjacent atom.
• the arylene group may be a Ce-io arylene group. In this context, the prefix (e.g. Ce-w) denotes the number or range of ring atoms.
• the arylene group may be monocyclic, or it may comprise two or more rings. Examples of monocyclic arylene groups include 1 ,4-phenylene. Examples of bicyclic arylene groups include 2,6-naphthylene.
• the heteroarylene group may be a Ce-w heteroarylene group.
• the prefix e.g. Ce-io
• the heteroarylene group may be monocyclic, or it may comprise two or more rings. Examples of monocyclic heteroarylene groups include pyrrolylene and pyridylene.
• Preferred linkers comprise groups selected from alkylene and heteroalkylene. More preferred linkers comprise heteroalkylene groups. Even more preferred linkers comprise alkylene ether groups. Most preferred linkers comprise ethylene oxide groups (e.g. derived from polyethylene glycol, PEG). In a preferred embodiment, the linker is or comprises a group represented by formula (L-l): formula (L-l) where:
• L 1 is a covalent bond or a C1-2 alkylene group
• L 2 is a C1-6 alkylene group or a C1-6 heteroalkene group
• L 3 is a C1-6 alkylene group; n is 1 to 8;
• Suitable C1-2 alkylene groups include methylene (methanediyl), ethylene (ethane-1 ,2-diyl).
• Suitable C1-6 alkylene groups include methylene (methanediyl), ethylene (ethane-1 ,2-diyl), propylene (propane-1 , 3-diyl), butylene (butan-1 ,4-diyl), pentylene (pentan-1 ,5-diyl) and hexylene (hexan-1 ,6-diyl).
• L 1 is a covalent bond or methylene.
• L 3 is CM alkylene. More preferably L 3 is ethylene.
• n 2 to 5.
• Suitable C1-6 heteroalkene groups include alkylene ether group such as ethylene oxide (-CH2CH2O-), propylene oxide (-CH2CH2CH2O-) and tetramethylene oxide (-CH2CH2CH2CH2O-).
• L 2 is ethylene oxide.
• the linker is or comprises a group represented by formula (L-ll): formula (L-ll) where L 1 , L 3 , n, * and ** are as described for formula (L-l), and the same preferences apply.
• the linker is or comprises a group represented by formula (L- III): formula (L-lll) where:
• L 4 is a C1-6 alkylene group
• L 5 is a C1-6 alkylene group or a C1-6 heteroalkene group
• L 6 is a covalent bond or a C1-2 alkylene group; m is 1 to 8;
• L 4 is CM alkylene. More preferably L 3 is ethylene.
• L 6 is methylene or ethylene.
• m is 2 to 5.
• L 5 is ethylene oxide.
• the linker is or comprises a group represented by formula (L-IV): formula (L-IV) where L 4 , L 6 , m, * and ** are as described for formula (L-lll), and the same preferences apply.
• the binding group of the degrader comprises a group capable of binding to the target nucleic acid molecule.
• the binding group binds to the target nucleic acid molecule through non- covalent bonding.
• Certain small-molecule ligands including are known to non-covalently bind to nucleic acids, and thus may form the basis of the non-covalent binding group.
• the non-covalent binding group is not a polynucleotide (e.g. nucleic acid) group.
• the covalent binding group is not or does not comprise a nucleotide.
• the non-covalent binding group is not an antibody.
• the non-covalent binding group has molecular weight of 1 ,000 kDa or less.
• the non-covalent binding group has a molecular weight of 800 kDa or less.
• the non-covalent binding group binds to a secondary or tertiary structure within the target nucleic acid.
• Suitable secondary or tertiary structures include quadruplexes, pseudoknots, triplexes, tetraloops, step-loops and hairpin loops.
• the non-covalent binding group binds to a quadruplex or pseudoknot.
• the non-covalent binding group selectively binds to a secondary or tertiary structure within the target nucleic acid.
• the non-covalent binding group preferentially binds to a secondary or tertiary structure within the target nucleic acid in comparison to linear or unstructured nucleic acid.
• the non-covalent binding group selectively binds to a quadruplex or pseudoknot.
• the non-covalent binding group selectively binds to a ribonucleic acid (RNA).
• RNA ribonucleic acid
• the non-covalent binding group may be known as a non-covalent RNA binding group.
• the non-covalent binding group may bind to the target nucleic acid through electrostatic interactions, such as ionic interactions, hydrogen-bonding and halogen bonding; van der Waals interactions such as permanent dipole-dipole interactions, dipole-induced dipole interactions, and induced dipole-induced dipole interactions; and TT-effects such as TT-TT interactions, TT-cation interactions and polar-TT interactions.
• electrostatic interactions such as ionic interactions, hydrogen-bonding and halogen bonding
• van der Waals interactions such as permanent dipole-dipole interactions, dipole-induced dipole interactions, and induced dipole-induced dipole interactions
• TT-effects such as TT-TT interactions, TT-cation interactions and polar-TT interactions.
• the non-covalent binding group may be based on the following small molecule nucleic acid binding molecules:
• the non-covalent binding group may be attached to the linker at any suitable position.
• a heteroatom such as O or NH
• the binding group is selected from formulae (B-l) to (B-lll).
• the interaction between the degrader and the target nucleic acid can be quantified using the dissociation constant (k D ).
• the dissociation constant between a degrader comprising a given non-covalent binding group and a nucleic acid may be known or it may be determined using standard techniques such as surface plasmon resonance (SPR), for example Biacore (Santos, et al., 2021 ). Suitable systems for measuring the dissociation constant include Biacore T200.
• the degrader binds to the target nucleic acid with a dissociation constant (ko) of 10,000 nM or less, such as determined by SPR.
• ko dissociation constant
• the degrader binds to the target nucleic acid with a ko of 1 ,000 nM or less, more preferably 500 nM or less, even more preferably 200 nM or less, and most preferably 100 nM or less.
• the non-covalent binding group of degrader typically binds to a secondary or tertiary structure within the target nucleic acid. Accordingly, the degrader typically binds to a secondary or tertiary structure with a dissociation constant (ko) of 10,000 nM or less, such as determined by SPR. Preferably, the degrader binds to the secondary or tertiary structure with a ko of 1 ,000 nM or less, more preferably 500 nM or less, even more preferably 200 nM or less, and most preferably 100 nM or less.
• ko dissociation constant
• the degrader binds to a quadruplex with a dissociation constant (ko) of 10,000 nM or less, such as determined by SPR.
• the degrader preferably binds to the quadruplex with a ko of 1 ,000 nM or less, more preferably 500 nM or less, even more preferably 200 nM or less, and most preferably 100 nM or less.
• the degrader binds to a pseudoknot with a dissociation constant (ko) of 10,000 nM or less, such as determined by SPR.
• the degrader preferably binds to the pseudoknot with a ko of 1 ,000 nM or less, more preferably 500 nM or less, even more preferably 200 nM or less, and most preferably 100 nM or less.
• the degrader binds to the target nucleic acid with a dissociation constant (ko) of 100 mM or less, such as determined by microscale thermophoresis (MST).
• the ko may be 50 mM or less, such as 25 mM or less, such as 20 mM or less, such as 15 mM or less, such as 10 mM or less, such as 9 mM or less, such as 8 mM or less, such as 7 mM or less, such as 6 mM or less, such as 5 mM or less, such as 4 mM or less, such as 3 mM or less, such as 2 mM or less.
• the degrader binds to the target nucleic acid with a dissociation constant (ko) of 10 mM or less, more preferably 5 mM or less.
• the k D may be determined using standard techniques such as by microscale thermophoresis (MST), such as described in the Examples below. Measurements may be carried out using a fluorescence-tagged nucleic acid, such as FAM-functionalised nucleic acid which is incubated with the degrader.
• the nucleic acid may be at a concentration of 50 nM, and may be provided in a buffer such as a HEPES buffer at pH 7.4.
• the degrader may be tested in serial dilution, such as at a highest concentration of up to 8 mM, such as up to 250 pM. Measurements may be made at a temperature of 25 °C. MST measurements may be carried out at an MST power of 30%. Suitable systems for measuring the dissociation constant include NanoTemper Monolith NT.115.
• the degrader binds to a secondary or tertiary structure within the target nucleic acid with a ko of 20 nM or less, more preferably 15 nM or less, more preferably 10 mM or less, more preferably 5 mM or less.
• the degrader binds to a pseudoknot with a dissociation constant (ko) of 20 mM or less, more preferably 15 mM or less, more preferably 10 mM or less, even more preferably 5 mM or less, and most preferably 2 mM or less.
• ko dissociation constant
• the interaction between the degrader and the target nucleic acid can be quantified using the half maximal effective constant (ECso).
• the ECso may be the same as the dissociation constant (ko) or the ECso may be different. Preference for the ECso is as described above for the the dissociation constant (ko).
• the half maximal effective constant (ECso) between a degrader comprising a given non-covalent binding group and a nucleic acid may be known, or it may be determined experimentally, for example using a fluorescence quenching assay (see e.g.
• Suitable systems for measuring the ECso include systems for measuring fluorescence, such as a plate reader, such as BMG CLARIOstar.
• the nucleic acid may be a fluorescence-tagged nucleic acid which may be treated with the degrader for a time period such as 40 minutes, such as with incubation at a temperature of 4 °C. Measurements may be carried out at a temperature of 25 °C.
• the concentration of the nucleic acid may be 50 nM, and optionally the nucleic acid may be in a buffer such as a HEPES buffer at pH 7.4.
• the concentration of the degrader may be up to 10 pM, for example where the degrader is tested in serial dilution from around 2 nM to around 10 pM.
• the degrader binds to the target nucleic acid with a half maximal effective constant (ECso) of 10,000 nM or less, such as determined by a fluorescence quenching assay.
• ECso half maximal effective constant
• the degrader binds to the target nucleic acid with an ECso of 1 ,000 nM or less, more preferably 500 nM or less, even more preferably 200 nM or less, and most preferably 100 nM or less.
• the non-covalent binding group of degrader typically binds to a secondary or tertiary structure within the target nucleic acid. Accordingly, the degrader typically binds to a secondary or tertiary structure with a half maximal effective constant (ECso) of 10,000 nM or less. Preferably, the degrader binds to the secondary or tertiary structure with an ECso of 1 ,000 nM or less, more preferably 500 nM or less, even more preferably 200 nM or less, and most preferably 100 nM or less.
• ECso half maximal effective constant
• the degrader binds to a quadruplex with a half maximal effective constant (ECso) of 10,000 nM or less, such as determined by a fluorescence quenching assay as described herein.
• the degrader preferably binds to the quadruplex with a ECso of 1 ,000 nM or less, more preferably 500 nM or less, even more preferably 200 nM or less, and most preferably 100 nM or less.
• the degrader binds to a pseudoknot with a half maximal effective constant (ECso) of 10,000 nM or less, such as determined by a fluorescence quenching assay as described herein.
• the degrader preferably binds to the pseudoknot with a ECso of 1 ,000 nM or less, more preferably 500 nM or less, even more preferably 200 nM or less, and most preferably 100 nM or less.
• the degrader binds to the target nucleic acid with a half maximal effective constant (ECso) of 100 mM or less, such as 50 mM or less, such as 25 mM or less, such as 20 mM or less, such as 15 mM or less, such as 10 mM or less, such as 9 mM or less, such as 8 mM or less, such as 7 mM or less, such as 6 mM or less, such as 5 mM or less, such as 4 mM or less, such as 3 mM or less, such as 2 mM or less.
• the degrader binds to the target nucleic acid with a half maximal effective constant (ECso) of 10 mM or less, more preferably 5 mM or less.
• the degrader binds to a secondary or tertiary structure within the target nucleic acid with a half maximal effective constant (ECso) of 20 nM or less, more preferably 15 nM or less, more preferably 10 mM or less, more preferably 5 mM or less.
• ECso half maximal effective constant
• the degrader binds to a pseudoknot with a half maximal effective constant (ECso) of 20 mM or less, such as 15 mM or less, more preferably 10 mM or less, even more preferably 5 mM or less, and most preferably 2 mM or less.
• ECso half maximal effective constant
• the non-covalent binding group of the degrader preferably selectively binds to a secondary or tertiary structure within the target nucleic acid.
• the binding selectivity can be quantified using the ratio between the dissociation constant for binding to a given secondary or tertiary structure in compression to the dissociation constant for binding to linear or unstructured nucleic acid, such as linear or unstructured RNA.
• the comparison linear or unstructured nucleic acid is prepared by mutating one or more residues within the secondary or tertiary structure of interest such that the secondary or tertiary structure no longer forms, while the remainder of the sequence is maintained.
• the selectivity of binding to an RNA G quadruplex can be assessed by using comparison RNA in which one or more GGG motifs are exchanged for AUC motifs.
• the binding selectivity between a given secondary or tertiary structure and linear or unstructured nucleic acid is 5:1 or greater.
• 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, even more preferably 50:1 or greater, and most preferably 100:1 or greater.
• the binding selectivity between a quadruplex and linear or unstructured nucleic acid is 5:1 or greater.
• selectivity between a quadruplex and linear or unstructured nucleic acid is 10:1 or greater, more preferably 20:1 or greater, even more preferably 50:1 or greater nM or less, and most preferably 100:1 or greater.
• the binding selectivity between a quadruplex and linear or unstructured nucleic acid is 5:1 or greater.
• selectivity between a quadruplex and linear or unstructured nucleic acid is 10:1 or greater, more preferably 20:1 or greater, even more preferably 50:1 or greater nM or less, and most preferably 100:1 or greater.
• the degrader is selected from compounds Deg-I to Deg-V.
• the degrader of formula (I) may be provided in free base form.
• the degrader of formula (I) may be provided in the form of a salt, preferably a pharmaceutically acceptable salt.
• degraders disclosed herein may be provided as salts in a protonated form together with a suitable counter anion.
• Suitable counterions include both organic and inorganic anions.
• Example of inorganic anions include those derived from inorganic acids, including chloride (Cl-), bromide (Br), iodide (I-), sulfate (SO 4 2 '), sulfite (SOs 2 '), nitrate (NOs'), nitrite (NO?'), phosphate (PO4 3 '), and phosphite (POa 3 ').
• organic anions examples include 2-acetoxybenzoate, acetate, ascorbate, aspartate, benzoate, camphorsulfonate, cinnamate, citrate, edetate, ethanedisulfonate, ethanesulfonate, formate, fumarate, gluconate, glutamate, glycolate, hydroxymalate, carboxylate, lactate, laurate, lactate, maleate, malate, methanesulfonate, oleate, oxalate, palmitate, phenylacetate, phenylsulfonate, propionate, pyruvate, salicylate, stearate, succinate, sulfanilate, tartarate, toluenesulfonate, and valerate.
• suitable polymeric organic anions include those derived from tannic acid and carboxymethyl cellulose.
• degraders disclosed herein may be provided as salts in a deprotonated form together with a suitable counter cation.
• Suitable counterions include both inorganic and organic cations.
• suitable inorganic cations include alkali metal ions such as Na + and K + , alkaline earth cations such as Ca 2+ and Mg 2+ , and other cations such as Al 3+ .
• suitable organic cations include the ammonium ion (i.e., NH 4 + ) and substituted ammonium ions (e.g., NHaFT, NH?R2 + , NHR3 + , NR 4 + ).
• 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).
• TEMED tetramethylethylenediamine
• An example of a common quaternary ammonium ion is N(CH3) 4 + .
• the degrader of formula (I) may be provided in the form of a solvate (a complex of solute (e.g., compound, salt of compound) and solvent).
• solvates include hydrates, for example, a mono-hydrate, a di-hydrate and a tri-hydrate.
• the degrader of formula (I) may be provided in desolvated form, for example, in dehydrated form.
• the invention provides a method for cleaving a target nucleic acid molecule.
• the method comprises: contacting the target nucleic acid molecule with a degrader of formula (I): C-L-B (I) where -C is a cleavage group that is imidazole, optionally substituted with 1 to 3 C1-6 alkyl groups, -L- is a linker and -B is a non-covalent binding group, such that the degrader non-covalently binds to the target nucleic acid molecule, and; allowing the degrader to cleave the target nucleic acid molecule bound thereto.
• the target nucleic acid molecule may be contacted with the degrader in solution.
• the target nucleic acid molecule may be contacted with the degrader within a cell (i.e. intracellularly).
• the cell may be in vitro and may be an isolated cell, for example an isolated cell line or cell isolated from an individual (from a tissue sample, such as a biopsy).
• Suitable cells may include mammalian, preferably human cells.
• Cells may include somatic and germ-line cells and may be at any stage of development, including fully or partially differentiated cells or non-differentiated or pluripotent cells, including stem cells, such as adult or somatic stem cells, foetal stem cells or embryonic stem cells.
• stem cells such as adult or somatic stem cells, foetal stem cells or embryonic stem cells.
• cells may include neural cells, including neurons and glial cells, contractile muscle cells, smooth muscle cells, liver cells, hormone synthesising cells, sebaceous cells, pancreatic islet cells, adrenal cortex cells, fibroblasts, keratinocytes, endothelial and urothelial cells, osteocytes, and chondrocytes.
• cells may be associated with a disease condition, for example cancer cells, such as carcinoma, sarcoma, lymphoma, blastoma or germ-line tumour cells, and cells with the genotype of a genetic disorder, such as Huntington’s disease, cystic fibrosis, sickle cell disease, phenylketonuria, Down syndrome or Marfan syndrome.
• a disease condition for example cancer cells, such as carcinoma, sarcoma, lymphoma, blastoma or germ-line tumour cells, and cells with the genotype of a genetic disorder, such as Huntington’s disease, cystic fibrosis, sickle cell disease, phenylketonuria, Down syndrome or Marfan syndrome.
• the target nucleic acid molecule may be an endogenous nucleic acid that is present in the cell.
• the degrader may be an exogenous molecule.
• a method may comprise introducing the degrader into the cell and allowing it to bind to the target nucleic acid molecule.
• the target nucleic acid molecule may be a DNA or RNA molecule.
• Suitable target RNA molecules may include mRNA and long non-coding RNA (IncRNA).
• the RNA molecule may comprise intronic and intergenic regions.
• the target nucleic acid molecule may comprise a secondary or tertiary structure. Suitable secondary and tertiary structures include quadruplexes, pseudoknots, tetraloops, step-loops and hairpin loops. Preferably, the target nucleic acid molecule comprises a quadruplex or pseudoknot.
• a method for cleaving a target nucleic acid comprising a secondary or tertiary structure may comprise: contacting the target nucleic acid molecule with a degrader of formula (I):
• C-L-B (I) where -C is a cleavage group that is imidazole, optionally substituted with 1 to 3 C1-6 alkyl groups, -L- is a linker and -B is a non-covalent binding group that interacts with the secondary or tertiary structure to non-covalently bind the degrader to the target nucleic acid molecule, and; allowing the degrader to cleave the target nucleic acid molecule bound thereto.
• -C is a cleavage group that is imidazole, optionally substituted with 1 to 3 C1-6 alkyl groups
• -L- is a linker
• -B is a non-covalent binding group that interacts with the secondary or tertiary structure to non-covalently bind the degrader to the target nucleic acid molecule, and; allowing the degrader to cleave the target nucleic acid molecule bound thereto.
• the secondary or tertiary structure is a quadruplex.
• the method may comprise: contacting a target nucleic acid molecule comprising a quadruplex with a degrader of formula (I):
• C-L-B (I) where -C is a cleavage group that is imidazole, optionally substituted with 1 to 3 C1-6 alkyl groups, -L- is a linker and -B is a non-covalent binding group that interacts with the quadruplex to non-covalently bind the degrader to the target nucleic acid molecule, and; allowing the degrader to cleave the target nucleic acid molecule bound thereto.
• the secondary or tertiary structure is a pseudoknot.
• the method may comprise: contacting a target nucleic acid molecule comprising a pseudoknot with a degrader of formula (I):
• C-L-B (I) where -C is a cleavage group that is imidazole, optionally substituted with 1 to 3 C1-6 alkyl groups, -L- is a linker and -B is a non-covalent binding group that interacts with the pseudoknot to non-covalently bind the degrader to the target nucleic acid molecule, and; allowing the degrader to cleave the target nucleic acid molecule bound thereto.
• the degrader When non-covalently bound to the target nucleic acid molecule, the degrader cleaves the target nucleic acid. Typically, the degrader cleaves one or more phosphodiester bonds in the target nucleic acid.
• the cleavage group When non-covalently bound to the target nucleic acid molecule, the cleavage group may abstract a protein from the 2’OH of a nucleotide in the target nucleic acid. Cleavage of the phosphodiester backbone may occur by intramolecular attack on the phosphate group at the 3’ position.
• the cleavage When non-covalently bound to the target nucleic acid molecule, the cleavage may bind to one or more transition metals (e.g. copper).
• the degrader may cleave the target nucleic acid through copper-mediated nucleic acid degradation.
• Binding of the degrader to the target nucleic acid may proceed via an intermediate species. That is, a target nucleic acid molecule may be cleaved as described herein by a method that comprises binding the target nucleic acid molecule to a degrader to produce an intermediate having the formula:
• C1-6 alkyl groups -L- is a linker, -B is a non-covalent binding group, ⁇ is a non-covalent interaction and NA is the target nucleic acid; and allowing the degrader to cleave the target nucleic acid molecule.
• a method may comprise identifying the target nucleic acid molecule. This may be useful for example in the mapping of sites comprising secondary or tertiary structures within the nucleic acid.
• the method may also comprise determining the abundance or amount of one or more nucleic acid molecules in a population of nucleic acids. A reduction in the abundance or amount of a nucleic acid molecule in the population relative to control is indicative that the nucleic acid molecule is the target nucleic acid molecule that has been selectively cleaved by the degrader.
• a suitable control may be a population of nucleic acids that has not been treated with the degrader.
• the invention provides a method for identifying a secondary or tertiary structure within a target nucleic acid molecule, the method comprising: providing first and second populations of nucleic acid molecules, each population comprising the target nucleic acid molecule; introducing into the first population of nucleic acid molecules a degrader of formula (I): C-L-B (I) where -C is a cleavage group that is imidazole, optionally substituted with 1 to 3 C1-6 alkyl groups , -L- is a linker and -B is a non-covalent binding group, such that the degrader non-covalently binds to the target nucleic acid molecule; allowing the degrader to cleave the target nucleic acid molecule present in the first population; and identifying nucleic acid molecules which are present in a reduced amount in the first population relative to the second population.
• Preferred embodiment of the degrader of formula (I) are set out above.
• the non-covalent binding group may bind to a secondary or tertiary structure within the target nucleic acid molecule.
• Suitable secondary or tertiary structures include quadruplexes, pseudoknots, tetraloops, step-loops and hairpin loops.
• the secondary or tertiary structure is a quadruplex or pseudoknot.
• the first and second populations of nucleic acid molecules may independently be isolated (ex vivo) populations of nucleic acid molecules. Alternatively, one or more of the populations of nucleic acid molecules may be present within a cell.
• the method may comprise extracting the total nucleic acid, such as total DNA or total RNA, from a cell.
• the nucleic acid may be further analysed, for example to determine the abundance or amount of one or more nucleic acid molecules.
• the extracted total nucleic acid may be sequenced, and the sequence reads analysed.
• RNA-seq RNA-sequencing
• NGS next generation
• nanopore sequencing RNA-sequencing
• a method may comprise extracting nucleic acid molecules from the cell, sequencing the extracted nucleic acid molecules and determining the number of sequence reads (i.e. read count) for each extracted nucleic molecule to determine the abundance or amount of each nucleic acid molecule in the cell.
• the raw read count may be normalised and expressed in 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.
• the selective cleavage of a target nucleic acid molecules by the degrader described above may alter downstream effects of the target nucleic acid molecule. This may be useful, for example, in the treatment or prophylaxis of a disease mediated by the target nucleic acid molecule.
• the present invention provides a degrader 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 (e.g., a disease).
• a method of treatment for example, a method of treatment of a disorder (e.g., a disease), comprising administering a therapeutically-effective amount of a degrader of formula (I) to a subject in need of treatment.
• Another aspect of the present invention pertains to use of degrader of formula (I) in the manufacture of a medicament for use in treating a disorder (e.g., a disease).
• the medicament comprises the degrader of formula (I).
• the treatment is treatment of a bacterial or viral infection.
• the viral infection is an infection with an RNA virus (e.g., a virus in which the viral genome comprises single- or double-stranded RNA).
• an RNA virus e.g., a virus in which the viral genome comprises single- or double-stranded RNA.
• RNA virus e.g., a virus in which the viral genome comprises single- or double-stranded RNA.
• Many pathogenic viruses exploit a -1 ribosomal frameshifting as a mechanism for correct translation of proteins and this phenomenon is enabled by secondary RNA structures such as stem-loops and pseudoknots. Accordingly, targeting these secondary RNA structures with a degrader of formula (I) can cleave and inactivate the viral RNA, and treat the viral infection.
• RNA viruses examples include (+)ssRNA viruses such as coronaviruses, picornaviruses and togaviruses; (-)ssRNA viruses such as orthomyxoviruses and rhabdoviruses; and dsRNA viruses such as reoviruses.
• the virus is a (+)ssRNA virus, more preferably a coronavirus.
• coronaviruses include alphacoronaviruses such as transmissible gastroenteritis virus, feline coronavirus, 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, SARS-CoV-2); gammacoronavirus such as avian coronavirus; and deltacoronavirus such as bulbul coronavirus HKU11 and porcine coronavirus HKU15.
• alphacoronaviruses such as transmissible gastroenteritis virus, feline coronavirus, canine coronavirus
• betacoronaviruses such as middle east respiratory syndrome-related coronavirus (MERS-CoV), murine coronavirus (M-CoV) and severe acute respiratory syndrome-related cor
• the bacterial infection may be an infection with a Gram-negative or Gram-positive bacterium.
• Both classes of bacteria contain a bacterial ribosome, which is a riboenzyme comprising both protein and RNA units. Accordingly, targeting the RNA units with a degrader of formula (I) can cleave and inactive the bacterial ribosome, and treat the bacterial infection.
• Gram-negative bacteria examples 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)
• the Gram-negative bacterial species is selected from the group consisting of E. coli, E. cloacae, H. pylori, S. enterica, H. influenzae, K. pneumoniae, L. pneumoniae, L. pneumophila, P. aeruginosa and N. meningitidis.
• 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).
• the Gram-negative bacterial genus is selected from the group consisting of actinomyces, bacillus, Clostridium, corynebacterium, enterococcus, erysipelothrix, listerial nocardia, staphylococcal, and streptococcal.
• the treatment is treatment of a respiratory tract infection, a urinary tract infection, or gastroenteritis.
• the treatment is treatment of cancer.
• the cancer may be a cancer where the associated oncogene is known, or suspected to have, or be suitable for forming, a secondary or tertiary structure, or an oncogene expressing a nucleic acid having a secondary or tertiary structure, such as the quadruplex or pseudoknot structures described herein.
• the disease for treatment may be associated with the expression of, or altered regulation of (such as upregulation), neuroblastoma RAS (NRAS), metastasis associated lung adenocarcinoma transcript 1 (MALAT 1 ), 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), amongst others.
• NRAS neuroblastoma RAS
• MALAT 1 metastasis associated lung adenocarcinoma transcript 1
• EWSR1 EWS RNA Binding Protein 1
• TERRA telomeric repeat containing RNA
• BCL-XL B-cell lymphoma-extra large
• FGFR fibroblast growth factor receptor
• MIR21 MicroRNA 21
• the treatment is administered to a subject in need of treatment.
• the subject in need of treatment may be a chordate, a vertebrate, a mammal, a placental mammal, a marsupial (e.g., kangaroo, wombat), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an a rodent (
• the subject in need of treatment may be an adult or juvenile.
• the subject in need of treatment is a human, more preferably an adult human.
• the subject in need of treatment is a non-human animal used in laboratory research.
• the non-human animal is a rodent (e.g., a guinea pig, a hamster, a rat, a mouse).
• the treatment is administered by any convenient route of administration, whether systemically/peripherally or topically (i.e. , at the site of desired action).
• the routes of administration may be oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, sub
• the degrader of formula (I) is administered alone.
• a pharmaceutical formulation e.g., composition, preparation, medicament
• the degrader in a pharmaceutical formulation (e.g., composition, preparation, medicament) comprising at least one degrader as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.
• the formulation may further comprise other active agents, for example, other therapeutic or prophylactic agents.
• the present invention further provides pharmaceutical compositions, and methods of making a pharmaceutical composition
• a pharmaceutical composition comprising mixing at least one degrader described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the compound.
• pharmaceutically acceptable pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, 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.
• Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 5th edition, 252005.
• the formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the degrader with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly mixing the degrader with a carrier (e.g., a liquid carrier, a finely divided solid carrier, etc.), and then shaping the product, if necessary.
• a carrier e.g., a liquid carrier, a finely divided solid carrier, etc.
• the formulation may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof.
• Formulations may suitably be in the form of liquids, solutions (e.g., aqueous, nonaqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, mouthwashes, drops, tablets (including, e.g., coated tablets), granules, powders, losenges, pastilles, capsules (including, e.g., hard and soft gelatin capsules), cachets, pills, ampoules, boluses, suppositories, pessaries, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols.
• solutions e.g., aqueous, nonaqueous
• suspensions e.g., aqueous, non-aqueous
• emulsions
• Formulations may suitably be provided as a patch, adhesive plaster, bandage, dressing, or the like which is impregnated with one or more compounds and optionally one or more other pharmaceutically acceptable ingredients, including, for example, penetration, permeation, and absorption enhancers. Formulations may also suitably be provided in the form of a depot or reservoir.
• the degrader may be dissolved in, suspended in, or mixed with one or more other pharmaceutically acceptable ingredients.
• the compound may be presented in a liposome or other microparticulate which is designed to target the compound, for example, to blood components or one or more organs.
• the treatment comprises administering a therapeutically-effective amount of a degrader of formula (I) to a subject in need of treatment.
• appropriate dosages of the degraders described herein, and compositions comprising the degraders can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects.
• the selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular degrader, the route of administration, the time of administration, the rate of excretion of the degrader, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the disorder, and the species, sex, age, weight, condition, general health, and prior medical history of the patient.
• the amount of degrader and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.
• Administration can be effected 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 dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.
• a suitable dose of the degrader is in the range of about 10 pg to about 250 mg (more typically about 100 pg to about 25 mg) per kilogram body weight of the subject per day.
• the compound is a salt, an ester, an amide, a prodrug, or the like
• the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.
• RNA oligo (20 pM) was added to a pH 7.5 HEPES (20 mM) buffer supplemented with KCI (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 mixtures were analyzed by LC-MS or gel electrophoresis.
• Oligonucleotides were analyzed by LC-MS following the method of Mikutis et al., 2020.
• Oligomers were analysed using a Xevo G2-S TOF mass spectrometer coupled to an Acquity UPLC system using an Acquity UPLC BEH C18 1.7pm column.
• the system utilises electronspray (ESI) ionisation.
• 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, with a flow rate of 0.200 mL/min.
• Calibration curves for the RNA species were based either on A260 or intensities of specified negative m/z signals. Intensities of integrated peaks were calculated using native modules of KNIME software platform (33).
• SARS-CoV-2 stocks used to infect Vero CCL-81 cells were established from passage 4 of SARS-CoV-2 isolated from a Portuguese patient (internal reference: 606_IMM ID_5452) at approximately 1.7x10 6 PFU/mL, after 4 days in Vero CCL-81 culture.
• Stock titers were calculated by plaque assay. Briefly, approximately 8x10 5 CCL-81 cells/well were seeded in 6-well plates and allowed to grow to confluence for 24 h. Medium was removed, and 500 pL of 10-fold serial dilutions of virus-containing supernatants were adsorbed in duplicate for 1 h, at 37 °C. Plates were rocked manually to redistribute inoculum every 15 minutes.
• CMC carboxymethylcellulose
• Vero CCL-81 cells at 80% confluency were incubated with 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 harvested.
• SARS-CoV-2 RNA 500 ng was incubated with or without 100 pM of MTDB-deg 16a in 1x HEPES buffer for 2 h at 37 °C with mild agitation. The samples were then prepared for sequencing following the manufacturer’s protocol for Direct RNA Sequencing (SQK-RNA002, ONT). The prepared libraries were loaded on FLO-MIN 106D flow cells (ONT) and sequenced on a MinlON Mk1C device (ONT). Genomic sequence of the Wuhan-hu1 strain of SARS-CoV-2 (GenBank: MN908947.3) and the genomic annotation (NC_045512.2) were downloaded from the NCBI database.
• SQK-RNA002, ONT Direct RNA Sequencing
• Sequence reads were aligned to the Wuhan-hu1 genome using minimap2 (Li et al., 2018). with parameters “-ax splice -N32 -un -k13”. CIGAR strings of the alignments were processed by customized scripts. Reads were flagged as leader if the splice junction within the read starts between the first 60-120 bp of the genome. Reads were assigned to individual transcript if it covers more than 90% of the annotated transcript or more than 90% or the read sequence lies within the transcript.
• MTDB-deg 16a Increasing concentrations of MTDB-deg 16a (ranging from 0.07 to 25 pM) were tested to determine the 50% inhibitory concentration (IC50).
• Vehicle (H2O) control and control molecules were included in parallel. Cells were seeded in 96 well-plates at approximately 40% confluency 24 h before infection. MTDB-deg 16a, MTDB or TDB-deg 16b were added either 1 h before infection or 1 h after infection. SARS-CoV-2 cryopreserved stocks, were thawed at room temperature and used to infect cells at a 0.05 multiplicity of infection (MOI). Inhibition of viral growth was measured by harvesting cells at 24 h after infection. Viral growth was assessed by measuring viral loads by PCR targeting the E gene and pseudoknot region.
• MOI multiplicity of infection
• RNA was extracted by using a NZY Viral RNA Isolation kit (NZYtech) and cDNA was synthesized by using NZY First-Strand cDNA Synthesis kit (NZYtech) following manufactures’ instructions.
• the quantitative RT-PCR was then performed by using Powerllp SYBR Green Master Mix (BIO-RAD), set up by Applied Biosystems RT-PCR 7500Fast machine with default SYBR green program.
• the primers used for detecting SARS-CoV-2 were:
• mice Ten to twelve week-old specific pathogen-free Hemizygous for Tg(K18-ACE2)2Prlmn (Strain B6.Cg-Tg(K18-ACE2)2Prlmn/J, the Jackson laboratory strain 034860) mice were used in this study. Mice were intranasally infected with 1 x 10 4 PFU of SARS-CoV-2 in 50 ti ⁇ of PBS. Compounds were administrated intranasally 1 hour pre-infection and 3 hours post-infection.
• MTDB-degrader 16a 25 mg/kg
• MTDB 10 mg/kg
• TDB-degrader 16b 25 mg/kg
• samples were treated with vehicle (H 2 O) or MTDB-deg 16a (6 mM) for 24 hours.
• whole left lung from mice was homogenized in 3 mL of DMEM and 750 pL was transferred to an equal volume of whole cell lysis buffer, supplemented as above. Protein concentrations were accessed using Bradford Assays (BioRad).
• Western Blot experiments were performed using the following antibodies: anti-beta actin (Abeam, 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) (Abeam, ab205719) and goat-anti rabbit HRP (Abeam, ab6721).
• 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 hours, then cooled down to room temperature and stirred over-night. The mixture was washed with brine and dried over MgSC . To remove DMF, toluene was added and the solvent was evaporated under reduced pressure. The crude product was purified via column chromatography (EtOAc: Hexane, 1 :1). Yield: 54% (colourless oil).
• 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 placed under N2 and stirred for 18 h at 55 °C.
• the solvent was removed in vacuo, and the products were purified via flash column chromatography (3:1 Pet. EthenAcOEt to 1 :1 Pet.
• Propargylic chelidamic acid 6 (0.12 g, 0.54 mol) was dissolved in 1.2 mL DCM. Then Ghosez reagent (170 pL, 1.3 mmol) was added dropwise at 0 °C. The orange solution was then stirred at RT for 2h. The 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 DCM and then added dropwise to the mixture. The mixture turned brown-red and was stirred under argon overnight.
• the crude protected product 8a (not shown) was precipitated from hot MeCN as a red solid.
• the red solid 8a was then dissolved in DCM.
• a 2:1 mixture of DCM:TFA was added to acidify the solution and remove the N-boc protection.
• the solvent was removed in vacuo and the product was purified via HPLC (gradient 100% H 2 O, 0.1% FA to 100% MeCN, 0.1% FA). Lyophilization afforded an off-white solid Alkyne-Pyridostatin 8 (51 mg, 86 pmol, 16%).
• Alkyne-Pyridostatin 8 (15 mg, 25 pmol) was dissolved in 2.5 mL of a 2:1 mixture of H 2 O: tBuOH. A solution of copper sulfate pentahydrate (250 pL, 100 mM, 25 pmol) was added followed by a solution of sodium ascorbate (1.3 mL, 100 mM, 130 pmol). The cloudy yellow solution was degassed and stirred for 10 mins. A solution of the appropriate azido-imidazole (3a, 3b or 3-azidopropionic acid) (3.8 mL ,10 mM) was then added. The solution was stirred under argon for 2h. The organic solvent was removed in vacuo. Then the product was purified via HPLC (gradient 100% H 2 O, 0.1 % FA to 100% MeCN, 0.1 % FA). The product was obtained as a white or an off-white solid.
• 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 at N2. The mixture was stirred for 12 h. The solvent was removed in vacuo to give a TFA salt of compound 12a (23.0 g, crude) as a red oil.
• reaction was stirred at room temperature for 1 h, after which they reation mixture turned clear yellow.
• the reaction was quenched with disodium EDTA dihydrate (9.3 mg, 25 pmol, 1 equiv.), the organic solvent was removed in vacuo and the mixture was purified via HPLC.
• the fractions containing the product were lyophilised, resulting in TDB-deg (15b) as a yellow-brown oily solid (10.2 mg, 14 pmol, 54% yield).
• RNA G-guadruplex Two degraders were rationally designed to target the RNA G-guadruplex (rG4) by joining the known G4 binder pyridostatin with azido-imidazoles of different lengths (9A, 9B).
• the copper-induced azide-alkyne cycloaddition (CuAAC) used to join the two components tolerates a vast array of substrates and results in triazole, a bioisostere of an amide and a moiety well-tolerated in biological systems.
• CuAAC copper-induced azide-alkyne cycloaddition
• the two rG4 degraders and a binding control were tested in vitro and in cellular systems.
• rG4 degraders can degrade the genome of SARS-CoV-2 and gain insight into the mechanism of degradation
• viral RNA from VERO cells infected with SARS-CoV-2 and treated it with PDS-deg6 (9A), then analysed it via direct RNA seguencing.
• SARS-CoV-2 genome has several putative rG4s sites (Zhao et al., 2021 ) and was shown to be tightly packed hence most of it in close proximity to an rG4 (Ziv et al., 2020), we hoped our degrader would induce wide-spread damage.
• the viability of cells after 24 hours of incubation with increasing concentrations of the G4-degrader was assessed using a traditional cell viability kit (e.g. CellTiter Blue assay), according to the manufacturer’s protocol.
• a traditional cell viability kit e.g. CellTiter Blue assay
• transgenic K18hACE2 mice (expressing hACE2 protein) were administered PDS-deg4 (9B) and PDS-deg6 (9A) at intranasally 25 mg/kg 40 minutes before infection, and again at 3 h and 18 h after infection (Fig- 4).
• Mice were infected with SARS-CoV-2 intranasally (with 2.5-5 x 10 4 PFUZ mouse in 50 pl of PBS on Day 0) and monitored on a daily basis for body weight, morbidity and mortality (found dead or euthanized in extremis) and clinical signs of infection. On day 5, all mice were sacrificed, and the left lung was collected for viral load quantification by plaque assay. Right lung, heart, liver, kidney and spleen were harvested for histopathological analysis.
• mice had to be sacrificed on Day 0. Organs were collected histopathological analysis. Mice administered with PDS-deg4 (9B) showed 10% loss of body weight in the first day after infection (Fig 4a). However, body weight stabilized between Day 1 and Day 3, after which decreased again at the same rate as vehicle controls. Animals treated with PDS-deg4 (9B) showed a significant decrease in lung viral load (Fig. 4b).
• MTDB-deg (16a) A non-covalent degrader molecule, MTDB-deg (16a), was rationally designed to target an RNA pseudoknot by joining the known pseudoknot binder MTDB with the azido-imidazole 3a (Fig. 5a).
• MTDB contains an ethyl ester moiety, which was exchanged for an amide to increase stability and for use as a handle for the attachment of the degrader.
• azidoimidazole 3a having a linker consisting of 6 PEG subunits, and which we previously found to be more effective than its shorter counterparts for RNA degradation of alkynyl-tagged RNAs (Mikutis et al., 2020).
• CuAAC CuAAC as the reaction to couple binder and degrader fragments because it is robust, easy to carry out, highly modular, and allows us to change the structures of the two fragments without altering the coupling step.
• RNA 69-er with a sequence that corresponds to the coronaviral pseudoknot and thus is predicted to form it.
• TDB-deg (16a) the parent binder molecule that is not capable of degradation
• TDB-deg (16b) a degrader 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 towards the pseudoknot (Fig.
• MTDB-deg (16a) is functional and can cut full-length coronaviral RNA
• MTDB-deg (16a) cuts the viral RNA, and to get a more precise picture of where the cut occurs, we analyzed the cut genomic RNA (gRNA) by direct RNA Nanopore sequencing. As expected, the region around the pseudoknot was affected the most (Fig. 6a). Interestingly, the pseudoknot flanks were more degraded than the pseudoknot itself. Indeed, a study on SARS-Cov-2 RNA interactome found that the region around the frameshifting element forms extensive short- and long-range interactions with the neighboring ORF1a and especially 0RF1 b; it is likely that the proximity of these elements to the pseudoknot enable MTDB-deg (16a) to efficiently cut them (Fig.
• control molecules MTDB and TDB-deg (16b) did not exhibit an antiviral effect, despite MTDB being known to disrupt frameshifting in SARS-CoV-2 (Kelly et al., 2020). Additionally, we found that none of the compounds were cytotoxic to host cells, which indicates that the observed effect on viral replication was a result of the specific antiviral activity of the compound (Fig. 8e). Curiously, the degrader was less active at concentrations higher than 6 pM (Fig. 9c), although no colloidal aggregation that could justify these readouts was seen in dynamic light scattering screens.
• MTDB-deg (16a) is an efficient antiviral agent against SARS-CoV-2 and is specific against coronaviral three-stemmed pseudoknots with irreversible impact.
• a SARS-CoV-2 mouse model of infection was used to determine the in vivo antiviral activity of the MTDB-deg 16a (Fig. 12a).
• Animals administered with MTDB-deg 16a showed a significant reduction of lung viral load relative to the vehicle control group by plaque assay (Fig. 12b).
• Fig. 12b plaque assay
• the chloramphenicol binder site was produced by peptide coupling of propargylic acid to (1R,2R)-(-)-2-Amino-1-(4-nitrophenyl)-1 ,3-propanediol using HATU and DIPEA, and followed by the coupling step using copper click chemistry.
• CuAAC copper-induced azide-alkyne cycloaddition
• the degradation activity was measured in an in vitro assay, targeting the E. coli ribosome. 200 pM ribozyme were incubated with degrader in different concentration, ranging from 15 mM to 0.47 mM, for 18 hours at 37 °C. Evaluation was made by agarose gel analysis.
• RNA was converted to cDNA using SuperScriptTM VILOTM Master Mix.
• the levels of genomic and sub-genomic SARS-CoV-2 transcripts were analysed on a QuantStudioTM 5 real-time PCR machine (Applied Biosystems) using PowerllpTM SYBRTM Green Master Mix (Applied Biosciences) according to the manufacturer’s instructions. All samples, including the template controls, were assayed in triplicates. The relative quantification of target gene expression was performed using the comparative cycle threshold (CT) method.
• CT comparative cycle threshold
• Table 1 Primer sequences used for RT-qPCR study on SARS-CoV-2.
• oligonucleotides were dissolved at an appropriate buffer to a final concentration of 100 nM, then incubated at 95 °C with shaking for
• Cy5-tagged oligonucleotide corresponding to G4 structure found on 5’UTR of NR AS mRNA were dissolved in 20 mM HEPES pH 7.4 buffer supplemented with KCI (100 mM) and MgCI? (10 mM) and plated on a 96-well plate. Oligonucleotides were treated with various concentrations of PDS family ligands as indicated, ranging from 5 nM to 10 pM, or a water vehicle control, followed by 30 minute incubation at 4 °C.
• Fluorescence corresponding to Cy5 fluorophore was then measured for each oligonucleotide-small molecule/vehicle control combination using a plate reader (BMG CLARIOstar) at 25.0 °C. For all the molecules, 12 serial dilutions with a dilution ratio 1 :1 were used, with the highest concentration tested being 10 pM. Fluorescence was normalised to a vehicle control and binding curves for each molecule were obtained via a sigmoidal fit by setting the Hill Slope coefficient to 1.
• FAM-tagged oligonucleotide (final concentration 50 nM) was dissolved in 20 MM HEPES pH 7.4 buffer supplemented with KCI (100 mM) and EDTA (10 mM). Oligonucleotide solutions were then treated with various concentrations of small molecules and analysed via MST according to the manufacturer's instructions (NanoTemper Monolith NT.115). For these MST measurements the following programme was used: 5 seconds laser off, 30 seconds laser on, 5 second laser off; 20% LED (blue) power, 30% MST power; measurements were carried out at 25.0 °C.
• Table 2 Sequences of oligonucleotides used for binding assays.
• This example investigates the interaction between MTDB-deg and a pseudoknot in an infection model of SARS-CoV-2.
• VERO cells infected with SARS-CoV-2 were treated with either with MTDB-deg or a vehicle control, followed by RNA extraction and qPCR analysis on 14 loci of the SARS-CoV-2 genome so to get a broad genomic coverage and reveal which segments are affected the most.
• PDS is a known fluorescence-quencher, thus a fluorescence quenching assay was used to evaluate compounds derived from this scaffold (Di Antonio et al., 2012).
• microscale thermophoresis was utilised to evaluate the binding affinities for this family of RNA binders.
• MST microscale thermophoresis
• a FAM-functionalised oligomer corresponding to the coronaviral pseudoknot is incubated with one of the binders, with the fluorescence of the bound oligomer exhibiting different temperature-dependence compared to the non-bound oligomer.
• the perturbed pseudoknot has one of its stems changed into a random sequence of the same length and thus is no longer able to form the full pseudoknot structure.
• MTDB was found to be not soluble enough for KD determination using MST although the trend of the curve indicated noticeable binding at concentrations 187.5 pM and above (Fig. 14b). No appreciable binding was observed for the mutated (perturbed) pseudoknot (Fig. 14c).
• MTDB-deg and TDB-deg have KD values of 1.86 and 2.88 mM, respectively (Fig. 14b).
• the KD values fell to 5.13 mM and 6.91 mM, respectively (Fig. 14c).
• the shape of the MTDB-deg binding curve indicates noticeable binding with concentrations above 134 pM, indicating that functionalization with the degrader did not compromise the binding affinity of this molecule.
• the degrader-linker component 3a (Click-Degrader 1) was also tested for bind to the pseudoknot oligonucleotides. With the concentrations tested, the binding was too weak to obtain a KD value, suggesting that this molecule is a much poorer binder than molecules derived from MTDB or TDB (Fig. 14b,c).

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